LC-ESI-MS-MS Analysis of Non-enzymatic Posttranslational ...Der größte Dank geht an meinen Mann...

LC-ESI-MS-MS Analysis

of Non-enzymatic Posttranslational

Protein Modifications

Der Naturwissenschaftlichen Fakultät

der Friedrich-Alexander-Universität Erlangen-Nürnbe rg

zur

Erlangung des Doktorgrades Dr. rer. nat.

vorgelegt von

Dima Aldiab

aus Damaskus

Als Dissertation genehmigt von der Naturwissenschaftlichen

Fakultät der Friedrich-Alexander-Universität

Erlangen-Nürnberg

Tag der mündlichen Prüfung: 30. 5. 2011 Vorsitzender der Promotionskommission: Prof. Dr. Rainer Fink

Erstberichterstatterin: Prof. Dr. Monika. Pischetsrieder

Zweitberichterstatter: Prof. Dr. Thomas. Drewello

Danksagung

Mein ganz herzlicher Dank gilt meiner Doktormutter Frau Prof. Dr. M. Pischetsrieder

für die Betreuung der Arbeit und für die freundliche und wissenschaftliche

Unterstützung während des Aufenthaltes in Deutschland. Mein Dank gilt weiterhin

dem Hochschulministerium in Syrien sowie der Tishreen-Universität, mit deren

Unterstützung die Durchführung dieser Abeit in Deutschland erst ermöglicht wurde.

Des Weiteren möchte ich mich bei Herrn Prof. Dr. T. Drewello für die Übernahme

des Zweitgutachtens bedanken, sowie Herrn Prof. Dr. M. Heinrich und Herrn PD Dr.

T. Göen für die Übernahme der Doktorprüfung.

Außerdem bedanke ich mich bei Herrn Dr. R. Waibel sowie dessen Mitarbeiterinnen

für die Aufnahme der NMR-Spektren.

Sehr herzlich danke ich Frau C. Meißner für die freundliche Hilfe während des

Aufenthaltes in Deutschland.

Für die Freundschaft und Hilfsbereitschaft bedanke ich mich herzlich bei meinen

jetztigen und ehemaligen Kollegen im Arbeitkreis. Liebe Kollegen, ich danke euch:

Andrea Wühr, Artur Kessler, Bianca Meyer, Florian Baum, Gregor Vollmer, Ingrid

Weigel, Jasmin Meltretter, Kerstin Augner, Leonie Atzenbeck, Martin Tutsch,

Mellanie Deckert, Nina Zänglein, Nadine Schneider, Rainer Bäuerlein, Sabrina

Gensberger, Sarah Elschenbroich, Stefan Mittelmaier, Tanja Sauer, Tobias Hoch,

Ulla Müller und Viola Breyer.

Der größte Dank geht an meinen Mann Eyad und meine Eltern, sowie an meine

Freunde in Syrien.

Schließlich gilt ein besonderer Dank meiner verstorbenen Mutter. Danke Mama,

ohne deine Liebe und Unterstützung wäre diese Arbeit und viele andere wichtige

Dinge nicht möglich gewesen.

I

Table of contents

1 Introduction .................................... ....................................................................... 1

1.1 The Maillard reaction ........................................................................................ 1

1.2 The phases of the Maillard reaction ................................................................. 2

1.3 The effects of Maillard reaction ........................................................................ 4

1.3.1 Nutrition and food safety ........................................................................... 4

1.3.2 Advanced Glycation End-Products (AGEs) ............................................... 5

1.3.3 Physiological effects of dietary MRPs ....................................................... 6

1.3.4 Beneficial Maillard products ...................................................................... 8

1.4 Prevention of the Maillard reaction ................................................................... 9

1.5 Milk ................................................................................................................. 10

1.5.1 Milk proteins ............................................................................................ 11

1.5.1.1 Casein .............................................................................................. 12

1.5.1.2 Whey protein .................................................................................... 13

1.5.2 Heat treatment of milk ............................................................................. 14

1.5.3 Effects of heat treatment on milk ............................................................. 15

1.5.3.1 Effects of heat treatment on milk protein.......................................... 15

1.5.3.2 Effect of heat treatment on lactose .................................................. 17

1.5.3.2.1 Lactose isomerisation/degradation ........................................... 17

1.5.3.2.2 Lactose and Maillard reaction ................................................... 17

1.5.3.3 Effects of heat treatment on milk fat ................................................ 20

1.5.3.4 Effects of heat treatments on vitamins ............................................. 21

1.5.3.5 Other effects of heat treatment ........................................................ 21

1.5.3.5.1 Flavor ........................................................................................ 21

1.5.3.5.2 Allergenicity .............................................................................. 21

1.5.4 Indicators for milk heat treatment ............................................................ 22

1.6 Liquid Chromatography Electro Spray Ionization Tandem Mass Spectrometry (LC-ESI-MS-MS) .................................................................................................. 23

1.7 Aims of the work: ............................................................................................ 26

2 Results and discussion .......................... ............................................................ 27

2.1 Selection of the marker compounds for protein modification .......................... 27

2.2 Standards synthesis ....................................................................................... 33

2.2.1 Synthesis of LacLys ................................................................................ 33

2.2.1.1 Affinity chromatography ................................................................... 36

2.2.1.1.1 Boronic acid affinity chromatography ........................................ 37

2.2.1.2 Hydrophilic interaction liquid chromatography (HILIC) ..................... 38

2.2.1.2.1 ZIC-HILIC column ..................................................................... 39

2.2.1.3 Identification of LacLys by 1H-NMR ................................................. 42

2.2.1.4 Determination of the purity of LacLys by 1H-NMR............................ 43

2.2.2 Synthesis of CML .................................................................................... 44

2.2.3 Synthesis of MeSO ................................................................................. 45

II

2.2.4 Trial to synthesize lysine aldehyde ......................................................... 47

2.2.5 Trial to synthesize Cys-SOH ................................................................... 48

2.2.6 OH-Trp .................................................................................................... 49

2.2.7 Discussion ............................................................................................... 49

2.3 Method development for the analysis of glycation and oxidation products in milk by LC-ESI-MS-MS ........................................................................................ 51

2.3.1 Introduction ............................................................................................. 51

2.3.2 ESI-MS-MS conditions ............................................................................ 52

2.3.2.1 Flow-dependent MS parameters ...................................................... 53

2.3.2.2 Compound-dependent MS parameters ............................................ 53

2.3.3 MS2 fragmentation of the investigated analytes ..................................... 55

2.3.4 Chromatographic seperation of CML, LacLys, MeSO and 5-OH-Trp using a C18 column ................................................................................................... 57

2.3.5 Glycation of α-LA in a milk model mixture ............................................... 59

2.3.6 Oxidation of α-LA in a milk model mixture ............................................... 64

2.3.7 Discussion ............................................................................................... 66

2.4 Detection of glycation and oxidation products in milk ..................................... 68

2.4.1 Analysis of MeSO and LacLys ................................................................ 69

2.4.1.1 Formation of MeSO during sample work-up .................................... 71

2.4.2 Analysis of 5-OH-Trp .............................................................................. 72

2.4.3 Analysis of CML ...................................................................................... 73

2.4.4 Separation of 5-OH-Trp, MeSO, CML and LacLys using a ZIC-HILIC column ............................................................................................................. 74

2.4.5 Comparison of the signal intensities using C18 or ZIC-HILIC columns ... 75

2.4.6 Analysis of milk samples by LC-MS-MS using a ZIC-HILIC column ....... 76

2.4.7 Discussion ............................................................................................... 78

2.5 Analysis of ornithine in milk proteins .............................................................. 81

2.5.1 Introduction ............................................................................................. 81

2.5.2 LC-ESI-MS-MS analysis of ornithine ....................................................... 82

2.5.3 Discussion ............................................................................................... 85

2.6 Method optimization for the detection of MeSO, CML and LacLys in proteins of processed milk ..................................................................................................... 86

2.6.1 Optimization of protein hydrolysis ........................................................... 86

2.6.2 Results .................................................................................................... 87

2.6.3 Discussion ............................................................................................... 93

2.7 Method validation for the quantification of LacLys, CML and MeSO in milk proteins ................................................................................................................ 97

2.7.1 Introduction ............................................................................................. 97

2.7.2 Quantitative analysis by standard addition .............................................. 98

2.7.3 Results .................................................................................................. 100

2.7.3.1 Within-day repeatability .................................................................. 100

2.7.3.2 Between-day repeatability .............................................................. 101

2.7.3.3 Recovery ........................................................................................ 102

2.7.3.4 Limit of detection and limit of quantitation ...................................... 105

2.7.4 Discussion ............................................................................................. 106

2.8 Quantitation .................................................................................................. 109

2.8.1 Introduction ........................................................................................... 109

2.8.2 Results .................................................................................................. 109

2.8.3 Discussion ............................................................................................. 111

3 Materials and methods ........................... .......................................................... 114

III

3.1 Materials and apparatus ............................................................................... 114

3.1.1 General materials .................................................................................. 114

3.1.2 Apparatus .............................................................................................. 114

3.1.3 Materials ............................................................................................... 115

Synthesis ................................................................................................... 115

Hydrolysis .................................................................................................. 116

Materials and equipments for liquid chromatography and mass spectrometry ................................................................................................................... 116

Oxidation and glycation in milk models ...................................................... 117

3.2 Buffers and solutions .................................................................................... 117

Milk resembling Phosphate Buffer Saline (PBS), pH 6.8 ............................... 117

Solutions for enzymatic hydrolysis (Hasenkopf et al) ................................. 118

Solutions for enzymatic hydrolysis (Delatour et al) .................................... 118

Solutions for the ninhydrin assay ................................................................... 119

Solutions to isolate lysyl oxidase .................................................................... 119

Standard`s concentration to determine the fragments of LacLys, CML, MeSO and 5-OH-Trp ................................................................................................. 120

Aqueous mobile phases for liquid chromatography ....................................... 120

Solutions for affinity chromatography ............................................................. 120

3.3 Methods ....................................................................................................... 121

3.3.1 Lactulosyllysine synthesis ..................................................................... 121

3.3.1.1. Trial to synthesize lactulosyllysine ................................................ 121

3.3.1.2 Chromatographic conditions for the separation of FMOC-LacLys . 121

3.3.1.3 Removal of the blocking group of FMOC-LacLys .......................... 122

3.3.1.4 Trial to purifiy LacLys using m-Aminoboronic acid ......................... 122

3.3.1.5 Synthesis of FMOC-LacLys ........................................................... 122

3.3.1.5.1 Fractionation of FMOC-LacLys on a ZIC-HILIC column ......... 123

3.3.1.5.2 Determination of the purity of LacLys by 1H-NMR .................. 123

3.3.2 Synthesis of Cbz-CML .......................................................................... 123

Removal of the blocking group of Cbz-CML .............................................. 124

3.3.3 Synthesis of methionine sulfoxide (MeSO) ........................................... 124

3.3.4 Trial to synthesize lysine aldehyde ....................................................... 124

3.3.5 Trial to synthesize cysteine sulfenic acid (Cys-SOH) ............................ 126

3.3.6 The conditions of liquid chromatography for the seperation of CML, MeSO, LacLys and 5-OH-Trp on a C18 column ............................................ 126

3.3.7 The conditions of liquid chromatography for the seperation of CML, MeSO, LacLys and 5-OH-Trp on a ZIC-HILIC column ................................... 127

3.3.8 Stimulation of glycation in a milk model mixture .................................... 127

3.3.9 Stimulation of oxidation in a milk model mixture ................................... 127

3.3.10 Preparation of milk samples for LC-MS-MS analysis .......................... 128

3.3.10.1 Milk defatting ................................................................................ 128

3.3.10.2 Removal of lactose and minerals from milk and milk model mixture ................................................................................................................... 128

3.3.10.3 Enzymatic protein hydrolysis (Hasenkopf et al) ........................... 128

3.3.10.4 Purification of protein hydrolyzate by ultrafiltration ....................... 128

3.3.10.5 Enzymatic protein hydrolysis (Delatour et al) ............................... 129

3.3.10.6 Acidic protein hydrolysis (Delatour et al) ...................................... 129

3.3.10.7 Purification of protein hydrolyzate by solid phase extraction ........ 130

3.3.10.7.1 The standards MeSO, LacLys and CML on a SPE column .. 131

3.3.11 Trial to detect ornithine in milk protein ................................................ 131

IV

3.3.12 Investigation the effect of sodium borohydride on MeSO .................... 131

3.3.13 Ninhydrin assay .................................................................................. 132

3.3.14 Method validation ................................................................................ 132

3.3.14.1 Within-day repeatability (intra-day variation) ................................ 132

3.3.14.2 Between-day repeatability (inter-day variation) ............................ 133

3.3.14.3 Recovery ...................................................................................... 134

3.3.14.4 LOD and LOQ .............................................................................. 135

LOD and LOQ of LacLys and MeSO ..................................................... 135

LOD and LOQ of CML ........................................................................... 136

3.3.15 Qantitation ........................................................................................... 137

3.3.15.1 Determination protein content by Kjeldahl ................................... 138

4 Summary ......................................... ................................................................... 139

5 Zusammenfassung ................................. ........................................................... 144

Bibliography ...................................... ................................................................... 150

List of Tables .................................... .................................................................... 159

List of Figures ................................... ................................................................... 161

V

Nomenclature

AP amadori product

AGEs advanced glycation end products

amu atomic mass unit

BSA bovine seum albumin

Caco2 colon adenocarcinoma cell

Cbz-Lys carbobenzyloxylysine

CAD collision activated dissociation gas

CUR curtain gas flow

CML Nε-Carboxymethyllysine

cps count per second

CRP C-reactive protein

CYP1A2 cytochrome P450 1A2

Cys-SOH cysteine sulfenic acid

CE collision energy

CV coefficient of variation

CXP collision cell exit potential

DM diabetes mellitus

DMF dimethylformamide

DNP 2, 4 dinitrophenylhydrazine

1DG 1-desoxyglucosone

3DG 3-desoxyglucosone

DP declustering potential

EP entrance potential

ESI electrospray ionization

ESRD end stage renal disease

EtOH ethanol

FMOC-Lys flurenylmethyloxycarbonyl lyisne

Gal galactose

HA formula hypoallergenic formula

VI

1H-NMR Proton Nuclear Magnetic Resonance

h hour

H2O2 hydrogen peroxide

HILIC hidrophilic interaction liquid chromatography

HMF hydroxymethylfurfural

HMW high molecular weight

HPLC high performance iquid chromatography

IBD inflammatory bowel disease

igG immunoglobulin G

IQ imidazaquinoxaline

IS ion spray voltage

KD kidney disease

Lac lactose

LacLys lactulosyllysine

LC liquid chromatography

LC-ESI-MS-MS liquid chromatography electrospray ionization

tandem mass spectrometry

LDL low density lipoprotein

LF lactoferrin

LMW low molecular weight

LOX losyl oxidase

LP Lactoperoxidase

m multiplett (NMR)

m/z mass to charge

MAOs Mono Amino Oxidase enzymes

MAP-kinase Mitogen-Activated Protein kinase

mAU milli absorbance unit

Me methionine

MeOH methanol

MeSO methionine sulfoxide

MeSOO methionine sulfone

MGO methylglyoxal

min minute

MRM multireaction monitoring

VII

MRPs Maillard reaction products

MS-MS tandem mass spectrmetry

NAT2 N-acetyltransferase

NF-kappaB nuclear factor kappa of activated B cells

NFPA nonafluoropentanoic acid

NPLC normal phase liquid chromatography

5-OH-Trp 5-Hydroxytryptophan

O2. superoxide

OH. hydroxyl radicals

PBS phosphate buffer saline

Pd palladium

PP proteose-peptones

ppm part per million (NMR)

RAGE receptors for advanced glyacation end products

ROS reactive oxygen species

RP reversed phase

RT retention time

s singlet (NMR)

S/N signal to noise

t triplet (NMR)

Temp temperature

TFA trifluoroacetic acid

TIC total ion chromatogram

TNF alpha tumor necrosis factor alpha

UHT ultra high temperature

VCAM vascular cell adhesion molecule

WP whey protein

α-LA alpha lactalbumin

β-LG beta lactoglobin

δ chemical shift (NMR)

J J-coupling (NMR)

CHAPTER 1. INTRODUCTION 1

1 Introduction

1.1 The Maillard reaction

The Maillard reaction or non-enzymatic browning comprises a series of chemical

reactions between amino groups and carbonyl compounds leading to the formation

of a variety of Maillard reaction products (MRPs). Controlled browning is often used

to develop desirable flavor, odor or color in food including coffee, bread and soy

bean sauce (Friedman 1996).

First studies of the Maillard reaction were focused on the reaction of

monosaccharides, especially glucose and fructose, and disaccharides such as

maltose and lactose. However, work has also been carried out on polysaccharides

(Gerrard 2002). Additionally, many studies elucidated the role of fat and

carbohydrate breakdown products such as methylglyoxal in Maillard chemistry

(Thornalley 1994).

Maillard reactions were first reported in 1912. Since then, food scientists studied the

mechanism of browning and its effects on food appearance, safety and nutritional

quality, while in medical science the relationship between browning reactions in vivo,

diseases and aging has only be explored for about 30 years (Friedman 1996).

Initially it was discovered that human hemoglobin contains protein-bound Amadori-

products that are particularly formed in diabetic patients with elevated blood glucose

levels. Measurement of fructosylated hemoglobin is now widely used as an index of

glycemia in diabetes (Schleicher 1991).


1.2 The phases of the Maillard reaction

The Maillard reaction can be divided into three phases. The primary reactions were

described by L.C.Maillard himself and are shown in figure 1. The condensation

between amine and carbonyl groups leads to the formation of labile Schiff-base,

which undergoes tautomerism yielding the relatively stable aminoketose, the

Amadori product (AP). Formation of AP is reversible and it is considered an

important intermediate in Maillard chemistry (Gerrard 2002). The products which

formed in this phase show neither color nor absorbance in the UV range (Weigel

2004).

Figure 1 : The early phase of the Maillard reaction

In the intermediate phase, Amadori products undergo many characteristic reactions

such as enolisation, elimination, autoxidation, decarboxylation, or aldol reaction

leading to a huge variety of products in varying yields (Gerrard 2002). These

products can show yellow to brown color and may absorb light in the UV range

(Weigel 2004). Many products were isolated from this stage such as

hydroxymethylfurfural (HMF), which has been used as indicator for heat damage

(Friedman 1996; Fernandez-Artigas, Guerra-Hernandez et al. 1999). Rearrangement

of the Amadori product can also produce highly reactive dicarbonyl compounds such

as methylglyoxal (MGO), 3-desoxyglucosone (3DG) and 1-desoxyglucosone (1DG)


which react again with free amino groups leading to cross-linking (Niwa, Katsuzaki et

al. 1997).

Many low molecular weight compounds formed during the early and intermediate

phases react with each other to form heterogeneous polymers during the late phase.

These products have a dark brown color and are called melanoidins. Additionally,

many Maillard products have been characterized that remain protein-bound such as

pentosidine, pyrraline. These moieties could be formed in human body between

protein-bound amines and sugars and called advanced glycation end-products or

AGEs (Gerrard 2002). The relationship between the early and late phases of the

Maillard reaction is exemplified in figure 2.

Figure 2 : Phases of the Maillard reaction


1.3 The effects of Maillard reaction

1.3.1 Nutrition and food safety

The loss of nutritional quality and food safety during the Maillard reaction is attributed

to the degradation of amino acids, a decrease in protein digestibility and to the

production of antinutritional and toxic compounds. Some studies showed that the

loss of nutritional quality of heat-treated casein, casein-glucose and casein-starch

mixtures can be related to decreased protein digestibility rather than to the

destruction of essential amino acids (Friedman 1992). The blockage of the ε-amino

group of lysine during the Maillard reaction will decrease the activity of trypsin and

reduce the global protein digestibility. Additionally, the intermediate products of the

Maillard reaction can react with amino acids leading to enzyme-resistant cross-links.

Furthermore, some heterocyclic compounds formed in the later stage of Maillard

reaction can inhibit carboxypeptidase. These changes impair the intestinal

absorption of amino acids and as a result impair the nutritional quality of protein

leading to growth inhibition (Mauron 1990).

Mutagenic and carcinogenic products in cooked, protein-rich food are formed by

several mechanisms including carbohydrate caramelization, protein pyrolysis, amino

acid-creatinine reaction and the Maillard reaction. Mutagens formation is dependent

on heating-time and processing temperature. Heterocyclic amines are the most

potent mutagens formed by the Maillard reaction. They induce a variety of tumors in

rodents. Of special interest is their formation in heat processed fish and meat. Many

reactions take place during these processes such as cyclization and dehydration to

give pyrole, pyrazine, and pyridine derivatives. In the next step, the heterocycles

pyrazine and pyridine undergo further transformation with the participation of

aldehydes and creatinine to produce heterocyclic amines such as

imidazaquinoxalines (IQ). The high temperature recommended to destroy pathogens

in meat may lead to the formation of greater amounts of the heterocyclic amines. It

was found that the heterocyclic amines are activated in the human organism by

cytochrome P450 1A2 (CYP1A2) and by N-acetyltransferase 2 (NAT2) to give

mutagens and carcinogens (Friedman 1996).


Another class of the heterocyclic amines, called carbolines, are formed when free or

protein-bound tryptophan is exposed to heat under food processing conditions.

These products show binding to the benzodiazepine receptor, premutagencity,

inhibitory activity towards mono amino oxidase enzymes (MAOs) and psychtropic

effects (Ronner, Lerche et al. 2000).

1.3.2 Advanced Glycation End-Products (AGEs)

The Maillard reaction takes place in the body which makes the formation and the

generation of AGEs in vivo an inevitable process. AGEs stimulate cellular responses

mediated by specific receptors (RAGE) (Drinda, Franke et al. 2002) and can lead to

tissue damage through alterations of tissue protein structure and function, such as

crosslinkage. Cross-linked proteins exhibit decreased solubility and increased

proteolysis resistance (Smith, Taneda et al. 1994).

The accumulation of AGEs such as pentosidine, pyrraline and CML (figure 3) in

different tissues has been implicated in the process of aging (Ahmed, Frye et al.

1997) and in the pathogenesis of complications associated with diabetes mellitus

such as nephropathy, retinopathy and neuropathy (Monnier, Sell et al. 1992). AGEs

were also found at elevated level in the serum and synovial tissue of patients with

rheumatoid arthritis (Drinda, Franke et al. 2002). Furthermore, Alzheimer`s disease

(Smith, Taneda et al. 1994), cataract (Lyons, Silvestri et al. 1991), and

atherosclerosis (Nagai, Hayashi et al. 2002) were related to the formation of AGEs.

Figure 3 : Some structures of AGEs


1.3.3 Physiological effects of dietary MRPs

The structural similarities between MRPs and AGEs lead to question the impact of

dietary MRPs consumption on the induction of systemic effects similar to the effects

of the analogues AGEs. The effects of MRPs depend on their absorption,

metabolism, and excretion. It is still little known about the effects of small intestinal

enzymes and those of large intestinal bacteria on melanoidins; additionally, the

metabolism of MRPs is still under investigation. The intestinal absorption of MRPs

was investigated in different animals showing that most of the ingested MRPs were

not absorbed but excreted in the feces, probably due to the resistance to enzymatic

and acidic hydrolysis in the gastrointestinal tract. The absorbed MRPs were either

excreted in the urine or retained in different tissue over time. Exemplarily, rats fed

heated egg white-glucose mixture excreted 3.2% of the ingested MRPs in urine and

73.6% in the feces (Valle-Riestra 1970). In another study, the metabolic transit of

casein-14C-glucose derived melanoidins was investigated after fractionation into high

molecular weight melanoidins (HMW >10 kDa) and low molecular weight

melanoidins (LMW< 10 kDa). 27% of LMW melanoidins were excreted with the urine,

while 61% were excreted in the feces. Just 4.3 % of the HMW melanoidins were

excreted in the urine versus 87 % in the feces indicating that the absorption of HMW

melanoidins is lower than this one of LMW melanoidins. Both of them are used or

retained in the body only in little amount, since the radioactivity remained in the

carcass was very low (Finot and Magnenat 1981).

The experiments have been expanded to involve humans. It was demonstrated for

example that the concentration of both, CML and fluorescent AGEs, in the plasma of

healthy omnivores were lower compared with those in the plasma of healthy

vegetarians (vegetarian diet duration 7-8 years). The anticipated mechanism

involves an enhanced dietary AGEs flux in vegetarians caused by a higher intake of

both whole grain products and technological processed grain products (Sebekova,

Krajcoviova-Kudlackova et al. 2001). One study on human has shown that a diet rich

in MRPs, which was prepared from egg white and fructose, induced an elevation of

AGEs concentration in serum and urine. Serum level of AGEs increased proportional

to the ingested amount of MRPs, while the urinary excretion correlates inversely with

the degree of kidney disease (KD) (Koschinsky, He et al. 1997). Additionally, the

serum concentrations of glycated LDL were higher in the patients with both diabetes


mellitus (DM) and end stage renal disease (ESRD) followed by those with only

ESRD compared to the healthy control. Thus, it can be assumed that a diet rich in

MRPs increases the risk factors for disease development in patient with KD (Bucala,

Makita et al. 1994). Some of the consumed MRPs can survive the digestive process

and transported into the circulation resulting in elevation of AGEs concentration in

serum. Consequently, they induce an increase in the level of the inflammatory

mediators such as CRP, VCAM and TNFα as well as glycated LDL (Vlassara, Cai et

al. 2002). In vitro, the thermally generated AGEs promote a dose-dependent

activation of MAP kinase in Caco2 cells through the binding to RAGE. This activation

may result in cellular reactions such as inflammatory response, cellular proliferation,

tumor growth and metastasis (Zill, Bek et al. 2003).

Maillard products which cannot pass into the circulation can affect the mucosa of the

large bowel. It is well known that the activation of nuclear transcription factor NF-κB

in both, mucosal macrophage and T lymphocytes, plays an important role in the

pathogenesis of inflammatory bowel diseases (IBD) such as Crohn`s disease and

ulcerative colitis. Therefore, the aim of several new treatment strategies of IBD is to

achieve a specific blockage of NF-κB, but all of these drugs must be cautiously used

since NF-κB is involved in cell growth and apoptosis. The activation of NF-κB can be

induced by variable stimuli like oxidative stress, cytokines, bacteria and viruses

(Dijkstra, Moshage et al. 2002). Additionally, it can be induced by MRPs via the

generation of H2O2. For example, one study demonstrated that the stimulation of

macrophages with coffee-derived or lysine-ribose-derived melanoidins induced a 13

and 18 fold increase of NF-κB activation, respectively (Muscat, Pelka et al. 2007).

Thus, the consumption of MRPs-rich diet may affect the intestinal immune function

and participate in the pathogenesis of IBD.

The consumption of food-derived MRPs is implicated in the progression of many

other diseases. Exemplarily, the long-term dietary intake of MRPs by rats induced an

increase in the renal and hepatic magnesium concentration and a decrease in the

femoral magnesium concentration which could affect the bone health (Delgado-

Andrade, Seiquer et al. 2007). MRPs cause also a hypertrophy of organs like liver or

stomach and they affect the activities of the intestinal pancreatic enzymes such as

chymotrypsin. Additionally, they have adverse effects on the mineral metabolism

(Ca, Mg, Cu, and Zn) and variable effects on both allergic response and cholesterol


metabolism. The extent of the severity of these effects is influenced by the severity

of heat treatment, by the content and the structure of different Maillard and cross-

linked products as well as by the health and susceptibility of the consumer (Finot,

Aeschbacher et al. 1990).

1.3.4 Beneficial Maillard products

Beside the undesirable effects of MRPs, they may also have health promoting

effects such as antimutagenic activity. The available information about the mutagenic

effect of MRPs is contradictory. Some studies describe MRPs as mutagenic, others

as slight or not mutagenic. Wagner et al have reported that MRPs from heated

fructose-cysteine mixtures have antimutagenic effect in the presence of H2O2 and S9

(Wagner, Reichhold et al. 2007). S9 are microsomes derived from the membrane of

endoplasmic reticulum and considered as source for metabolizing enzymes,

especially cytochrome P450. The antimutagenic mechanism of the Maillards

products can include inhibition or reduction of the hepatic microsomal activity, or

scavenging of free radicals by melanoidins. Antimutagens are classified into

desmutagens and bioantimutagens. Desmutagens inactivate mutagens by chemical

or enzymatc modifications, while bioantimutagens suppress the mutagenesis after

DNA modification by the mutagen (Kada, Inoue et al. 1986). Furthermore,

melanoidins may be able to reduce the absorption of mutagens (Powrie, Wu et al.

1986).

Melanoidins, which are formed during the late phase of the Maillard reaction,

possess further antioxidative activity. This antioxidative activity is related in most

cases to poorly characterized melanoidins. Scavenging of reactive oxygen species

such as hydroxyl radicals, binding of metal ion and decomposing of primary radical

to non radical compounds could be the important mechanisms of their antioxidative

effect (Morales and Jimenez-Perez 2004). It is well known that oxidized LDL plays

an important role in the development of atherosclerosis. Some studies have shown

that glucose-lysine, arginine or glycine derived Maillard products can inhibit copper-

induced oxidation of human LDL in vitro. Thus, they could reduce the risk of

cardiovascular disease in vivo (Dittrich, El-Massry et al. 2003).


Other studies have demonstrated that fructose-tryptophan derived Maillard products

prevent lipid oxidation in sardin, while glucose-tryptophan derived Maillard products

inhibit linoleic acid oxidation (Friedman 1996) .

Maillard reaction products show antibiotic effects against many strains of gram-

positive bacteria, such as bacillus subtilus, lactobacillus and staphylococcus. The

growth of different Escherichia strains was inhibited to different extent, while the

strains of salmonella were not at all inhibited. Products with higher molecular weight

(higher than 1000 Da) had a greater inhibitory activity than those with lower

molecular weight (Einarsson, Snygg et al. 1983). Potential targets of the antibacterial

activity of MRPs include bacterial membrane, genetic material, bacterial enzymes

and reduced oxygen uptake as a result of its interaction with iron (Einarsson, Eklund

et al. 1988; Friedman 1996). A variety of casein-lactose derived melanoidins have

shown a strong inhibition of the adherence of urease to gastric mucin and so

suppressed Helicobacter pylori colonization, which induces gastric pathologies in an

animal model and in human subjects (Hiramoto, Itoh et al. 2004). Additionally,

melanoidins can stimulate the growth of the nutritional gut flora in vitro such as

bifidobacteria, lactobacilli, bacteroides and clostridia. Thus, melanoidins may affect

the growth of these bacteria in vivo which improve the health of lower gut (Ames,

Wynne et al. 1999).

1.4 Prevention of the Maillard reaction

Many approaches can be used to prevent or minimize food browning and its

antinutritional and toxicological consequences.

Thiols inhibit browning reactions and, under certain conditions, they may be as

effective as sodium sulfite in preventing nonenzymatic browning. Acetylation of

lysine amino group by transglutaminase will transform it to amide group and thus will

prevent its participation in the browning reaction. Some studies revealed that oxygen

seems to be required for non-enzymatic browning, consequently, antioxidants should

suppress the browning in food (Friedman 1996). Finally a variety of substances such

as aminoguanidine, acetylsalicylic acid, or pyridoxine have been reported as

inhibitors of the Maillard reaction (Matsuura, Aradate et al. 2002).


1.5 Milk

The presence of lactose and proteins as well as the neutral pH value render milk as

a suitable medium for the Maillard reaction during processing and storage. This

leads to alteration of the nutritional value of milk concurrently with the formation of

various products like lactulosyllysine and CML.

Milk is produced in mammary glands in mammals. There are different sources of

milk for human nutrition such as cattle, goats and sheep, but cow is the major source

of milk in the world. Nowadays, the word milk refers to cow milk. Milk and milk

products represent important dietary components because of their high nutritional

value (Belitz. H. D 2009).

Milk is a complex liquid food with 87 % water. It contains numerous nutrients such as

saturated and unsaturated lipids, proteins including caseins and whey proteins,

carbohydrates mainly lactose as well as different vitamins and minerals. The

important milk components for human nutrition and their concentrations are

illustrated in table 1.

In addition to its nutritional value, some studies suggested physiological functions of

milk proteins that may promote health. It has been reported for example that some

milk peptides have antihypertensive effects through the inhibition of angiotensin-

converting enzymes (Nakamura, Yamamoto et al. 1995). Other peptides derived

from κ-casein have antithrombotic effect due to its ability to inhibit both, the

aggregation of ADP-activated platelets and the binding of fibrinogen to ADP-

activated platelets (Jolles, Levy-Toledano et al. 1986).

Other studies reported that milk consumption may stimulate insulin release, an effect

which may be caused by whey proteins (Nilsson, Holst et al. 2007). Fresh milk is a

good source of glutathione which is a tripeptide of cysteine, glycine and glutamic

acid. Glutathione acts as an antioxidant, whereby it reduces the levels of reactive

oxygenspecies (ROS) in cells (Haug, Hostmark et al. 2007).

Milk is also a source of selenium and the vitamins A and E. These three nutrients

exhibit also antioxidant activity and promote the human health (Lindmark-Mansson

and Akesson 2000). Many factors can affect milk composition such as cattle breed,

age, stage of lactation, health status of the udder, energy balance and feeding


regime. For example, milk concentration of selenium, vitamin A and E reflex their

intake by feed (Haug, Hostmark et al. 2007).

Cow milk differs from human milk in many aspects. It has about 3 and 7 times higher

levels of minerals and casein, respectively, than human milk. Additionally, in human

milk there is no β-lactoglobulin (β-LG), immunoglobulin G (IgG) is replaced by

immunoglobulin A (igA), while lactoferrin (LF) and lysozyme present the two major

proteins. Despite all of these differences, cow milk is still the major ingredient for

infant formulas (Wit 1998)

milk component

content in 1L whole milk

milk component

content in 1L whole milk

total fat 33 g magnesium 0.1 g

total saturated fatty acids

19 g zinc 4 mg

oleic acid 8 g selenium 37 µg

omega 6 fatty acid

1.2 g vit E 0.6 mg

omega 3 fatty acid 0.75 g vit A 280 µg

lactose 53 g vit B12 4.4 µg

proteins 32 g riboflavin 1.83 mg

calcium 1.1 g folate 50 µg

Table 1 : Milk components and their concentrations in whole milk (Haug, Hostmark et al. 2007)

1.5.1 Milk proteins

The total protein content of one liter milk is about 33 g which is divided approximately

into 80% of caseins and 20% of whey proteins. These proteins represent a source

for essential amino acids and have a high nutritional value.


1.5.1.1 Casein

Up to 10% of the total casein fraction is present as monomers, the other part exists

in colloidal particles known as casein micelles. The principal casein fractions are αS-

casein, β-casein, γ-casein and κ-casein. These fractions are present in water in

colloidal form, except of κ-casein which is soluble. Caseins differ in their properties

from whey proteins and among each other. Casein micelles consist of small units

called submicelles which bind together through calcium phosphate bridges. Each

submicelle contains about 30 casein molecule. There are two different types of

submicelles. Submicelles with κ-casein occupy the surface of the micelles and

prevent the aggregation of submicelles by steric repulsion. Submicelles without κ-

casein are localized in the interior position of the micelles. Casein is not very

susceptible to coagulation. It coagulates only when heated at high temperature for

long time (e.g. 5 hours at 120 °C).

However, temperature and low pH can strongly affect the structure of the micelles.

For example, caseins coagulate at pH 4.6; this coagulation is enhanced by

increasing heat treatment. On the other hand, the coagulation temperature drops

with decreasing pH (Belitz. H. D 2009).

One biological function of casein is to carry calcium and phosphate. Two thirds of the

total minerals in milk are bound to casein micelles, in which 44% of the total

inorganic phosphate and 90% of the calcium in milk are bound as colloidal calcium

phosphate. The components of casein micelles are shown in table 2. Another

function of casein, especially of αS-casein, is the production of the clot in the

stomach of infants for efficient digestion (Wit 1998).

micelle component %

micelle component %

casein 93.20 phosphate (org) 2.3

Ca 2.9 phosphate (anorg)

2.9

Mg 0.10 citrate 0.40

Na 0.10 K 0.30

Table 2: Composition of casein micelles (Belitz. H. D 2009)


1.5.1.2 Whey protein

Whey is the liquid which remains after casein has been precipitated during cheese

production. Whey proteins are globular proteins which represent about 20 % of the

total protein content in milk. Whey proteins are rapidly digested proteins which

provide high concentrations of essential amino acid, especially lysine and

tryptophan. Therefore they have a high nutritional value. The term whey proteins

comprises different proteins, namely β-lactoglobulin (β-LG), α-lactalbumin (α-LA),

bovine serum albumin (BSA), immunoglobulin G (IgG), lactoferrin (LF),

lactoperoxidase (LP), proteose-peptone (PP) and different enzymes (Haug,

Hostmark et al. 2007). These proteins differ in their biological function as well as in

their abundance in milk, as seen in table 3.

whey proteins Concentration in milk

g/L Biological function

β-LG 3.2 carier for provitamin A

α-LA 1.2 lactose synthesis

BSA 0.4 fatty acid transfer

IgG 0.8 passive immunity

LF 0.2 bacteriostatic agent

LP 0.03 antibacterial agent

Enzymes 0.03

Proteose-peptones 1.3 opioid activity

Table 3: Composition, abundance and biological function of whey proteins. Adapted from (Wit 1998)

There are clear differences in the sizes of casein micelles and whey proteins. The

radius of casein micelle is 50 nm. The radius of whey proteins vary between 1.8 nm

for α-LA, 3.5 nm as diad axis of β-LG. Radius of BSA is 4 nm and tetrad axis of IgG

is 6 nm (Wit 1998).

Whey proteins have high nutritional value due to the high content of essential amino

acids. Additionally, whey proteins are widely used in food industry because of their


functional properties. For example, they are used in confectionery, dairy and bakery

products as replacement of egg protein to induce and stabilize foaming, as well as

for their emulsifying properties. Moreover, desalted whey is used to produce infant

formulas. In this case, cow milk is supplemented with whey proteins to reduce the

ratio between casein and whey protein from 80/20 to 40/60 in infant formula.

Furthermore, the content of lactose, fat, minerals and vitamins of infant formuls is

adapted to human milk (Wit 1998).

Besides the nutritional and functional properties of whey proteins, some studies have

reported that milk whey proteins may promote bone formation in healthy adult

women (Aoe, Toba et al. 2001). Additionally, they inhibit the formation of gastric

mucosal ulcerative lesions in the stomach of rats caused by absolute ethanol

ingestion (Rosaneli, Bighetti et al. 2002). Other studies reported that whey proteins

exhibit antiviral activity (Andersen, Jenssen et al. 2003) in animal studies in addition

to anti cancer effect in the patients with metastatic carcinoma (Kennedy, Konok et

al. 1995). Furthermore, different effects on the metabolism and the immune system

were observed (Krissansen 2007).

1.5.2 Heat treatment of milk

Milk heat treatment is classified into several categories:

1. Thermization: In this process, milk is heated under conditions milder than

pasteurization (57 °C) which reduces the number of bacteria and preserves

milk taste.

2. Pasteurization: For pasteurization, either high temperature is applied for short

time like 85 °C for 2-3 s and 72-75 °C for 15-30 s, or low temperature for long

time while stirring (e.g. 63-66 °C for 30-32 min). Pasteurization destroys all

pathogens that are present in milk.

3. Ultrahigh temperature treatment (UHT) eliminates all spores and

microorganisms. It is applied either indirectly by coils or plates at 136-138 °C

for 5-8 s, or directly by live steam injection at 140-145 °C for 2-4 s followed by

aseptic packaging.

4. Bactotherm process is a combination of centrifugal sterilization in bactofuges

at 65-70 °C and UHT treatment of the separated sedi ment followed by

recombination.


5. Sterilization: Sterilized milk is usually produced in 2 steps consisting of

heating in an autoclave at 107-115 °C for 20-40 min followed by heating in

bottle at 120-130 °C for 8-12 min (Belitz. H. D 200 9).

Heat treatment in general is applied to kill all disease-causing microorganisms

ensuring safety of milk consumption as well as to increase the products shelf live.

Additionally, heat treatment is important to achieve the functional uses of whey

proteins. However, during thermally treatment, several milk constitutes can be

negatively affected.

1.5.3 Effects of heat treatment on milk

1.5.3.1 Effects of heat treatment on milk protein

During heat treatment of milk, many reactions can take place which involve only

protein or protein with the other macronutrients. These reactions include:

• Protein reactions: Lysinoalanine (LAL) is formed by the reaction of lysine with

dehydroalanine. The latter is produced by β-elimination of cysteine and serine

(Mauron 1990). The content of LAL in sterilized and condensed milk was 13

and 18 fold higher than the content in fresh milk (Haagsma and Slump 1978).

• Protein-oxidized lipid interactions: During these reactions, unsaturated lipids

are oxidized resulting in different products. These products can be classified

into primary products (hydroperoxide), secondary products (aldehyde, ketone,

carbonyls) and stable products like carboxylic acids. Methionine is rapidly

oxidized by hydroperoxides to methionine sulfoxide, while lysine reacts with

the secondary products like aldehyde and ketone in a Maillard-type reaction

(Nielsen, Loliger et al. 1985).

• Protein-polyphenol interactions: These processes include the reaction of

lysine or methionine with quinones to form lysine-quinones adducts and

methionine sulfoxide, respectively (Mauron 1990). Such reaction might be

relevant to choco-milk products.

• Protein-lactose reactions: This reaction is called Maillard reaction or protein

glycation and it is considered as the most frequent reaction in milk leading to

protein damage during processing.


• Protein oxidation: In parallel to glycation, milk proteins are subjected to

various oxidation reactions under processing conditions (Mauron 1990). In

milk products, tryptophan, methionine and cysteine are particularly sensitive

to oxidative modification. For example, tryptophan oxidation can lead to an

important number of derivatives generated by opening of the indole ring.

Oxidation converts methionine to methionine sulfoxide and further to

methionine sulphone under stronger oxidative conditions (Guy and Fenaille

2006). Cysteine could be oxidized to cysteine sulfenic acid (Meltretter, Seeber

et al. 2007). Other residues, such as lysine, proline, arginine, histidine and

tyrosine can also be subjected to oxidation. Oxidation of the latter amino acids

is often characterized with the formation of carbonyl groups within their

structures (Guy and Fenaille 2006). Carbonyl formation is also a result of the

reaction between the amino group and the reactive dicarbonyl compounds

generated from Maillard reaction and/or lipid oxidation. In addition, free radical

produced by metal-catalyzed oxidation in infant formulas can yield carbonyl

groups. Therefore, the protein carbonyl (PRC) content is used as an indicator

for protein oxidation (Fenaille, Parisod et al. 2005). Lysine aldehyde is an

example for a carbonyl product formed in milk proteins during heat treatment

(Meltretter, Seeber et al. 2007).

• Denaturation: Heat treatment can change the secondary and tertiary

structures of milk proteins, mainly whey proteins causing denaturation

(Mauron 1990). The order of heat stability of whey protein is : α-LA > β-LG >

BSA > IgG (Mortier L 2000). During denaturation sulfhydryl groups are

cleaved, especially in β-LG, leading to the formation of sulfur compounds, like

methyl sulfide, which contribute to the characteristic flavor of milk (Calvo

1992).

The previous interactions lead to the degradation of essential amino acids as well as

to a decrease of the protein digestibility. Consequently they reduce the nutritional

value of milk (Mauron 1990).


1.5.3.2 Effect of heat treatment on lactose

1.5.3.2.1 Lactose isomerisation/degradation

Lactose is the main natural disaccharide in milk consisting of glucose and galactose.

During heat treatment of milk, lactose is subjected to isomerisation and degradation.

It has been shown that the activation energy for isomerisation at low temperature

(<100 °C) is higher than the early Miallard reactio n indicating that the participation of

lactose in the early Miallard reaction is more favored than its isomerisation. On the

other hand, lactose isomerisation and degradation is more important at high

temperatures (120 °C). It was found for example tha t 80% of lactose has undergone

isomerisation at sterilization temperature versus 20% of lactose participated in the

Maillard reaction. The products of lactose isomerisation are lactulose and epilactose.

Lactulose is a disaccharide of galacatose and fructose which is normally not found in

raw milk, while epilactose is a disaccharide of galacatose and mannose (van Boekel

1998).

Epilactose is detected in small amounts only in sterilized milk. Lactulose is detected

in higher amounts in heated milk like sterilized and UHT milk as well as in

pasteurized milk (Olano, Calvo et al. 1989). In condensed milk, for example, about

10 % lactose is isomerized to lactulose (Belitz. H. D 2009). Lactulose is widely used

as indicator for heat treatment of milk, especially to distinguish UHT milk from

sterilized milk, because its content in sterilized milk is 5 times higher than in UHT

milk (Olano, Calvo et al. 1989; Luzzana, Agnellini et al. 2003).

Many products arise from lactulose degradation such as galactose, formic acid and

others. Formic acid is formed in relatively large amounts and is mainly responsible

for the pH decrease in heated milk. The degradation products of lactose can undergo

further Maillard reaction (van Boekel 1998). Studies have shown that although the

amount of galactose increases proportionally to the severity of heating, this marker

could not clearly differentiate between different types of heat treatment (Olano, Calvo

et al. 1989).

1.5.3.2.2 Lactose and Maillard reaction

In the early stage, lactose reacts with the ε-amino group of protein-bound lysine

forming the Schiff’s base which is transformed into lactulosyllysine (LacLys) via


Amadori product rearrangement (figure 1). In the advanced stage, LacLys is

fragmented to give CML, erythronic acid and galacatose (van Boekel 1998).

Figure 4 : Early stage of the Maillard reaction in milk

CML is formed only in a little amount in heated milk and is described as a useful

indicator for the advanced stage of the Maillard reaction in milk products (Fenaille,

Parisod et al. 2006).

Later, it has been shown that CML is formed by two additional mechanisms. During

autoxidative glycation, CML is formed by the reaction between lysine and glyoxal

which results from metal-catalysed glucose autoxidation. In the third mechanism,

which is also called Namiki pathway, the Schiff’s base is degraded resulting in C-2

fragments like glyoxal and glycoaldehyde, which subsequently react with lysine to

form CML (Glomb and Monnier 1995; Ferreira, Ponces Freire et al. 2003). Figure 5

illustrates the three pathways for the formation of CML.


Figure 5: Formation of CML through different pathways (Ferreira, Ponces Freire et al. 2003)

Besides the oxidative cleavage, LacLys is subjected to further degradation (figure 6),

mainly via 2, 3-enolization leading to 1-deoxyosone, which is a very reactive

intermediate. 1-Deoxyosone then undergoes further reactions like enolization or

cyclization leading to 3-furanone and β-pyranone, respectively. β-Pyranone

isomerizes to cyclopentenone and both of them may convert into

galactosylisomaltose. These products may further react with proteins to form cross-

linked proteins.

There are two other breakdowns routes for degradation of LacLys. The 3-

deoxyosone-pathway via 1,2-enolization is favored under acidic conditions and leads

to the formation of HMF, pyrraline as well as pentosidine. Both lysylpyrraline and

pentosidine are formed in very little amounts in milk and could be used as indicators

for severe heat treatment. The third breakdown route is the 4-deoxosone-pathway

which is favored under more alkaline conditions leading to 4-deoxyaminoreductone.

The last two degradation routes are probably not important in milk because of its

neutral pH (van Boekel 1998). In the final stage, melanoidins are formed which are

bound to milk proteins. Melanoidins in milk are not very well characterized (van

Boekel 1998).


Figure 6: Degradation of Amadori product under acidic, neutral or alkaline conditions

1.5.3.3 Effects of heat treatment on milk fat

Milk fat are present as globules surrounded by a complex membrane called milk fat

globule membrane (MFGM) which contains a mixture of unsaturated phospholipids,

proteins, glycoproteins, cholesterol, enzymes and other minor components. These

globules are emulsified in the aqueous phase. Milk fat contributes to the flavor and

color of milk. However, it is one of the least chemically stable compounds in milk

which can readily undergo autoxidation, especially under the conditions of

processing and storage. Unsaturated fatty acids are the main target of oxidation, in

particular unsaturated phospholipids in the MFGM. Milk cholesterol can also be

oxidized under the conditions of heat treatment and drying forming more than 60

cholesterol products. Some of them can induce the onset of atherosclerosis (Guy

and Fenaille 2006).


Lipid oxidation deteriorates the fat itself and produces oxidative fragments, some of

which are volatile and can cause a rancidic off-flavor. Furthermore, some lipid

oxidation products like dicarbonyl compounds can react readily with food proteins to

form Advanced Lipidoxidation End-products (ALEs). Consequently, lipid oxidation

has a negative impact on color, flavor, functional/physical properties and nutritional

value (Guy and Fenaille 2006).

1.5.3.4 Effects of heat treatments on vitamins

During heat treatment, the water-soluble vitamins B1, B6, B12, folic acid and vitamin C

are prone to degradation. About 10-30% of these vitamins are lost in UHT milk.

Sterilization destroys ca 50% of the vitamins B1, B6, folic acid and up to 100% of

vitamin C and B12 (Belitz. H. D 2009). Fat soluble vitamins are also susceptible to

degradation. For example, UHT milk has lost up to 52% of α-tocopherol after 3

months storage at 40 °C. α-Tocopherol levels in milk powder decreased by 17% after

storage for 20 days at 20 °C (Guy and Fenaille 2006 ).

1.5.3.5 Other effects of heat treatment

1.5.3.5.1 Flavor

The flavor of fresh milk is caused by a numerous natural components. During heat

treatment, milk flavor is altered due to the formation of new compounds. These new

compounds can be generated from proteins, lipids, lactose and others. For instance,

sulfur compounds which give milk the cooked flavor are formed due to denaturation

of proteins, mainly of β-LG, and subsequent cleavage of sulfhydryl compounds.

Fat are also important contributors to milk flavor due to the formation of methyl

ketones, aldehyde and hexanal during heat treatment of milk. Additionally, lactose

degradation products like benzaldehyde and furanoids can contribute to the flavor of

heated milk (Calvo 1992). Strong heat treatment of milk, like sterilization, results in

the accumulation of volatile Maillard products like methylpropanal which affect milk

flavor (Belitz. H. D 2009).

1.5.3.5.2 Allergenicity

It was observed that milk is less antigenic in vivo after heat treatment. The antigenic

sites of protein may be selectively altered by the reaction with reducing sugar under


mild heat treatment. This chemical and structural modification could be responsible

for the observed reduction in antigenicity (Friedman 1996). In contrast, another study

conculded that the modified proteins, especially those that are resistant to digestion

tend to increase the allergenicity of milk, because they may represent the major

allergens in food (Fenaille, Parisod et al. 2005).

1.5.4 Indicators for milk heat treatment

Two types of chemical reactions may be used as indicators to evaluate the heat

treatment of milk. The first group includes degradation, denaturation and inactivation

of heat labile components like whey proteins and enzymes. The activity of negative

alkaline phosphatase and lactoperoxidase, for example, are used as indicators for

monitoring the heat damage of pasteurized and highly pasteurized milk, respectively.

The whey protein nitrogen index (WPNI), which expresses the amount of

undenaturated whey proteins, is also used as indicator for heat treatment.

The second group includes the formation of new substances like lactulose and the

Amadori product (Mortier L 2000). The Amadori product (LacLys) can be determined

by the furosine method, which is based on the acidic hydrolysis of LacLys at 110 °C

for 24 hours leading to the formation of furosine and pyridosine as well as to

regeneration of some lysine. However, when food is subjected to severe heat

treatment, the Amadori product is transformed into advanced glycation end-products.

Therefore furosine is not suitable as indicator for severely heated milk. Instead

advanced end-product such as CML must be applid as indicator for heat treatment

(Charissou, Ait-Ameur et al. 2007).


1.6 Liquid Chromatography Electro Spray Ionization

Tandem Mass Spectrometry (LC-ESI-MS-MS)

Liquid chromatography is a physical separation method base on the distribution of

the analytes between two phases. The first one is the stationary phase and the

second phase is a liquid phase that moves through the stationary phase in a defined

direction. Many detectors are in routine use together with liquid chromatography

such as UV, fluorescence, conductivity as well as mass spectrometry. Mass

spectrometry allows the differentiation of compounds which have similar retention

time independent on their chromatographic resolution (Ardrey 2003).

Mass spectrometry is an excellent qualitative tool for the determination of a

compound`s molecular weight. Moreover, tandem mass spectrometry (MS-MS)

induces characteristic fragments of the analyte which allows the elucidation of its

structure. Additionally, MS-MS is considered a very selective and sensitive detector

for the quantitation of analytes even in complex matrixes such as blood, plasma and

food. The greater selectivity of MS-MS compared to simple MS is the dual mass

selection, first by the selection of precursor ion from different ions in the matrixes,

and second by the selection of products ions, which arose from the pre-selected

precursor ion.

Many techniques are used to induce the ionization of the analytes in the ion source

under atmospheric pressure such as atmospheric pressure chemical ionization

(APCI) and electro spray ionization (ESI). For ESI, the eluate from the HPLC, is

directed into the ion source of the mass spectrometer through a capillary maintained

at high voltage and atmospheric pressure. The application of high voltage on the

electrospray needle disperses the liquid stream forming a mist of highly charged

droplets which are desolvated during their pass through the atmospheric pressure

region. Desolvation is assisted by a stream of drying gas, usually nitrogen.

The ionization in ESI takes place directly in the solution during spraying. The lower

temperature which is applied compared to APCI, makes ESI a softer ionization

technique, since no/or hardly any thermal decomposition of thermally labile

molecules is formed during ionization. Ionic, ionizable and polar compounds are

easily ionized by ESI, while non polar molecules have very poor response in ESI.


The ionization is followed by the evaporation of the solvent assisted by heat at

elevated flow rates (Zimmer 2003).

In positive-ion mass spectrometry, ions are formed by the addition of various

species, mostly protons, to the analyte molecule. In negative-ion mode, the

molecules are usually deprotonated to form ions. The number of charges on the ion

is related to the number of added species. Thus, the measured mass to charge ratio

(m/z) of one ion is dependent on the molecular weight of the analyte itself as well as

on the number of the added species, as shown by the following equation:

m/z = (M+nH)/n

M is the molecular weight of the analyte, H is the molecular weight of the added

species (1 for hydrogen, 18 for ammonium ion and 23 for sodium) and n is the

number of charges which are carried by the ion. Ions are generated outside of the

mass analyzer and are then directed into its entrance.

The triple quadrupole is the most widely used mass analyzer in MS-MS. It consists of

three sets of quadrupole rods: two mass analyzer, MS1 and MS3 (Q1 and Q3), and

the collision cell (Q2) (Ardrey 2003). Figure 7 illustrates the set up of a triple

quadrupole tandem mass spectrometer.

The molecular ion [M+H]+ (precursor ion) is selected in the first mass analyzer (Q1),

and is then transmitted into the collision cell (Q2), where fragmentation is carried out.

In the collision cell, the ions collide with a wall of gas molecules, usually nitrogen,

which is called collision gas or collisionally activated dissociation gas (CAD gas).

The generated fragment ions f+1, f+2, f+3, f+4 (product ions) are focused into the

second mass analyzer (Q3), where usually only the product ions of the highest

intensity are chosen and monitored (f+1,f+

3).

This technique is called selected reaction monitoring (SRM), because it monitors the

fragmentation of a selected precursor ion to a selected product ion. It is considered

as one of the most widely used MS-MS experiments. The term multiple reactions

monitoring (MRM) refers to simultaneous monitoring of several pairs of products ions

and precursor ions derived from different analytes (Zimmer 2003).

Other widely used MS-MS experiments are the product ion-scan, the precursor ion

scan and the constant neutral loss scan.

A single product ion is usually inadequate to identify the analyte of interest. If

chromatography is involved, the combination of retention time and a single product

ion may be considered adequate, especially if this ion is generated by soft ionization


technique. However, in order to ensure the identity of the analyte, it is recommended

to monitor at least two product ions of the analyte (Ardrey 2003). In such cases, the

product ion which gives a chromatographic peak with the highest intensity is called

the quantifier fragment (f+3), because it is used for quantification. The other

fragments are used to confirm the analyte`s identity and are called the qualifier

fragments (like f+1). Mass spectrometry is commonly used for the quantitation of low

molecular weight molecules below 1000-2000 Da (Zimmer 2003).

Figure 7: Schematic illustration of tandem mass spectrometry in MRM mode [adapted from (Zimmer 2003)]


1.7 Aims of the work:

Untargeted peptide mapping indicate that lactulosyllysine, ε-carboxymethyllysine,

methionine sulfoxide, lysine aldehyde and cysteine sulfenic acid or

hydroxytryptophan are the major protein modifications which are formed during the

heating of a milk model. So far however, the relevance of these products in heated

milk is not clear. Therefore, the aims of the present work were the quantification of

the major protein modification in heated milk by LC-ESI-MS-MS analysis of the

modified amino acids after enzymatic protein hydrolysis. For this purpose the

following steps were carried out:

• The synthesis of standard compounds lactulosyllysine, ε-carboxymethyllysine,

methionine sulfoxide, lysine aldehyde and cysteine sulfenic acid, which

represent the major modified amino acids in milk.

• Development of a method to detect lactulosyllysine, ε-carboxymethyllysine,

methionine sulfoxide, lysine aldehyde, cysteine sufenic acid as well as 5-

hydroxytryptophan and ornithine in milk proteins via LC-ESI-MS-MS in MRM

mode.

• Development of a method to release lactulosyllysine, ε-carboxymethyllysine

and methionine sulfoxide from milk proteins by enzymatic hydrolysis.

• Validation of the method

• Quantification of lactulosyllysine, ε-carboxymethyllysine and methionine

sulfoxide in different heated milk products.

CHAPTER 2. RESULTS AND DISCUSSION 27

2 Results and discussion

2.1 Selection of the marker compounds for protein

modification

The goal of the present study was the development of a LC-ESI-MS-MS method to

separate and quantify the major protein modifications, which are formed during

processing of milk. Tryptophan, methionine and cysteine are particularly sensitive to

oxidation in milk products. Other residues like lysine could also be subjected to

oxidation. Furthermore, lysine is one of the preferential sites for glycation. Thus,

lactulosyllysine (LacLys) and ε-carboxymethyllysine (CML) are described as

indicators for early and advanced stage of the Maillard reaction, respectively.

LacLys is the first stable product of the reaction between lactose and protein-bound

lysine in the early Maillard reaction. Several methods are in routine use to detect

LacyLys in milk products; among those, the furosine method is the most commen

one. This method is based on acid hydrolysis (HCl 6N) of LacLys at 110 °C for 24

hours which leads to the formation of furosine and pyridosine as well as to

regeneration of some lysine. Furosine can then easily be determined either by ion-

exchange chromatography with ninhydrin detection (Henle, Walter et al. 1991) or by

HPLC-UV detection at 280 nm (Guerra-Hernandez, Corzo et al. 1999).

The problem of the furosine method is the conversion factor which is used to

calculate concentrations of LacLys and other Amadori products. This factor is

uncertain and tightly dependent on the reaction conditions. Baptista et al have found,

for example, that the acid hydrolysis of LacLys yielded approximately 32 %, 40%,

and 28% of furosine, lysine and pyridosine, respectively (Baptista. Jose 2004).

Another literature reported 50 % yield of furosine (Penndorf, Biedermann et al.

2007). Furthermore, furosine formation differs depending on the sugar involved in

the formation of the Amadori product. After acidic hydrolysis, for instance, the molar

yield of furosine was 32% from fructosyllysine, 34 % from lactulosyllysine and 42%

from tagatosyllysine (Krause, Knoll et al. 2003).


For indirect estimation, LacLys is oxidized by periodic acid which yields CML,

erytronic acid and galacatose. The content of CML is then determined by GC-MS

(Badoud, Fay et al. 1991). However, this method may lead to overestimation of

LacLys, due to CML is already present in the milk proteins.

Another indirect method was used to estimate LacLys in milk products. This method

induced the formation of hydroxymethylfurfural (HMF) from LacLys by heating in the

presence of oxalic acid. Subsequently, HMF was determined by colorimetric

methods. However, HMF can also be formed during lactose degradation (van Boekel

1998).

Figure 8 : Degradation of LacLys via oxidative cleavage, acid hydrolysis and heating in the presence of oxalic acid


Recently, MALDI-TOF-MS has been used as a new specific method for the detection

of LacLys as well as other Maillard products after partial enzymatic hydrolysis

(Meltretter, Seeber et al. 2007). Absolute quantification however was not possible by

any of these methods. Therefore, it is important to develop a method for direct

analysis of LacLys.

CML is a well characterized and extensively studied MRP in vivo as well in food and

it is widely used as an indicator for the advanced Maillard reaction. At present,

different analytical approaches have been employed for the determination of CML in

milk and dairy products. For example, CML was measured in infant formula by

ELISA (Birlouez-Aragon, Pischetsrieder et al. 2004). CML was also determined after

acidic hydrolysis by GC-MS (Charissou, Ait-Ameur et al. 2007). In recent years,

complete protein hydrolysis followed by LC-ESI-MS-MS in MRM mode with isotopic

dilution assay has been widely used for the determination of CML in different milk

types like pasteurized, UHT, skimmed and condensed milk (Hegele, Buetler et al.

2008) as well as in raw milk, infant formula, evaporated whole milk and cheese

(Assar, Moloney et al. 2009).

Methionine can be oxidized during heating of milk as well as by the treatment with

H2O2, which is used to sterilize whey, milk and milk containers. Additionally,

methionine is oxidized by H2O2 which is generated during lipid oxidation. Oxidation

converts methionine mainly to methionine sulfoxide (MeSO), while stronger oxidative

conditions lead to the formation of methionine sulfone (figure 9) (Guy and Fenaille

2006). The oxidation of methionine to MeSO, which has a more polar side chain,

may decrease the bioavailability of the sulphur amino acid and may affect the

functionality of milk protein resulting in a change of the emulsifying capacity (Baxter,

Lai et al. 2007). MeSO may be used as good marker for oxidation, but it is not often

quantified, mainly because of its lability during protein hydrolysis (Guy and Fenaille

2006).


Figure 9: Products of methionine oxidation

Lysine aldehyde is identified as the main carbonyl compound in proteins oxidized by

by metal catalysis. Metal-catalysed oxidation involves the generation of H2O2 and

subsequent reduction of Fe3+ and Cu2+ to Fe2+ and Cu1+. Both, Fe2+ and Cu1+, bind

to specific metal binding sites on proteins and react with H2O2 to generate the highly

reactive OH. radicals. The latter attacks neighbouring amino acids forming carbonyl

compounds (Requena, Chao et al. 2001; Akagawa, Sasaki et al. 2006). Additionally,

lysine aldehyde is formed by Strecker-type degredation during the Maillard reaction

(Suyama, Akagawa et al. 2002)

Figure 10: Lysine oxidation to form lysine aldehyde

Lysine aldehyde is also formed by the action of lysyl oxidase (LOX). LOX catalyzes

the oxidative deamination of the ε-amino group of lysine residues, especially in

collagen and elastin, to yield lysine aldehyde which is also referred to as allysine.

Once generated, allysine condenses with another allysine molecule or with the ε-

amino group of lysine leading to crosslinkage and decreased protein functionality

(Akagawa, Wako et al. 1999; Sell, Strauch et al. 2007). Lysine aldehyde and other

carbonyl compounds are measured conveniently by the use of 2, 4-

dinitrophenylhydrazine (DNP) which reacts with carbonyl groups to generate

dinitrophenylhydrazone. The latter displays characteristic UV-absorbance at 360-


390nm. Specific methods were developed to detect lysine aldehyde such as the use

of MALDI-TOF-MS after partial enzymatic hydrolysis, where lysine aldehyde is

identified by the mass difference of -1Da (Meltretter, Seeber et al. 2007). Another

specific method was reported which has used sodium borohydride to reduce lysine

aldehyde to hydroxyaminocaproic acid prior to the analysis by GC-MS (Requena,

Chao et al. 2000).

It is well known that many amino acids in proteins are susceptible to oxidation by

several forms of reactive oxygen species (ROS), such as superoxide (O2.), hydroxyl

radical (OH.) and hydrogen peroxide (H2O2) (Reddie, Seo et al. 2008). Some studies

indicate the formation of hydroxytryptophan (OH-Trp) in whey proteins. The oxidation

of the thiol group of cysteine can result in a wide range of products. The major

product of the reaction with H2O2 is cysteine sulfenic acid (Cys-SOH), which is a

highly reactive product. It is assumed that cysteine sulfenic acid is a transient

intermediate to form more stable products such as cysteine sulfinic acid and cysteine

sulfonic acid. Cysteine sulfenic acid may also bind to neighouring cysteine to form a

disulfide bond giving cystine (Saurin, Neubert et al. 2004). Furthermore, two

molecules of sulfenic acid can yield the thiosulfinate derivative (figure 11). However,

despite of its high reactivity, cysteine sulfenic acid was identified in some enzymes

(Claiborne, Miller et al. 1993). Milk proteins are also suceptible to oxidation during

processing and storage. Some studies indicate the formation of cysteine sulfenic

acid in whey proteins via MALDI-TOF-MS (Meltretter, Seeber et al. 2007).

Additionally, formation of ornithine from arginine may occur in food during heat

treatment.


Figure 11: Some cysteine oxidation products

The aims of the present work were the quantification of the compounds LacLys,

CML, MeSO, 5-OH-Trp, Cys-SOH, lysine aldehyde and ornithine in milk by LC-ESI-

MS-MS analysis in MRM mode after complete protein hydrolysis. Since most of

these modified amino acids are not commercially available, the first step of this work

was to develop methods to synthesize the reference compounds in order to use

them as standards for identification and quantification.


2.2 Standards synthesis

2.2.1 Synthesis of LacLys

LacLys was synthesized according to a method described in literature to synthesize

Nε-(1-deoxy-D-fructose-1-yl)-L-lysine which is the Amadori product from glucose

(fructosyllysine, FL) (Smith and Thornalley 1992; Vinale, Fogliano et al. 1999). In

order to prevent lactosylation at the α-amino group of lysine, Nα-

fluorenylmethyloxycarbonyl-lysine (Nα-FMOC-Lys) was used. After refluxing FMOC-

Lys with lactose, the reaction mixture was analyzed by LC-MS using a RP-18

column.

The total ion current (TIC) of the synthesized product showed two peaks, one after

11.34 min which is unreacted FMOC-Lys. The second peak appeared after 10.7 min

indicating a higher polarity than FMOC-Lys. Lactosylation of whey proteins causes a

decrease in the retention time of the modified whey proteins compared to the native

one due to the highly hydrophilic lactose moieties (Czerwenka, Maier et al. 2006).

Additionally, both, FMOC-Lys and the unknown compound, had the same UV-

absorption maximum at 272 nm. Furthermore, the molecular ion [M+1]+ of the

unknown compound appeared at m/z 693 corresponding to m/z of FMOC-LacLys.

These data refer to a possible formation of FMOC-LacLys which should be

confirmed by 1H-NMR. However, using a RP-18 column it was difficult to isolate this

unknown compound from the reaction mixture due to its similar retention time

compared to FMOC-Lys as well as due to its low yield (about 10% of FMOC-Lys has

undergone reaction).


Figure 12: The chromatogram of the reaction mixture of FMOC-lys and lactose shows the formation of a new product with m/z of 693 indicating the formation of FMOC-LacLys

After the removal of the blocking group (FMOC), the resulting yellowish powder was

analyzed by mass spectrometry and showed the molecular ion [M+1]+ of m/z 471.2

corresponding to protonated lactulosyllysine (MW 470.211). Additionally, MS2 of the

putative lactulosyllysine yielded the fragments m/z 471.2→ 225.1, m/z 471.2→ 128.1

and m/z 471.2→ 84.1, which are identical to the fragments of fructosyllysine, which

is the analogous product of LacLys (Hegele, Parisod et al. 2008).

Figure 13 : MS2 spectrum of the synthesized LacLys

The observed fragments can be explained by the following path way. The

protonation of the alcohol function on C5 of fructose moiety of LacLys (m/z 471.2) is

followed by a loss of water and possibly nucleophilic attack of Nε of lysine to the

same carbocation leading to the formation of a six-membered ring (the fragment m/z

471.2→453). Another water moiety is then cleaved from the intermediately formed

ring with the alcohol function on C6 of the fructose moiety giving the fragment m/z


471.2→435. The fragment m/z 471.2→ 435 then either undergoes rearrangement of

a hydrogen atom and elimination of six-memberd ring involving the Nε of lysine giving

the qualifier fragment m/z 471.2→ 128.1. Alternatively, it eliminates galactose and

formaldehyde to yield the product ion m/z 471.2→ 225.1 which showed the highest

intensity of the LacLys fragments (the quantifier fragment). The product ion m/z

471.2→ 84.1 is generated from the fragment m/z 471.2→ 225.1 by the elimination of

both 3-hydroxypyridine and formic acid (Hegele, Parisod et al. 2008). The following

figure shows the fragmentation pathway of the protonated LacLys and the resulting

ion products.

Figure 14: Fragmentation pathway of lactulosyllysine


The synthesized LacLys showed a complex 1H-NMR spectrum due to the presence

of lysine, lactose and morpholine. This resulted in difficulties in interpretation of the

spectrum as well as the determination of the product purity. Therefore, trials were

carried out to purify LacLys as well as to improve its yield. In order to increase the

solubility of lactose, which shoud lead to a higher reaction yield, lactose was

dissolved in 1 ml water before it was added to the reaction mixture. Additionally, the

reaction mixture was refluxed for 5, 7 and 8 hours. At last, the precipitated lactose

was removed by filtration.

As a result, no real improvement was noted. Only 14% of FMOC-Lys reacted with

lactose. This result was not satisfied, considering that the synthesis required further

steps. Therefore, it was attemped to purify LacLys using affinity chromatography.

2.2.1.1 Affinity chromatography

Affinity chromatography separates biochemical mixtures on the basis of reversible

interactions between the analyte of interest and a specific binding molecule. It

enables the purification of a substance present at a low concentration in a crude

sample. The target substance can interact with a specific functional group as a result

of electrostatic or hydrophobic interaction, van der Waals forces and hydrogen

bonding. This interaction can be reversed, either specifically by a competitive

substance, or non specifically by changing the pH, ionic strength or the polarity.

The stages of affinity chromatography are summarized in figure 15 and include:

1- Equilibration of the affinity medium with the binding buffer to ensure that the

target molecule interacts with a specific binding substance, while all other

molecules are washed though the column.

2- The sample is applied, the target molecule binds specifically to the binding

molecule and the unbound substances elute through the column with the

binding buffer.

3- Conditions are changed to recover the target molecule, either specifically or not

specifically by the elution buffer.

4- Affinity medium is re-equilibrated using the binding buffer.


Figure 15: Illustration of affinity chromatography. [Adapted according to the handbook (Affinity Chromatography) from Amersham Biosciences]

2.2.1.1.1 Boronic acid affinity chromatography

Under alkaline conditions, boronic acids have a strong affinity towards cis-diol

containing compounds such as catechols, nucleic acids, some proteins and

carbohydrates (Frolov and Hoffmann 2008).

The Amadori product contains also cis-diol groups. m-Aminophenylboronic acid was

used, for example, to estimate glycated hemoglobin in normal and diabetic persons

in the early 1980 (Yue, McLennan et al. 1982). Furthermore, m-aminophenylboronic

acid was used to isolate glycated protein and peptides from human serum prior to

the analysis by LC-MS-MS (Zhang, Tang et al. 2007). Recently, Frolov et al have

optimized a method to enrich glycated peptides from bovine serum albumin followed

by the analysis by LC-ESI-MS-MS and MALDI-MS (Frolov and Hoffmann 2008).

In the current experiment, m-aminophenylboronic acid was used to purify the

Amadori product LacLys. The elution solutions were D-sorbitol and acetic acid. The

signal intensity of LacLys, which was purified by affinity chromatography, was


compared to the control which was not subjected to affinity chromatogaphy

(15µg/ml).

Figure 16: The interaction of m-aminophenylboronic acid with lactulosyllysine

Using of D-sorbitol as a specific eluent, the intensity of LacLys increased about 50%

compared to the control. By the use of acetic acid, no real improvement was

observed. In contrast, Frololov et al have found that the elution of glycated peptide

with acetic acid was as efficient as with D-sorbitol based on the peak areas in RP-

HPLC (Frolov and Hoffmann 2008). Boronic acid has high affinity towards cis-diols,

which inteacts with LacLys and lactose. The addition of sorbitol leads to a

competition between LacLys, lactose and sorbitol to bind to boronic acid.

This led to an enrichment of LacLys, but probably also of lactose. Another drawback

of the method is that after affinity purification, LacLys is present in a sorbitol solution

which needs to be removed by a further purification procedure. Therefore, a new

attempt was carried out to enrich FMOC-LacLys from the reaction mixture using a

ZIC-HILIC column

2.2.1.2 Hydrophilic interaction liquid chromatograp hy (HILIC)

Most of compounds formed during the initial stage of the Maillard reaction are

relatively polar. Therefore, they are poorly retained on conventional reverse phase

columns (Hao, Lu et al. 2007).


HILIC is a mode of chromatography which is similar to normal phase liquid

chromatography (NPLC) because it also uses a hydrophilic stationary phase. HILIC

separation employs different polar stationary phases such as unbound silica, amine

bonded phases, amide bonded phases and zwitterion bonded phases (like ZIC-

HILIC) (Guo and Gaiki 2005). In contrast to classic NPLC, the hydrophobic organic

mobile phase is replaced by an aqueous-miscible solvent such as acetonitrile or

methanol. The combination of high-organic solvent (more than 70 %) with a low

aqueous-solvent enables HILIC to be ideal for the compound ionization by ESI as

well as to provide a good retention for polar analytes (Schettgen, Tings et al. 2007).

The analyte retention on HILIC columns is proportional to its polarity and inversely

proportional to the mobile phase polarity. Several retention mechanisms are involved

in HILIC columns, such as hydrophilic interaction and ion exchange (Grumbach,

Wagrowski-Diehl et al. 2004; Schettgen, Tings et al. 2007).

Early HILIC application focused on the separation of peptides and carbohydrate

(Alpert 1990; Alpert, Shukla et al. 1994). Recently, the application of HILIC was

extended to include the analysis of amino acids (Hao, Lu et al. 2007), advanced

glycation end products (AGEs) (Schettgen, Tings et al. 2007) as well as drugs

(Grumbach, Diehl et al. 2003) and food like wheat gluten (Schlichtherle-Cerny,

Affolter et al. 2003).

2.2.1.2.1 ZIC-HILIC column

ZIC-HILIC columns share the main properties with the other HILIC columns. The

ZIC-HILIC column has a permanent and hydrophilic zwitterionic stationary phase

attached to porous silica. The weak electrostatic interaction between the charged

analyte and the neutral zwitterionic stationary phase results in a unique selectivity

especially for polar analytes which are poorly retained on reverse phase columns.

The zwitterionic stationary phase is stable and, according to the manufacture, it can

be operated in pH range 3-8 which allows changing the polarity of the analyte and

affecting the selectivity without changing the stability of the column.


Figure 17: Functional group packed in ZIC-HILIC columns

By the use of a ZIC-HILIC column, FMOC-LacLys eluted after 5.1 min. Unreacted

FMOC-Lys eluted after 3.93 min as shown in figure 15.

Figure 18: Chromatogram of the reaction mixture of FMOC-Lys and lactose separated on a ZIC-HILIC column

The appearing compound between FMOC-lysine and FMOC-LacLys showed the

molecular ion [M+1]+ of m/z 531.1 corresponding to protonated FMOC-fructosyllysine

and suggesting that lactose was hydrolysed during the synthesis.

After fractionation, one peak only appeared after 5.1 min. This peak showed the

molecular ion [M+1]+ of m/z 693 which indicates the presence of FMOC-LacLys, as

shown in figure 19. The synthesis of LacLys is illustrated in figure 20.


Figure 19: Chromatogram and mass spectrum of FMOC-LacLys obtained after fractionation of the reaction mixture of FMOC-Lys and lactose on a ZIC-HILIC column.

Figure 20: Synthesis of LacLys


2.2.1.3 Identification of LacLys by 1H-NMR

After the removal of the blockage group by morpholine, 1 mg of the synthesized

LacLys was dissolved in 1ml of D2O and then the structure of this product was

analyzed by 1H-NMR.

The synthesized LacLys showed the following 1H-NMR spectral data (600 MHz,

D2O) : δ=3.41 (t, 1H, αCH-Lys, J=5.8), δ =3.252 (s, 2H, Lys-CH2-lac), δ=3.01 (t, 2H,

εCH-Lys, J=7.5 ) δ=1.76 (m, 2H, βCH-Lys J not resolved), δ=1.7 (m, 2H, δCH-Lys,

J=7.6), δ=1.41 (m, 2H, γCH-Lys, J=6.6 ). δ=4.4 (d, 1H, CH-Gal (C1), J=3.4), δ=3.17

(t, 1H, CH-Glu (C4), J=6.3), δ=3.51 (t, 1H, CH-Gal (C4), J=5.5), δ=4.05-4.136 (m,

1H, CH-Gal (C5); 1H, CH-Glu (C5)), δ=3.53-3.85 (many H signals belonging to CH-

Glu (C3, C6);CH-Gal (C2, C3, C6); and all OH of glucose and galcatose)

The interpretation of the 1H-NMR spectrum of LacLys was achieved by comparison

to the 1H-NMR spectra of lysine and lactose, which were recorded separately.

Additionally, the interpretation was based on 1H-NMR spectral data of lactose and

lysine as well as fructosyllysine which were predicted by ChemDrawUltra11.01.

Furthermore, the spectral data of LacLys were compared to those of fructosyllysine

reported in literature (Smith and Thornalley 1992; Vinale, Fogliano et al. 1999).

It is expected that the resonance signal, which is assigned to the protons of the bond

between lactose and lysine, should appear as a singlet in 1H-NMR spectrum

because both neighboring atoms to this bond are not bound to protons (n = 0). Thus,

the multiplicity of the bond between lysine and lactose is 1. The multiplicity of the

bond is determined by the number (n) of magnetically equivalent protons on the

neighboring atoms and is given by the quantity n + 1 (Skoog 2005). This singlet

signal appeared between the proton signals of α- and ε-carbons of lysine as shown

in figure 21.


Figure 21: 1H-NMR spectrum of the synthesized LacLys shows the signal of α-C and ε-C of lysine as well as the expected signal of the bond between lactose and lysine

2.2.1.4 Determination of the purity of LacLys by 1H-NMR

The synthesized LacLys was measured quantitatively by 1H-NMR using DMF

(99.8%) as a standard to estimate the purity and acetone as a standard for axis

calibration.The 1H-NMR spectrum of lysine in D2O exhibited one triplet at δ=3.41

assigned to the proton at α-carbon of lysine (figure 21). The resonance signal of this

proton did not interfere with the other signals observed in the spectrum; therefore,

this signal was adopted to estimate the purity of LacLys by comparing its area to that

one of the aldehyde proton of DMF (δ=7.926). It might be better to estimate the

purity of LacLys depending on the resonance signal of the protons in the bond

position between lactose and lysine, but this signal was not completely resolved. The

purity of LacLys was 6.13%. Despite of this low purity, the synthesized product can

be used for quantification of LacLys in milk products, since the absolute content of

the standard could be determined by quantitative 1H-NMR. Thus, for first time

lactulosyllysine was synthesized and its purity was determined by quantitative 1H-

NMR. The main impurity was attributed to morpholine used to remove the FMOC-

group. Morpholine showed 3 signals at δ of 3.132, 3.294 and 3.95 as (figure 21).


Figure 22: Signal of the aldehyde proton of DMF which appeared at 7.926 ppm

2.2.2 Synthesis of CML

CML was prepared as reported in the literature using iodoacetic acid as alkylating

agent (Huber and Pischetsrieder 1998) with some modifications (figure 24). In order

to block the α-amino group and thus, to prevent the formation of a carboxymethyl

group on the α-amino group of lysine, Nα-carbobezyloxy-L-lysine (Cbz-lysine) was

used for the synthesis. Under the applied chromatographic conditions, the product

Nα-Cbz-CML eluted after 17 min as shown in figure 23.

Figure 23: Chromatogram of the reaction mixture of Cbz-lysine and iodoacetic acid shows the formation of Cbz-CML

The limited participation of Cbz-lysine in the reaction as well as the double alkylation

on the primary amino group (formation of α-Cbz-bis-CML) necessitated fractionation

by preparative HPLC to isolate α-Cbz-CML. After the removal of the blockage group

Cbz by hydrogenation, the synthesized product was analyzed by MS-MS. The mass


spectrum showed the molecular ion [M+1]+ of m/z 205.1 corresponding to CML.

Additionally, the mass spectrum showed the fragments m/z 205.1→ 84, m/z 205.1→

130 and m/z 205.1→ 56 which are identical to those reported in the literature (Yalcin

and Harrison 1996; Teerlink, Barto et al. 2004; Delatour, Hegele et al. 2009).

However, the synthesized CML was not completely pure; therefore, a CML standard

was purchased to validate the method as well as to quantify CML in milk products.

Figure 24: Synthesis of CML

2.2.3 Synthesis of MeSO

Methionine sulfoxide was synthesized using H2O2 as an oxidizing agent as reported

in literature (Brot, Werth et al. 1982; Fliss, Weissbach et al. 1983). The method was

further optimized to improve the yield.

The residue of the reaction mixture of methionine and H2O2 was loaded on a ZIC-

HILIC column. As it shown in figure 25, most of methionine (Me) was oxidized to

methionine sulfoxide (MeSO), which eluted after 6.7 min. A slight formation of

methionine sulfone was observed (MeSOO, retention time 6 min). The peak of

unreacted methionine appeared after 5.4 min.


Figure 25: Chromatogram of the reaction mixture of methionine and H2O2 which was incubated at 21 °C. The chromatogram shows the form ation of MeSO and MeSOO.

In order to improve the yield of MeSO, many trials were carried out in which different

amounts of methionine, H2O2 and HCl were incubated for 1-2 hours at 21 °C. The

trials are summarized in table 4.

trial methionine

0.25 M (µl)

HCl 0.5 M

(µl)

30% H2O2

(µl)

incubation time

(hour)

1 400 200 10 1

2 400 100 5 1

3 400 100 5 2

4 400 100 0 1

5 400 0 5 1

6 200 100 0 1

7 200 0 5 1

8 200 0 10 1

Table 4: Amount of methionine (0.25 M), HCl and H2O2 (30%) used to form MeSO

The best yield of MeSO was obtained by the incubation of 200 µl of 0.25 M

methionine and 10 µl of 30% H2O2 for 1 hour at 21°C. Methionine was totally

oxidized to methionine sulfoxide without the formation of methionine sulfone. The

synthesized product was then analyzed by LC-MS-MS showing the molecular ion

[M+1]+ of m/z 166.1 equivalent to the molecular ion of MeSO. In order to confirm the


product identity of the synthesized MeSO, high resolution electron impact mass

spectrometry (HREIMS) was carried out which showed that the synthesized product

had the same mass as calculated for MeSO (165.0460 Da). Additionally, the

synthesized MeSO revealed the same 1H-NMR spectral data as an authentic

standard. However, the synthesized MeSO had a lower purity than a commercial

standard (about 85%). This purity was estimated by the peak areas of both product

standards. Therefore, a commercial MeSO standard was used for method validation

as well as for quantitative analysis.

2.2.4 Trial to synthesize lysine aldehyde

Synthesis of lysine aldehyde was attempted using egg shell membrane (ESM) which

is an easily accessible source for LOX. LOX was isolated from ESM according to a

method reported in literature using potassium phosphate buffer containing NaCl and

urea (Kagan, Sullivan et al. 1979). The LOX extract was then incubated with Cbz-

lysine in the presence of urea at 37 °C to catalyse deamination of the ε-amino group.

The reaction mixture was analyzed by LC-MS.

Using a RP-18 column, unreacted Cbz-lysine eluted after 7.76 min. In the solution

which contained the LOX extract, an unknown compound appeared after 10.38 min,

as shown in figure 26. The unknown compound was detected first after 3 days. Its

yield increased gradually till the 9-th day. Additional formation was not observed in

the next 5 days. At the same time, Cbz-lysine decreased gradually till the 9-th day

when 18% had reacted. The molecular ion [M+1]+ of Cbz-lysine and the unknown

compound were 281.2 and 280.2 m/z, respectively, indicating a possible formation of

Cbz-lysine aldehyde.


Figure 26: Chromatogram and mass spectrum of the reaction mixture of Cbz-lysine and urea extract of ESM shows unreacted Cbz-lysine as well as the formation of unknown compound. The unknown compound showed m/z 280.2 corresponding to the protonated Cbz-lysine aldehyde with a mass difference of -1Da compared to Cbz-lysine.

The resulting reaction mixture contained additionally to the target compound, urea,

the enzyme extract as well as unreacted Cbz-lysine. Therefore, it was important to

isolate it before removal of the blockage group.

The isolation was achieved by HPLC fractionation using similar conditions to those

which were used previously for the detection. The 1H-NMR spectrum of this product

did not reveal any signal of an aldehyde proton, which appears generally about 10

ppm, although the analysis by mass spectrometry showed the molecular ion m/z

280.2. Furthermore, the molecular ion m/z 146.2 corresponding to protonated lysine

aldehyde was not detected after the removal of the blocking group. It is likely that

Cbz-allysine was formed, but it was not stable under the conditions of synthesis and

purification, probably because of the high reactivity of the aldehyde group.

2.2.5 Trial to synthesize Cys-SOH

The synthesis was attempted using Fenton`s reagent which is a mixture of hydrogen

peroxide and Fe2+, typically FeSO4. Fenton`s reagent is used to generate highly

reactive hydroxyl radicals which can react with the sulfhydryl group of the cysteine

residue to give a variety of oxidation products. This method has been applied on

some peptides of macrophage enzymes and resulted in a variety of oxidation

products which were identified by LC-MS-MS (Shetty, Spellman et al. 2007).

Cysteine was incubated with H2O2 at different temperatures as well as for different

time periods. In order to stop the oxidation of cysteine, methionine was added since


it is more susceptible to oxidation. A precursor ion scan of the reaction mixtures of

cysteine and hydrogen peroxide did not show the ions m/z 138, 154 and 172, which

are corresponding to protonated cysteine sulfenic acid, cysteine sufinic acid and

cysteine sulfonic acid, respectively. The reaction led to the formation of a new

product that eluted after 3.87 min and showed the molecular ion [M+1]+ of m/z 241.2

(figure 27). The product showed maximum absorption at 260-270 nm which is the

optimal wavelength for monitoring the disulfide bond (Ashby and Nagy 2007). These

data indicate the formation of cystine as the main product of cysteine oxidation (MW

240.3). Cysteine sulfenic acid was either not formed or was an unstable intermediate

which underwent further reactions.

Figure 27: Chromatogram of the reaction mixture of cysteine and H2O2 (recorded at 250 nm) shows the scavenger methionine and a new product with m/z 241.2 which was assigned to cystine

2.2.6 OH-Trp

OH-Trp was not synthesized. The compound 5-OH-Trp, which is commercially

available, was used as a standard compound.

2.2.7 Discussion

The use of tandem mass spectrometry to detect oxidation and glycation products in

milk protein requires first the preparation of standard compounds for identification

and quantification of the products in the samples. LacLys was synthesized by the

reaction of FMOC-lysine and lactose under nitrogen atmosphere to avoid the

oxidative cleavage of FMOC-LacLys to FMOC-CML. Due to the low reactivity of


lactose, only 10-14 % of FMOC-lysine underwent the reaction. Additionally, lactose

was hydrolysed during the reaction to give glucose and galactose. Both

monosaccharides have higher glycation activity compared to lactose (Chevalier,

Chobert et al. 2001). Consequently, glucose and galactose promote the formation of

other Amadori products thus reducing the yield of FMOC-LacLys. This low yield was

not sufficient for further synthesis; therefore FMOC-LacLys was isolated from the

reaction mixture by fractionation on a ZIC-HILIC column followed by the removal of

the blocking group. The identity of the resulting product was confirmed by MS-MS.

The content of the standard was determined by quantitative 1H-NMR using DMF as

an internal standard. DMF was chosen as a standard for quantitation because it is

not volatile. Additionally, the signal of the aldehyde proton did not interfere with the

other resonance signals in the 1H-NMR spectrum. The purity of LacLys was 6.13%.

The main impurity was identified as morpholine, which was used to remove the

blockage group (FMOC-group) and could not be removed from the mixture, because

both LacLys and morpholine showed similar solubility in water. Since the absolute

content of the standard could be determined by quantitative 1H-NMR, the

synthesized product could be used for quantification of LacLys in milk products.

The synthesis of CML and MeSO has been performed using an alkylating agent and

an oxidizing agent, respectively. The structures of the synthesized compounds were

confirmed by 1H-NMR and MS. For quantification however, commercial CML and

MeSO standards were applied, which were available in higher purity.

Both, lysine aldehyde and cysteine sulfenic acid could not be synthesized. Cys-SOH

is particular stable in the absence of other sulfhydryl groups, limited solvent access

and in association with apolar elements in the protein structure (Claiborne, Miller et

al. 1993). Since these conditions were not given during the synthesis, Cys-SOH can

undergo further reactions. For example, there is evidence for the transitional

formation of Cys-SOH in protein, where it rapidly react with thiols to form mixed

disulfides (Giles, Tasker et al. 2001).

In this part of work, LacLys, CML and MeSO as marker compounds for protein

modification in milk were succssefuly synthesized. Additionally, 5-OH-Trp is

commercially available. Therefore, it was decided to develop a method for the

detection and quantification of these compounds in milk products by LC-ESI-MS-MS.


2.3 Method development for the analysis of

glycation and oxidation products in milk by LC-ESI-

MS-MS

2.3.1 Introduction

During heat treatment, which is applied to improve food safety and sensory quality

as well as to increase the shelf life of milk products, the quality and the nutritional

value of milk proteins can be adversely affected by oxidative damage and Maillard

reaction. For example, the essential amino acid lysine is blocked by lactose during

the Maillard reaction leading to the formation of lactulosyllysine which is further

degraded to give different products like CML (figure 28). LacLys and CML are used

as indicators for the early and advanced stages of the Maillard reaction in milk,

respectively (van Boekel 1998). Milk proteins are also sensitive to oxidative

modifications which affect particularly methionine, lysine and tryptophan (figure 29).

As a result, the digestibility of milk protein and the bioavailability of these essential

amino acids are decreased (Mauron 1990).

The aim of the following part of work was to develop a method to detect the oxidation

products 5-OH-Trp and MeSO as well as the glycation products LacLys and CML,

which are formed during the heat treatment of α-lactalbumin in a milk resembling

system by LC-ESI-MS-MS.

Figure 28: Reaction of lysine with lactose during heating of whey protein


Figure 29: Some protein oxidation products

2.3.2 ESI-MS-MS conditions

Analysis was performed using the mass spectrometer Sciex 4000 QTRAP equipped

with electrospray ionization in positive mode by multi-reaction monitoring (MRM).

The mass analyzer was coupled to a Dionex UltiMate 3000 liquid chromatography.

The three most intense mass transitions of each compound were monitored. The

parameters of the mass analyzer which are dependent on the LC flow rate as well as

those depending on the compounds themselves were optimized to achieve the

highest signal intensity for the analytes. The data acquisition was performed with a

scan time of 200 ms.


2.3.2.1 Flow-dependent MS parameters

Source-dependent parameters are generally optimized at the flow rate of liquid

chromatography to get the highest intensities. These parameters are:

• GS1 (Gas 1) controls the nebulizer gas which helps to generate small droplets

from the sample flow.

• GS2 (Gas 2) controls the turbo gas which helps to evaporate the spray

droplets and prevent the solvent from entering the instrument.

• Temp (Temperature) controls the temperature of the turbo gas. Therefore it

helps to evaporate the solvent and to produce sample ions in the gas phase.

• CUR (Curtain gas flow): This parameter controls the curtain gas interface,

which controls the flow of gas between curtain plate and the orifice. The

curtain gas interface prevents the solvent`s droplets from entering and

contaminating the ion optic.

• IS (ion spray voltage) controls the voltage applied on the needle to ionize the

sample.

• CAD (Collision activated dissociation gas) controls the pressure of the

collision gas in the collision cell, which, for MS/MS scan, collides with the

precursor ions for fragmentation.

• Ihe (Interface Heater) switches the interface heater on and off. Heating the

interface helps to maximize the ion signal and prevents the contamination of

the ion optic (Manual user API 4000 QTRAP, 2008).

The optimized parameters are displayed in table 5.

2.3.2.2 Compound-dependent MS parameters

Compound-dependent parameters vary depending on the compound to be analyzed.

These parameters are:

• DP (Declustering potential) controls the potential difference between Q0 and

the orifice plate. It is used to minimize the solvent cluster ions which may

attach to the sample. Thus DP prevents the solvent from entering and

contaminating the ion optic.

• EP (Entrance potential) controls the entrance potential, which guides and

focuses the ion through Q0 region.


• CE (Collision energy) controls the collision energy received by the precursor

ion during the acceleration into the collision cell, where they collide with the

CAD gas and are fragmented.

• CXP (Collision Cell Exit Potential) controls the collision cell exit potential and

is used to focus and accelerate the ions out of Q2 towards Q3 (Manual user

API 4000 QTRAP, 2008).

The optimized parameters are displayed in table 6.

Figure 30 : Ion optic in the vacuum chamber of an MS-MS triple quadrupole system (Manual user API 4000 QTRAP, 2008). Q1: first quadrupole; Q2: collision cell; Q3: third quadrupole; ST: stubby lens between Q0 and Q1; ST2: stubby lens between Q1 and Q2; ST3: stubby lens between Q2 and Q3; RF: radio frequency; CEM: channel electron multiplier

CUR 30 (psi) IS 5500 (V)

Temp 500 °C GS1 60 (psi) GS2 75 (psi) CAD medium ihe on

Table 5: Optimized MS-parameters dependent on the LC-flow


compound precursor ion

(m/z) product ion

(m/z) DP (V) EP (V) CE (V) CXP (V)

LacLys 471.2 225.1 106 10 33 14 471.2 128.1 106 10 39 10 471.2 84.1 106 10 77 14

5-OH-Trp 221.1 162.1 116 8 25 6 221.1 134 116 8 35 4 221.1 77.1 116 10 73 6

CML 205.1 84.1 116 8 29 2 205.1 130.1 116 8 59 4 205.1 56.1 116 8 17 4

MeSO 166.1 74 46 8 33 8 166.1 56 46 10 19 4 166.1 102.1 46 8 11 4

Table 6: Monitored transitions of LacLys, 5-OH-Trp, CML and MeSO and the optimized MS-parameters dependent on these compounds

2.3.3 MS2 fragmentation of the investigated analyte s

The fragments of CML, MeSO, LacLyc and 5-OH-Trp were determined by product

ion scan, and then the three most intense fragments of each compound were chosen

for identification. The fragment which has the highest intensity was used as quantifier

fragment, as seen in table 6 where the quantifier fragments are printed in bold. The

other two fragments were used as qualifier fragments. However, since CML is

formed only in little amount in milk proteins, it was identified during the investigation

of glycation in milk samples by the quantifier fragment in addition to one of the

qualifier fragments (m/z 205.1→ 84.1 and m/z 205.1→ 130.1.1, respectively).

Otherwise, high concentrations of milk samples have to be used to detect the second

qualifier fragment (m/z 205.1→ 56.1). The predicted structures of these fragments

are shown in the figures 31, 32, 33 and 34.

The quantifier and qualifier fragments of CML (m/z 205.1→84.1 and m/z

205.1→130.1.1, respectively) were identical to those reported in literature (Delatour,

Fenaille et al. 2006; Schettgen, Tings et al. 2007). The qualifier fragments of MeSO

(m/z 166.1→56) (Sochaski, Jenkins et al. 2001) and (m/z 166.1→102) (Thornalley,

Battah et al. 2003) were reported in literature. The quantifier fragment of MeSO (m/z


166.1→74) has not been reported in literature. The quantifier fragment (m/z

471.2→225.1) and the qualifier fragments (m/z 471.2→128.1, m/z 471.2→84.1) of

LacLys were analogous to those of the Amadori product fructosyllysine (figure 14)

(Hegele, Buetler et al. 2008; Hegele, Parisod et al. 2008). The fragmentation of 5-

OH-Trp has not been reported in literature.

Figure 31: Product ion mass spectrum of CML and the corresponding fragments

Figure 32: Product ion mass spectrum of MeSO and the corresponding fragments


Figure 33: Product ion mass spectrum of LacLys and the corresponding fragments

Figure 34: Product ion mass spectrum of 5-OH-Trp and the corresponding fragments

2.3.4 Chromatographic seperation of CML, LacLys, Me SO

and 5-OH-Trp using a C18 column

In order to detect the compounds MeSO, 5-OH-Trp, LacLys and CML in milk

proteins, it was important to determine the retention time of these compounds.

Therefore, a mixture of the standard compounds MeSO, 5-OH-Trp, LacLys and CML

was analyzed under the optimized parameters of mass spectrometry (table 5 and 6).

The compounds were separated on a C18 column 150*2.1mm, particle size 5 µm,


protected with a pre-column 20*2.1mm. The column was housed in the oven at 20

°C. The flow rate and injection volume were set at 200 µl/min and 5 µl, respectively.

Since CML and LacLys are very polar compounds, they have only little retention on

RP-columns. To overcome this problem, ion pair reagents like nonafluoropentanoic

acid (NFPA) are used. NFPA was chosen due to its high compatibility with MS-MS

compared to other ion pair reagents like trifluoroacetic acid (TFA). Mobile phases

were acetonitrle (solvent B) and 5 mM NFPA in water (solvent A). The gradient was

optimized to achieve best separation of the four analytes. The gradient started with

10% acetonitrile, which increased in 3 min to 40% and in the next 3 min to 50%.

Acetonitrile was kept for 4 min at 50%, and then increased rapidly in 2 min to 100%

where it was kept for further 2 min. Acetonitrile decreased then in 6 min to the initial

conditions (10%) followed by the equilibration for 5 min before injecting the next

sample. All samples were dissolved in a mixture of water/acetonitrile (50/50%).

Methionine sulfoxide eluted after 4.1 min separately from the other compounds.

LacLys, 5-OH-Trp and CML eluted almost together after 7.2 to 7.6 min (figure 35). In

order to separate LacLys, 5-OH-Trp and CML from each other, the gradient was

systematically optimized but the separation of the compounds on the RP-18 column

could not be achieved. However, the method can be still applied because the

compounds LacLys, 5-OH-Trp and CML are separated by their specific mass

transitions. The detection of the three fragments of each compound depended on the

compound concentration. Thus, only one fragment was detected by the analysis of

low concentrations of the standard compounds. Therefore, high protein

concentrations were required for the investigation of oxidation and glycation products

in whey proteins as well as in milk products, since the identification of a modified

amino acid by MS-MS needs at least the detection of two fragments.


Figure 35: LC-MS-MS chromatogram of the standards mixture, obtained in MRM mode, on a C18 column. The standard mixture contained MeSO, 5-OH-Trp, LacLys and CML

An unknown peak appeared after 8.08 min which showed the quantifier fragment of

LacLys (m/z 471.2→225.1) but no qualifier fragment was detected. This peak

appeared during the analysis of standards, a mixture of water and acetonitrile

(50/50%), water alone and also during the analysis of milk samples. Additionally, it

appeared when the elution gradient was run on the C18 column without loading any

sample indicating that the presence of this peak is related to the mobile phases

themselves. Since the used acetonitrile was of MS grade while the used ion pair

reagent had a purity of 97%, it is expected that this peak belongs to a fragment of an

unknown compound present with the ion pair reagent.

2.3.5 Glycation of α-LA in a milk model mixture

α-Lactalbumin (α-LA) is the second major whey protein after β-LG in milk. It

represents about 30% of the total whey proteins and it is an important source for the

essential amino acid tryptophan. In order to study glycation in milk protein, a milk

model containing lactose and α-LA in the relevant concentrations diluted in a milk

salt matrix were heated at 60 °C. Three controls we re prepared: α-LA and lactose

without heat treatment, native α-LA as well as α-LA heated alone at 60 °C. After the

heat treatment, lactose and minerals were removed by dialysis to avoid artificial

modification during sample preparation. The lyophilized residue was then subjected


to multi-enzyme digestion to liberate the modified amino acids prior to the analysis

by LC-MS-MS. For qualitative experiments, the enzymatic digestion has been

applied as described in literature (Hasenkopf, Ronner et al. 2002) using three

enzymes for three days at 37 °C. For quantification , enzymatic protein hydrolysis has

been further optimized (see 2.8).

Pepsin, pronase and aminopeptidase were added after each other in 24 hours

intervals. Pepsin is an enzyme which cleaves the peptide bond preferably at the

carboxylic side of phenylalanine, tryptophan and leucine and methionine. Pepsin,

according to the supplier, has the optimum activity at a pH range of 1.8-2.2; therefore

the protein was dissolved in 0.02 M HCl (pH 2). Pronase is a mixture of non-specific

proteases, which digest proteins into individual amino acids. The molecular weight of

the various enzymes present in pronase ranges between 20-60 kDa and the

optimum pH value is 6-7.5. Aminopeptidase is an enzyme which cleaves the terminal

amino acids of peptides and proteins. The optimal pH value of aminopeptidase

activity, according to the supplier, varies between 7-7.5 up to 9; therefore it was

necessary to adjust the pH of the protein solution to 7.5 before adding pronase and

aminopeptidase. As a blank for the enzymatic hydrolysis, the enzymes and buffers

were added to 1ml of HCl 0.02 M without protein. The blank was treated and

analyzed exactly like the protein samples.

The hydrolyzate was separated from the enzymes by ultrafiltration using centrifugal

concentrator devices with a MWCO of 10 kDa (figure 36), since all used enzymes

have molecular weight higher than 20 kDa.

After lyophilization, the residue was reconstituted in a water/acetonitrile mixture

(50/50%). The concentration of 25µg/ml was enough for the detection of the modified

amino acids by MS-MS (25 µg of the sample before enzymatic hydrolysis).

Figure 36 : A centrifugal concentrator device which was used for the clean-up of the enzymatic hydrolyzate


Many modifications were detected after the incubation of α-LA and lactose in a milk

model mixture at 60 °C. The incubation temperature was chosen to provide a

sufficient extent of modification and to prevent at the same time the denaturation of

α-LA which occurs at temperatures higher than 65 °C (Czerwenka, Maier et al.

2006). After 3 days of incubation of α-LA with lactose at 60 °C, both MeSO and

LacLys were clearly detected by their three characteristic fragments. Because of the

low amount of CML in the samples, only the quantifier fragment (m/z 84.1) was

detected. By increasing the concentration of the sample, the second fragment of

CML was additionally detected (m/z 130.1). Moreover, the signal of CML increased

with increasing incubation time, where the highest signal was detected after 14 days

incubation. The highest signals of MeSO and LacLys, in contrast to CML, were

detected after 7 days and then decreased after 14 days of incubation. 5-OH-Trp was

not detected in any sample.

The peak of MeSO appeared after 3.81 min, which is 0.31 min earlier than the

standard MeSO. This early elution could be caused by other components of the

sample matrix which co-eluted at the same time. CML and LacLys eluted at the

same retention time as the standards (7.2 min and 7.6 min, respectively).

In the three controls neither LacLys nor CML were detected. This is expected since

no lactose was present in the native or the heated α-LA (the second and third

control). Additionally, this result confirms that lactose could be sufficiently removed

during dialysis from the unheated α-LA/lactose mixtures to prevent artificial formation

of LacLys during enzymatic hydrolysis.

Methionine sulfoxide was detected in all controls samples (heated and native α-LA).

The detection of methionine sulfoxide in native α-LA can be attributed either to

previous formation of methionine sulfoxide during the isolation of α-LA or to new

oxidation of methionine during sample preparation.

In order to test the latter hypothesis, 5 µg of methionine was subjected to the

conditions of the enzymatic hydrolysis, cleaned up and analyzed exactly like the

samples of milk model mixture, with one exception: The pepsin solution was

replaced by 50 µl of HCl 0.02 M, to avoid the autolysis of pepsin under the conditions

of enzymatic hydrolysis and thus, to avoid the overestimation of methionine

sulfoxide. The chromatograms showed only a minor formation of MeSO during


enzymatic hydrolysis suggesting that the artificial formation of MeSO during sample

preparation can be neglected. Compared to the heated control, the signal of MeSO

has clearly increased in the presence of lactose in the samples. This result leads to

the assumption that the presence of lactose can enhance the oxidation of methionine

during the heat treatment of α-LA. MeSO concentration decreased after 14 days of

incubation, whereas in the heated control MeSO was continuously formed during the

entire incubation process (figure 38).

Figure 37: Extracted ion chromatogram (XIC) of glycated α-LA shows the formation of CML (right). LC-MS-MS chromatogram of glycated α-LA obtained in MRM mode shows the formation of LacLys (left). α-LA and lactose were heated in a milk matrix at 60 °C for 3 (A), 7 (B) and 14 (C) days and subjected to enzymatic hydrolysis prior to LC-MS-MS analysis. CML formation increased during the whole incubation period, while the highest signal of LacLys was obtained after 7 days of incubation.


Figure 38: LC-MS-MS chromatogram of glycated α-LA obtained in MRM mode shows the formation of MeSO. α-LA and lactose were heated in a milk matrix at 60 °C for 3 (1 a), 7 (2 a) and 14 (3 a) days and subjected to enzymatic hydrolysis prior to LC-MS-MS analysis. MeSO was detected in native α-LA (standard) and in α-LA heated without lactose for 3 (1 b), 7 (2 b) and 14 (3 b) days


2.3.6 Oxidation of α-LA in a milk model mixture

The aim of this experiment was to investigate the formation of protein oxidation

products. Superoxide radicals, which are converted into hydrogen peroxide, may be

generated from glycated proteins such as the Amadori product (Mossine, Linetsky et

al. 1999). Thus, the superoxide radicals generated from glycated proteins in milk can

enhance the oxidation of methionine as well as tryptophan. Therefore, this

experiment aimed to study the formation of MeSO and 5-OH-Trp during the heat

treatment of the milk model under oxidative conditions.

In order to induce oxidation, α-LA was heated in the presence of H2O2 at 120 °C.

Native α-LA as well as α-LA heated without H2O2 served as controls.

5-OH-Trp was not detected neither in oxidized α-LA nor in the control. Methionine

sulfoxide was found in all samples, even in the control (native α-LA and α-LA heated

without H2O2). The signal intensity of MeSO increased gradually with increasing the

heat treatment in the presence of H2O2 compared to the control samples. This result

indicats that H2O2, as a relatively mild oxidant, promotes the oxidation of methionine.

Moreover, the heat treatment of α-LA at 120 °C without the addition of H 2O2 did not

enhance the oxidation of methionine; no real difference in the intensity was found

between the heated control samples (for 10, 20 and 30 min at 120 °C) and the native

α-LA (standard), as seen in figure 39.


Figure 39: LC-MS-MS chromatogram of oxidized α-LA obtained in MRM mode shows the formation of MeSO. 5-OH-Trp was not detected. α-LA and H2O2 were heated in a milk matrix for 10, 20 and 30 min at 120 °C (1a, 2a and 3a, respectively) and subjected to enzymatic hydrolysis prior to LC-MS-MS analysis. LC-MS-MS spectra obtained in MRM mode of native α-LA (standard) and α-LA heated for 10, 20 and 30 min at 120 °C (1b, 2b and 3 b, respectively) which were used as control


2.3.7 Discussion

The heat treatment of α-LA with lactose at 60 °C induced oxidation and gly cation

reactions and led to the loss of both, lysine and methionine. Most of LacLys was

formed during the first three days of incubation, and then the concentrations

increased slightly till the seventh day. During the next 7 days, a decrease of LacLys

was observed. Further studies are necessary to investigate if the degredation of

LacLys during advanced heat treatment is also relevant for milk processing.

LacLys has been identified as the most common modification in milk protein and has

been detected in various dairy products (Meltretter and Pischetsrieder 2008). The

decrease of LacLys during longer incubation in parallel to the increase of CML can

be explained by the oxidative degradation of LacLys to give CML as well as the

participation of degradation products of LacLys in the formation of CML (Ahmed,

Argirov et al. 2002). However, a decreased release of LacLys due to protein

insolubility and protease resistance, as a result of severe modification and cross-

linking, can not be fully excluded (Ahmed, Ahmed et al. 2005).

In general, heating of α-LA at 60 °C led to the formation of MeSO regardles s of the

presence of lactose which might result from oxidation by metal ions present in the

buffer. However, the signal of MeSO increased clearly in the presence of lactose.

This result indicates a role of lactose in oxidation reactions. The Amadori product

promotes the generation of the superoxide radical ion, which is converted into

hydrogen peroxide (Mossine, Linetsky et al. 1999). The latter then reacts with metal

ions to form the hydroxyl radicals which are highly reactive and attacks amino acids

residues leading to oxidation of proteins (Dean, Fu et al. 1997). Since it was shown

in the present study, that hydrogen peroxide leads readily to MeSO formation, this

mechanism could explain promotion of oxidation reaction by lactose. The decrease

of MeSO signal after 14 days of incubation at 60 °C could be caused by further

oxidation to methionine sulfone.

5-OH-Trp was not detected in glycated nor in oxidized α-LA. The absence of 5-OH-

Trp does not proof that tryptophan was resistant to oxidation in the samples. The

indole ring of tryptophan is oxidized in proteins in different ways leading to the

formation of various regioisomers of OH-Trp as well as to kynurenine and N-

formylkynurenine. These products were detected in actin of rats after exposure to

oxidative stress (Fedorova, Kuleva et al. 2010). Formation of tryptophan oxidation


products in milk protein is still not assured. The mass shift of 16 Da, which was

detected in some peptides of glycated whey proteins, suggested oxidation of

tryptophan residue. However, this assumption was equivocal since this mass shift

could also result from the oxidation of cysteine residue present at the same peptide

to give cysteine sulfenic acid. Furthermore, the mass shift of 32 Da detected at

another peptide was thought to be caused either by a single oxidation of both

tryptophan and methionine to give hydroxyltryptophan and methionine sulfoxide,

respectively, or a double oxidation of one of them leading to the formation of N-

formylkynurenine or methionine sulfone (Meltretter, Becker et al. 2008). Further

studies are required to determine if tryptophan was really oxidized in processed milk

or milk models.


2.4 Detection of glycation and oxidation products in

milk

Cow milk is processed to give different dairy products like butter, yogurt and cheese.

The modern industrial processes can produce more durable milk forms like UHT and

sterilized milk as well as easily transportable forms like milk powder. However, the

thermally treatment applied during the product manufacturing can induce different

modification for milk components similar to those, which have been detected in α-LA

in the previous experiments. The aim of this part of work was the detection of some

glycation and oxidation products in milk proteins formed during industrial processing.

The investigated milk samples were low fat UHT milk and sterilized milk as examples

for liquid long shelf life products.

Additionally, liquid and powdered hypo-allergenic infant formulas (HA-infant formula)

were investigated. Hypoallergenic infant formulas are hydrolyzed milk formulas

which are recommended for preventing or alleviating the atopy response of infants

with a familiar history of atopy. Hypoallergenic infant formulas are divided into

extensively and partially hydrolyzed formulas. Extensively hydrolyzed formulas are

mostly used in therapeutic application because they contain a mixture of amino acids

or low molecular weight peptides with a very low residual antigenicity. The partially

hydrolyzed formulas, which less extensively hydrolyzed, are used to prevent the

development of atopy response for infant with high risk for atopy (Nentwich,

Michkova et al. 2001).

For the analysis of glycation and oxidation products in the commercial milk samples,

protein preparation by dialysis, enzymatic hydrolysis and amino acids purification

were carried out as described for the glycated and oxidized α-LA samples.

Additionally, fat was separated from milk before the dialysis by centrifugation for one

hour at 4 °C. Finally, the samples were analyzed un der the same chromatographic

and MS-MS conditions which were used previously to detect the modification of α-

LA. For LC-MS-MS analysis, 25 µg of the enzymatically hydrolysed residue was

dissolved in 1 ml of a water/acetonitrile mixture (50/50%).


2.4.1 Analysis of MeSO and LacLys

The LC-MS-MS chromatograms of UHT milk, sterilized milk, liquid and powder HA-

infant formula have shown a clear formation of MeSO and LacLys (figure 40). As it

was observed for α-LA, MeSO eluted at 3.81 min, which is earlier than the standard

MeSO, while LacLys from samples eluted at the same retention time as the standard

(7.6 min).

Figure 40: LC-MS-MS chromatogram of UHT milk obtained in MRM mode shows the formation of MeSO and LacLys. After the removal of lactose and fat, milk proteins were subjected to enzymatic hydrolysis prior to LC-MS-MS analysis using a C18 column. LacLys and MeSO were identified by their specific mass transitions (LacLys: m/z 471.1→225.1, m/z 471.1→128.1, m/z 471.1→84.1; MeSO: m/z 166.1→74, m/z 166.1→56, m/z 166.1→102.1).

An unknown peak appeared at a retention time of 7 min and showed the ion

products m/z 166.1→74 and m/z 166.1→56, which are the characteristic fragments

of MeSO. To investigate the identity of this peak, MeSO (as standard) was added to

the protein hydrolyzate followed by LC-MS-MS analysis. The chromatograms

showed only an increase of the signal intensity of the peak with a retention time of

3.81 min, which is the peak of MeSO. For further investigation, UHT milk was heated

for 30 and 60 min at 120 °C and analyzed as describ ed before. Since the heat

treatment enhances glycation and oxidation reactions, the peak area of MeSO

increased gradually during the heat treatment. On the other hand, the area of the

unknown peak was almost stable. This means that the peak at RT of 7 min is not

derived from MeSO but is an interferent with lower polarity than MeSO. Later, by the


use of the ZIC-HILIC column, this unknown compound eluted before MeSO at the

retention time 4.5 min. This observation confirms its lower polarity than MeSO,

because the retention on a ZIC-HILIC column is proportional to the polarity

(Grumbach, Wagrowski-Diehl et al. 2004). The unknown compound is probably

generated from the milk matrix.

Figure 41: The increase of the MeSO signal and the stability of the unknown peak during heat treatment of milk

The peak areas calculated under the quantifier fragments of both, MeSO and LacLys

(m/z 166.1→74 and m/z 471.2→225.1, respectively), was higher in the samples of

HA-milk and sterilized milk compared to those of UHT milk indicating severe heat

damage in these products (figure 42). The high temperature used for sterilization

promoted the formation of LacLys compared to HA-milk treatment. In contrast, HA-

milk samples showed a higher signal of MeSO than sterilized milk. In order to draw

valid conclusions on the influence of industrial processes on product formation, the

development of a quantification method is necessary. Because of matrix effects on

ionization, the use of internal standard or standard addition for quantification is

required.


Figure 42 : Relative signal of MeSO and LacLys in different milk samples

2.4.1.1 Formation of MeSO during sample work-up

MeSO was detected in the blank in which enzymatic hydrolysis was carried out in the

absence of sample proteins. It could be excluded that the buffers and enzymes were

contaminated with MeSO. A reason for the MeSO background could be the autolysis

of the enzymes during enzymatic hydrolysis, especially of pepsin, under the alkaline

conditions.

In order to check this possibility, each of the three enzymes was removed from the

blank hydrolyzate after 1 day incubation and before the addition of the next enzyme.

The enzymes were removed by ultrafiltration using the centrifugal concentrator

devices used previously for the clean-up of protein hydrolyzate (MWCO of 10kDa).

This control was then analyzed by LC-MS-MS and the results were compared to

those of the blank containing the three enzymes untill the end of hydrolysis. The

chromatograms showed about 31% to 42% lower intensity of MeSO after the

removal of pepsin or both pepsin and pronase, respectively, indicating that the

hydrolysis of pepsin is the main source for MeSO in the blank (figure 43). The

presence of MeSO in the blank after the removal of pepsin is caused either by

inadequate removal of pepsin and thus further hydrolysis during the second and third

days, or by hydrolysis of the other enzymes. The intermediate removal of pepsin

during enzymatic hydrolysis of raw and UHT milk, led to the loss of some peptides

resulting in a 21-24 % lower signal intensity for MeSO and LacLys. Therefore, for

further investigation, the enzymes were not removed before the end of enzymatic

hydrolysis (3 days), but the overestimation of MeSO was determined quantitatively

and then subtracted from results of the milk samples.


Figure 43: Formation of MeSO during the different steps of enzymatic protein hydrolysis. A blank without sample protein was subsequently hydrolysed by pepsin, pronase and aminopeptidase. The enzymes remained in the samples throughout hydrolysis (A). Pepsin was removed before further hydrolysis (B). Pepsin and pronase were removed before further hydrolysis (C)

Figure 44: The decrease of MeSO and LacLys signal intensities after the removal of pepsin from milk hydrolysate after 24 hours of enzymatic hydrolysis

2.4.2 Analysis of 5-OH-Trp

5-OH-Trp was not detected in any of the samples, even if the sample concentration

was increased to 50 and 75 µg/ml.


2.4.3 Analysis of CML

CML was not detected in the samples of UHT milk; this could be related to the low

concentration of the analyzed sample (25µg/ml) and/or to the low modification during

the ultra-high temperature processing. In the other samples, a small peak appeared

after 7.35 min which showed the most intense product ion of CML (m/z 205.1→ 84.1)

indicating a possible formation of CML, as seen in figure 45.

Figure 45: LC-MS-MS chromatogram of sterilized milk obtained in MRM mode shows a clear formation of MeSO and LacLys and a possible formation of CML. After the removal of lactose and fat, milk proteins were subjected to enzymatic hydrolysis prior to LC-MS-MS analysis using a C18 column. MeSO and LacLys were identified by their specific mass transitions. Only one mass transition of CML was detected (m/z 205.1→ 84.1).

In order to ensure the identification of CML, it was necessary to increase the

sample’s concentration to the threshold at which two fragments of CML can be

detected. This could be problematic, especially for samples with low modification

rate like UHT and pasteurized milk, because the signals in concentrated samples are

more susceptible to matrix effects. Matrix effects, in particular signal suppression,

might lead to underestimation of the actual concentration and a higher limit of

detection (LOD) (Zimmer 2003). Additionally, the concentrated samples could be

damaging for the spryer needle of the LC-MS-MS apparat.

CML and LacLys are very hydrophilic analytes; therefore they have little retention on

reversed-phase columns. This problem was overcome by the use of the ion pair


reagent NFPA. The ion pair reagent is a large molecule which can act as a counter

ion to the analyte leading to the formation of a neutral pair. The neutral pair partitions

between the mobile and stationary phase, or the hydrophobic part of the ion pair

reagent adsorbs to the stationary phase to form an ion exchange surface. Thus, the

increase of the retention time on C18 column caused by the use of NFPA is

depending on the positive charge which is carried on CML and LacLys. Therefore it

will be difficult to separate CML from LacLys. Moreover, the co-elution of LacLys with

CML can cause matrix effects, especially ionization suppression which results in a

reduction of the signal intensity and a lower sensitivity. This effect can also result

from other polar and/or non-polar compounds in the matrix, which co-elute with CML,

even if these compounds are not defined in the MRM method. This loss of sensitivity

is more notable and negatively affected for CML detection due to its low formation

compared to LacLys.

A possibility to improve the method`s sensitivity for CML detection was to seperate

CML from LacLys and probably from other undefined matrix compounds, which have

similar retention time as CML. Since the separation of CML from LacLys could not be

achieved on the C 18 column despite of extended variation of the elution gradient, a

ZIC-HILIC column was used for further experiments.

2.4.4 Separation of 5-OH-Trp, MeSO, CML and LacLys

using a ZIC-HILIC column

HILIC is a mode of chromatography that uses a hydrophilic stationary phase with

organic water miscible solvent like acetonitrile and methanol. The use of an eluent

with a high portion of organic solvent (more than 70%) for HILIC chromatography is

ideal for the compound ionization by ESI-MS. Additionally, suitable retention times

for polar analytes are obtained (Schettgen, Tings et al. 2007). The analyte retention

on the HILIC columns is proportional to its polarity and inversely proportional to the

mobile phase polarity. Therefore, it is expected that CML can be separated form

LacLys by the use of HILIC columns such as ZIC-HILIC column. ZIC-HILIC columns

share the main properties with the other HILIC columns

MS-MS conditions were the same which were previously used for the analysis of 5-

OH-Trp, MeSO, CML and LacLys on the C18 column; however, the HPLC gradients

were changed to be compatible with the ZIC-HILIC column.


As it is seen in figure 46, a complete separation of the compounds 5-OH-Trp, MeSO,

CML and LacLys (mixture of standards) was achieved. These compounds eluted

after 4.9, 6.5, 10.5 and 25.6 min, respectively.

Figure 46: LC-MS-MS chromatogram obtained in MRM mode of 5-OH-Trp, MeSO, CML and LacLys (standard mixture) on a ZIC-HILIC column 150*2.1mm, 3.5 µm

2.4.5 Comparison of the signal intensities using C1 8 or

ZIC-HILIC columns

A standard solution containing 5-OH-Trp, MeSO, CML and LacLys was analyzed

once using a C18 column and then using a ZIC-HILIC column. The concentrations of

these compounds were 0.25, 0.008, 0.08 and 0.6µg/ml, respectively. The two trials

were carried out on two consecutive days. The chromatograms showed clear

differences in the intensities of the analytes between the two columns with a huge

advantage for the ZIC-HILIC column. The increase of the intensities using the ZIC-

HILIC column varied between 8, 17, 25, and 200 times for LacLys, MeSO, CML and

5-OH-Trp, respectively (figure 47). Thus, detection limits are considerably

decreased. This will be of special importance for CML which is present in the

samples in concentrations close to the detection limit.

Analysing UHT milk samples on the ZIC-HILIC column showed that the intensities for

CML as well as MeSO became only 3 times higher and for LacLys 5 times higher

compared to separation on the C18 column. Furthermore, the analytes intensities


showed a higher variation on the ZIC-HILIC column compared to the C18 column.

High variation was especially noted for LacLys in milk samples where the coefficient

of variation (CV) reached 40%. The reason for such imprecision could be the mass

spectrometer parameters of the ion source. They were optimized for an eluent ratio

of 50/50%, but LacLys eluted at ratio 20/80 of organic and aqueous solvents,

respectively.

Consequently, it was decided to analyze the compounds using two different methods

to achieve optimal results for quantification. The C18 column was chosen for further

investigation of LacLys. The ZIC-HILIC column was chosen for further investigation

of CML and 5-OH-Trp. Since MeSO is detected easily in milk samples and since the

variation of its signal intensity was similar on C18 and ZIC-HILIC columns, C18

column was chosen for further investigation of MeSO to avoid the long run time on

ZIC-HILIC (52 min).

Figure 47: Comparison of the signal intensities of the standards MeSO, CML, 5-OH-Trp and LacLys on C18 and ZIC-HILIC columns

2.4.6 Analysis of milk samples by LC-MS-MS using a ZIC-

HILIC column

UHT milk, sterilized milk and HA-infant formula were analyzed using a ZIC-HILIC

column. For this purpose, 50 µg of enzymaticlly hydrolyzed resdiue was dissolved in

1 ml of a water/acetonitrile mixture. Under these conditions, CML was detected


clearly in all milk samples at the retention time of 10.5 min. The formation of CML

was proportional to the thermal treatment. The highest signal was detected in the

sample of sterilized milk followed by the samples of HA infant formula with a

negligible difference between the liquid and powder forms. The mild heat treatment

of UHT milk resulted in the lowest signal for CML. Thus, the difficulty to detect the

low level of CML in UHT milk has been overcome by the replacement of the C18

column by a ZIC-HILIC stationary phase. However, an unknown peak appeared at a

retention time of 5 min, which showed the fragments of CML. In order to clarify the

origin of this peak, CML-standard was added to UHT milk samples before analysis.

The LC-MS-MS chromatograms showed an increase of the signal intensity of the

peak with a retention time of 10.5 min, which is the peak of CML. The signal of the

unknown peak, however, did not increase after spiking, indicating that the peak did

not originate from CML.

Figure 48: LC-MS-MS chromatogram of UHT milk obtained in MRM mode shows the formation of CML. Milk proteins were subjected to enzymatic hydrolysis prior to the LC-MS-MS analysis using a ZIC-HILIC column. An unknown peak with the same specific mass transitions (m/z 205.1→ 84.1, m/z 205.1→ 130.1) appeared after 5 min.

For further investigation of this peak, UHT milk was defatted and then heated for 30

and 60 min at 120 °C. CML was then analyzed as desc ribed before. Since the heat

treatment promotes the oxidation and glycation reaction, the peak of CML at 10.5

min increased gradually with increasing heat treatment. In contrast, the intensity of

the unknown peak was almost stable. This confirms that the compound at a retention

time of 5 min is not derived from CML and is not formed during the heat treatment.


More likely, the unknown compound is derived from the milk matrix. Despite the

higher sensitivity obtained by the use of ZIC-HILIC column compared to C18 column,

5-OH-Trp was not detected in any of the investigated samples.

Figure 49: The increase of CML signal and the stability of the signal of the unknown compound during heat treatment of milk

2.4.7 Discussion

The industrial production of dairy products requires thermal treatment in order to

ensure microbiological safety as well as to increase the shelf life of the products.

However, when exposed to heat, the amino acids in milk proteins may undergo

oxidation and glycation reactions leading to the formation of different compounds

such as lactulosyllysine, carboxymethyllysine, methionine sulfoxide,

hydroxytryptophan and others. In order to detect these modified amino acids in milk

proteins, a complete enzymatic hydrolysis has been applied using three enzymes

(pepsin, pronase and aminopeptidase) for 3 days at 37 °C. The release of the

modified amino acids was followed by the analysis by LC-MS-MS which represents a

powerful analytical technique to determine qualitatively and quantitatively low levels

of the target compounds with a high a degree of certainty.

By the use of LC-ESI-MS-MS, the analytes LacLys, CML and MeSO were detected

in UHT milk and sterilized milk as well as in hypoallergenic infant formula. 5-OH-Trp

was not detected in any of the investigated samples.

At present, different analytical approaches have been introduced to determine MeSO

in milk proteins. The partial enzymatic hydrolysis prior to the analysis by MALDI-

TOF-MS allowed for example the detection of MeSO in many peptides of whey


proteins (Meltretter and Pischetsrieder 2008). MeSO was also detected and

quantified in milk protein via reverse-phase HPLC system with UV detection after a

complete enzymatic hydrolysis for 20 hours at 37 °C (Baxter, Lai et al. 2007). In the

current study, MeSO was detected under similar chromatographic conditions after

complete enzymatic hydrolysis. However, MS-MS detection was used, which allows

product identification by three ion products (m/z 166.1→74, m/z 166.1→56 and m/z

166.1→102.1) additional to the retention time. Thus, higher certainty can be

achieved.

It is a common problem in the analysis of the polar compounds like CML and LacLys

that they have only little retention on reversed-phase columns due to their high

polarity. This problem has been overcome by the use of ion pair reagents such as

NFPA, which, in contrast to other ion pair reagent is volatile and better compatible

with ESI-MS-MS (Teerlink, Barto et al. 2004). The combination of C 18 column,

NFPA and MS-MS detection has been used successfully to detect advanced

glycation products in some human fluids (Teerlink, Barto et al. 2004; Gonzalez-

Reche, Kucharczyk et al. 2006) as well as detect early and advanced glycation

products in dairy products (Hegele, Buetler et al. 2008; Hegele, Parisod et al. 2008;

Delatour, Hegele et al. 2009).

The use of NFPA is more preferable for MS-MS, because it yields higher signal

intensity compared to other ion pair reagent like TFA (Assar, Moloney et al. 2009).

However, the possibility of ion suppression by NFPA can not be completely

excluded. Furthermore, the use of NFPA decreases the pH of the mobile phases

which accelerates the deterioration of RP-columns (Schettgen, Tings et al. 2007). In

the current study, moreover, the separation of CML and LacLys was not achieved on

the C18 column by the use of NFPA. Co-elution could weaken the signal intensity,

especially the signal intensity of CML, requiring the use of higher sample

concentrations. To avoid these problems, hydrophilic interaction liquid

chromatography was introduced as an alternative to reverse-phase chromatography.

The retention of the analytes on the ZIC-HILIC column was proportional to their

polarity enabling a complete separation of CML and LacLys (RT of 10.5 and 25.6

min, respectively). Additionally, the use of the ZIC-HILIC column enhanced clearly

the signal intensity of the analytes in the standard solutions as well as in milk

samples. However, a strong variation of the signal intensities of LacLys was

observed by the use of the ZIC-HILIC column. This imprecision could be caused by


the mass spectrometer parameters of the ion source, since these parameters were

optimized at an eluent ratio of 50/50 5mM ammonium acetate buffer and acetonitrile,

but the elution of LacLys was at ratio 80/20. Since the sensitivity of the method was

sufficient for LacLys and MeSO, further investigation of both analytes was performed

on a C18 column.

In general, sterilized milk showed a high modification rate which can be explained by

the high and prolonged heat treatment applied for sterilization (heating at 107-115 °C

for 20-40 s followed by heating in bottle at 120-130 °C for 8-12 min). This thermal

treatment enhanced glycation reactions and led to a high level of LacLys and CML.

The degradation of glycation products promotes oxidation reactions and explains the

high intensity of MeSO in sterilized milk. The level of MeSO in infant formula was

higher than the other investigated milk samples including sterilized milk.

However, since strong matrix effects like ion suppression are expected during the

analysis by ESI-MS-MS (Zimmer 2003), it is not recommended to quantify according

to peak areas. The quantification should be accomplished either by stable isotope-

labeled internal standard or by standard addition method.

5-OH-Trp was not detected in any of the investigated milk samples including infant

formulas. Infant formulas are generally supplemented with α-LA to resemble the

human milk as well as to compensate the deficiency of tryptophan in the whole

bovine milk proteins (Heine, Klein et al. 1991). The absence of 5-OH-Trp in infant

formula as well as in other milk samples is consistent with the results for glycated

and oxidized α-LA. This result does not mean, as discussed previously, the absence

of tryptophan oxidation products in the samples, because other OH-Trp regioisomers

and other oxidation products like kynurenine, N-formylkynurenine may be present.

However, the identification of these products requires further investigation.


2.5 Analysis of ornithine in milk proteins

2.5.1 Introduction

The formation of ornithine from arginine is well known. In vivo, ornithine is formed via

enzymatic catalysis by arginase in the urea cycle with concurrent production of urea.

Arginase is classified into arginase ו which is highly expressed in the liver and

arginase which is expressed in the kidney, prostate, brain and mamary glands

(Deignan, Livesay et al. 2007).

Ornithine is prepared conveniently from arginine under alkaline conditions.

Additionally, it can be gained from arginine in acidic solution at high temperature.

However, the latter reaction is very slow and leads to less than 1 % of ornithine

under the conditions usually used for the acidic hydrolysis of protein (HCl 6N, 24

hours at 110 °C) (Murray, Rasmussen et al. 1965)

Figure 50: Ornithine formation from arginine

Ornithine is not found in native proteins in vivo; however it has been detected in

long-live proteins like collagens and lens crystalline where the level of ornithine

increases during protein aging. The mechanism of ornithine formation is not known

but it may involve a conversion of the arginine residue into arginine-derived AGEs,

mainly argpyrimidine and pentosidine as well as Nδ-(5-hydro-4-imidazolon-2-yl)-

ornithine (G-H1) and Nδ-(5-hydro-5-methyl-4-imidazolon-2-yl)-ornithine (MG-H1),

which then are degraded into ornithine (Sell 2004). In food products, ornithine was

detected in sausages (Villmann, Sandmeier et al. 2007). Since glycation is very

pronounced during processing of milk, the aim of the following part of the work was

to analyze if ornithine is formed in milk proteins during heating.


Figure 51: Some derivatives of imidazolone formed from the reaction of arginine and dicarbonyls

2.5.2 LC-ESI-MS-MS analysis of ornithine

The chromatographic conditions (C 18 column, mobile phases and gradient) as well

as the mass spectrometric flow-dependent parameters were the same which were

used previously to detect LacLys and MeSO. The mass spectrometric compound-

dependent parameters were optimized to get the highest signal intensity for

ornithine.

Only two fragments of ornithine (m/z 133.1) were determined, which were the

quantifier fragment (m/z 133.1→ 70.1) and the qualifier fragment (m/z 133.1→ 116)

(figure 52). These fragments were the same as reported in literature (Argirov, Leigh

et al. 2005).

Precursor ion (m/z)

Product ion (m/z)

DP (V) EP (V) CE (V) CXP (V)

133.1 70.1 46 10 25 4

133.1 116 46 10 11 8

Table 7: Mass spectrometric compound-dependent parameters of ornithine


Figure 52: Product ion scan of ornithine shows the fragments and their proposed structures

The standard of ornithine eluted after 8.4 min and showed its characteristic

fragments (m/z 133.1→ 70.1 and m/z 133.1→ 116). In the samples of raw and UHT

milk proteins (25 µg/ml), a peak appeared at 8.3 min which showed only the

quantifier fragment (m/z 133.1→ 70.1), as seen in figure 53. Furthermore, no

difference was observed between the peak areas of raw milk and those of UHT milk

as well as of UHT milk heated for 10, 20, 30 and 60 min at 120 °C.


Figure 53: LC-MS-MS chromatogram of the standard ornithine (A) and UHT milk proteins (B) obtained in MRM mode. Milk proteins were subjected to enzymatic hydrolysis prior to LC-MS-MS analysis using a C18 column. Panel B shows a peak at the retention time of ornithine which showed only one of its characteristic fragments (m/z 133.1→ 70.1). The second fragment, which was characteristic for the standard (m/z 133.1→ 116), was not detected

Figure 54: Peak areas obtained for the quantifier fragment (m/z 133.1→ 70.1) of the compound detected at the retention time of ornithine


2.5.3 Discussion

In the present part of the work, the presence of ornithine in milk proteins was

investigated. In thermally heated milk proteins, a compound was detected which

eluted at the retention time of the standard ornithine and which showed one mass

transitions characteristic for the MS/MS fragmentation of ornithine (m/z 133.1→

70.1). The second important mass transition of ornithine (m/z 133.1→ 116),

however, was not detected. A single fragment is usually inadequate for the

identification of an analyte by MS-MS. When chromatography is involved, one

fragment in combination with the retention time may be considered adequate,

especially if the ions are generated by soft ionization technique. However, the use of

more than one fragment is still advisable (Ardrey 2003). In the current study, the

fragment m/z 70.1 was generated by the soft ionization technique (ESI) and it is

identical to the fragments reported in the literature. Additionally, the compound had

the same retention time as the standard ornithine. However, since two fragments of

ornithine (as standard) were detected under the same chromatographic and mass

spectrometric conditions, the detection of ornithine in milk proteins was questionable.

Furthermore, the fact that the concentration of the compound was independent from

the heat treatment of milk proteins indicates that it represents rather a matrix

compound of milk than ornithine.

It has been reported that the fragment m/z 133.1→ 70.1 can be generated from

arginine (Argirov, Leigh et al. 2005). In the current study, however, the compound

was not detected by the analysis of arginine standard or arginine that was subjected

to enzymatic hydrolysis. These results indicate that the detected fragment was not

generated from arginine. Further studies are therefore required to find out if the

compound indeed originates from milk matrix components or if the analytical

conditions prevented the detection of the second characteristic fragment of ornithine

in milk proteins.


2.6 Method optimization for the detection of MeSO,

CML and LacLys in proteins of processed milk

In the previous chapters, a method was established to detect MeSO, CML and

LacLys in milk proteins. A complete hydrolysis was accomplished by the application

of three proteases in the course of three days prior to analysis by LC-MS-MS. In the

present part of the work, the conditions of hydrolysis were optimized to obtain the

best release of the target analytes and, consequently, to achieve maximal sensitivity.

Moreover, the method was optimized to avoid overestimation or underestimation of

these analytes during the sample preparation.

The optimization process involved three steps which were the duration of the

enzymatic protein hydrolysis, the reduction of LacLys to prevent its conversion into

CML during protein hydrolysis and thus to avoid overestimation of CML. Additionally,

the optimization process involved purification of the hydrolyzate.

2.6.1 Optimization of protein hydrolysis

In order to determine the conditions which lead to a sufficient release of the modified

amino acids concurrently with minimal hydrolysis time, an another enzymatic

hydrolysis protocol was applied which takes 27 hours (Delatour, Hegele et al. 2009).

This new enzymatic hydrolysis method was compared to that one described by

Hasenkopf et al (72 hours). Additionally, some samples were subjected to acidic

hydrolysis and compared to the results of enzymatic hydrolysis. Acidic hydrolysis

requires reduction of LacLys prior to analysis to prevent its conversion into CML

during hydrolysis. The different hydrolysis methods were compared by the ninhydrin

assay, which measures the release of α-amino groups as an indicator for hydrolysis

efficiency. Additionally, the concentrations of the target analytes were monitored by

LC-MS-MS.

LacLys can be decomposed into CML by the oxidative cleavage of the carbohydrate

chain during the enzymatic digestion which results in an overestimation of CML

(Assar, Moloney et al. 2009; Delatour, Hegele et al. 2009). In order to prevent this


artificial formation of CML, LacLys was reduced in the present work by sodium

borohydride (NaBH4).

Figure 55: Reduction of lactulosyllysine with borohydride to form a product stable during acidic hydrolysis

The reduction of LacLys required the use of solid phase extraction (SPE) instead of

ultrafiltration to remove the reducing agents, which interfere with LC-MS-MS

analysis.

2.6.2 Results

The ninhydrin assay showed that the enzymatic hydrolysis for 72 hours of heated

UHT milk and sterilized milk resulted in a higher absorbance at 570 nm compared to

the samples hydrolyzed for 27 hours, even if higher concentrations were used (figure

56). These results were consistent with those obtained by MS-MS analysis where

longer hydrolysis duration resuted in higher signal intensities of MeSO, CML and

LacLys (figure 57). These findings indicate that the longer enzymatic hydrolysis

leads to higher release of the target analytes from proteins.


Figure 56: Protein samples hydrolyzed with a cocktail of enzymes for 72 hours or 27 hours were analyzed by ninhydrin assay. Enzymatic hydrolysis of sterilized milk and UHT milk heated for 10, 20, 30 and 60 min (120 °C) for 72 ho urs resulted in higher absorbance indicating a higher content of free amino acids

Figure 57: LC-MS-MS signal intensity of MeSO, CML and LacLys obtained after the enzymatic hydrolysis of sterilized milk sample for 72 hours or 27 hours

In order to investigate the influence of the purification step of the hydrolyzate on the

target analytes, SPE was introduced and compared to ultrafiltration. The first

experiment was carried out to investigate the target analytes recovery from the

stationary phase of the SPE column. A solution of the standards MeSO, LacLys and

CML was prepared and divided into two portions. The first one was loaded on a SPE

column. The eluate and the second part of the standard solution, which was used as

control, were lyophilized prior to LC-MS-MS analysis. The standards LacLys, CML

and MeSO which were loaded on a SPE column showed a decreased signal


intensity compared to the other part of the standards which was not loaded on the

SPE column. In particular, more than 50% of the signal intensity of LacLys was lost

during SPE (figure 58).

These results were consistent with those obtained by the analysis of milk samples

such as sterilized milk and UHT milk. Considerably lower peak intensities were

observed when samples were subjected to SPE after hydrolysis instead of

ultrafiltration. The observed loss was independent from the enzymatic hydrolysis

protocol applied.

Centrifugal vacuum concentrator (Speed-Vac) was used to concentrate some

samples after purification step instead of lyophilization. The typical application of

centrifugal vacuum concentrator is the removal of water and organic solvents such

as methanol, acetonitrile, chloroform or acetone. Additionally, centrifugal vacuum

concentrators are used to remove strong acids and bases from the samples. This is

of especial importance for acidic protein hydrolysis, since strong acids can not be

removed by lyophilization. A slight increase of CML was noted in the samples which

were dried by Speed-Vac after SPE compared to those dried bylyophilization. This

increase could be caused by LacLys degradation, since the drying by Speed-Vac

was carried out at 35 °C and lasted about 7 hours ( figure 59 and 60).

Figure 58: Decrease of the signal intensity of the standards MeSO, CML and LacLys after purification by SPE column compared to control. The samples were analyzed by LC-MS-MS. The highest loss was noted for LacLys


Figure 59: Signal intensity of MeSO, CML and LacLys detected by LC-MS-MS in UHT milk which was heated for 60 min at 120 °C. The sample w as purified either by ultrafiltration or by SPE, and then dried by Speed-Vac

Figure 60: Signal intensity of CML detected by LC-MS-MS in the samples of heated UHT milk which were dried either by lyophilization or Speed-Vac after purification

LacLys was reduced by 0.1 M NaBH4 prior to enzymatic protein hydrolysis to

investigate the artificial formation of CML during enzymatic hydrolysis. Some milk

samples, in which lactulosyllysine was reduced by NaBH4, showed a decrease of the

signal intensity of CML. This decrease did not exceed 25% compared to the signal

intensities obtained without reduction, independent of the enzymatic hydrolysis

protocol. Additionally, this decrease was not noted in all samples.


Figure 61: Decrease of the signal intensity of CML in heated UHT milk and sterilized milk in which LacLys was reduced by NaBH4 prior to enzymatic hydrolysis. The samples were analyzed by LC-MS-MS

In order to determine the conditions which lead to a sufficient release of the modified

amino acids, acidic hydrolysis was applied and compared to enzymatic hydrolysis

protocols by LC-MS-MS analysis as well as the ninhydrin assay. LacLys was not

detected in the acidic hydrolyzate of sterilized milk, heated UHT milk and HA-infant

powder formula. This result is expected, since LacLys was reduced by NaBH4 prior

to hydrolysis. However, the signal intensity of CML was about 400 % higher

compared to the samples subjected to enzymatic hydrolysis, independent if

enzymatic hydrolysis was carried out with or without the addition of NaBH4 (figure

62). The concentration of NaBH4 was increased to 2M in order to assure a complete

reduction of LacLys. However, the increase of NaBH4 concentration interfered with

analysis by LC-MS-MS so that the three analytes could not be detected anymore.

Remarkably lower MeSO intensities were found in the acidic hydrolyzates compared

to the enzymatic hydrolyzates. This decrease of intensities was observed when

NaBH4 was added prior to acidic hydrolysis. The analysis of a model mixture of

MeSO and NaBH4, which was heated under the conditions used previously for acidic

hydrolysis, showed a decrease of MeSO concentration, which was however not

accompanied by the formation of methionine. This result indicates that NaBH4 did

not reduce MeSO directly during acidic hydrolysis, but MeSO has undergone further

reaction. MeSO intensities in the enzymatic hydrolyzates did not decrease by the

addition of NaBH4.


Figure 62: LC-MS-MS signal intensity of the analytes MeSO, CML, and LacLys which were detected in (A) HA-infant powder formula and (B) sterilized milk. The diagrams show an increase of CML in the acidic hydrolyzate compared to both enzymatic hydrolyzates. LacLys was not detected after the reduction by NaBH4 in the acidic nor the enzymatic hydrolyzates. Clear decrease of MeSO signal was found in the acidic hydrolyzate

The enzymatic hydrolysis (72 hours) of heated UHT milk, sterilized milk, HA-infant

powder formula and lactalbumin resulted in a higher absorbance in the ninhydrin

assay compared to those which were acidicly hydrolyzed (figure 63). Before the

ninhydrin assay, acidic hydrolysates were buffered to a pH between 5 to 5.5, so that

an influence of the pH value on the color reaction can be excluded.


Figure 63: Protein samples of UHT milk heated for 10, 20, 30 and 60 min (120 °C), sterilized milk, HA-infant powder formula and lactalbumin. The samples were analyzed by ninhydrin assay after acidic or enzymatic hydrolysis for 72 hours. The absorbance at 570 nm of the enzymatic hydrolyzate (3µg protein) was higher compared to the acidic hydrolyzate (10µg protein)

2.6.3 Discussion

The experiments were carried out to determine the optimal conditions to release

MeSO, CML and LacLys from milk proteins as completely as possible. The analysis

by LC-MS-MS showed that a longer duration of enzymatic hydrolysis led to higher

signal intensities of these analytes: Signal intensities increased about 5 times after

enzymatic hydrolysis for 72 hours compared to 27 hours.

It may be hypothesized that the increase of LacLys is caused by an artificial

formation during the hydrolysis as a result of the reaction between the released

lysine residue and remaining lactose. It may be also assumed that the prolonged

hydrolysis for 72 hours can enhance the oxidative cleavage of LacLys and thus, will

increase the artificial formation of CML. Additionally, both, LacLys degradation and

the prolonged sample incubation, may enhance the oxidation of methionine.

However, during the sample preparation, lactose is removed by dialysis, so that

artificial lactosylation is less likely. Moreover, the reduction of LacLys prior to

enzymatic hydrolysis led only in some samples to a decrease of CML intensity,

which was always lower than 25% compared to the samples which were not


reduced. The results indicate that LacLys is relatively stable during enzymatic

hydrolysis so that the artificial formation of CML during prolonged enzymatic

hydrolysis should play only a minor role. The stability of LacLys under enzymatic

hydrolysis reduces the possibility of methionine oxidation, since methionine is

oxidized by hydroxyl radicals generated from LacLys decomposition (Mossine,

Linetsky et al. 1999). Furthermore, the incubation of methionine under the conditions

of enzymatic hydrolysis did not result in methionine oxidation, as it was seen

previously (see 2.3.5), so that the artificial formation of MeSO can be excluded.

These results in addition to those of the ninhydrin assay, which showed a higher

absorbance after enzymatic protein hydrolysis for 72 hours compared to 27 hours,

refer to a higher hydrolysis efficacy. Consequently, these results support a higher

release of the modified amino acids rather than their artificial formation.

The analysis of the modified amino acids by LC-MS-MS showed that the method

which is used to purify the samples affect the recovery of the target analytes. Lower

signal intensity was found after the purification by solid phase extraction compared to

samples purified by ultrafiltration. The lower recovery can be caused by the

adsorption of the analytes on the stationary phase. This is particularly the case for

LacLys, which showed about 50% loss during SPE. LacLys may also be degraded

during drying by the centrifugal vacuum concentrator. This step was carried out at 35

°C and lasted 7 hours which explains the slight inc rease of CML signal compared to

the samples dried by lyophilization.

Additionally, acidic hydrolysis was used to release the modified amino acids prior to

sample cleanup by SPE and LC-MS-MS analysis. Several authors report that the

Amadori product is not stable and can undergo spontaneous oxidative cleavage

during hydrolysis in 6 M HCl at elevated temperature. However, these conditions are

required to completely release amino acids from the proteins (Guy and Fenaille

2006; Hegele, Buetler et al. 2008).

To ascertain that CML was not formed artificially from LacLys, milk protein samples

were reduced by NaBH4 prior to acidic hydrolysis. It is well known that acidic

hydrolysis yields a better protein cleavage (20-25% more) than enzymatic hydrolysis

(Sell 2004; Delatour, Hegele et al. 2009). Therefore, an increase of CML intensity

was expected using acidic hydrolysis. However, the analysis by LC-MS-MS showed

that CML intensity in the acidic hydrolyzate was 4 times higher instead of only 25%

higher compared to the enzymatic hydrolyzate. This result was also in contrast to a


previous study which reported that the enzymatic hydrolysis resulted in an artificial

formation of CML, where CML concentrations in the enzymatic hydrolyzate were 3

times higher compared to the acidic hydrolyzate (Delatour, Hegele et al. 2009).

Another study did not observe any overestimation of CML when the proteins were

subjected to acidic hydrolysis without prior reduction by NaBH4 (Charissou, Ait-

Ameur et al. 2007).

The high level of CML in the acidic hydrolyzate led to doubt the sufficiency of the

reduction step and indicated that the absence of LacLys has resulted from the

reduction by NaBH4 as well as from the oxidative cleavage of LacLys. This

degradation of LacLys might lead then to an artificial formation of CML and explain

the increase of the signal intensity of CML.

The results of the ninhydrin assay could not completely confirm or falsify those

obtained by LC-MS-MS analysis. All samples of the acidic hydrolyzates developed

less color compared to the enzymatic hydrolyzates. Two factors are important to

develop the maximum color in the ninhydrin assay. The first is a sufficient reducing

agent to convert ninhydrin to the reduced hydantoin which is essential to develop the

color. The other factor is the temperature which must approach the boiling point over

the heating period. In the current experiment, a concentration of stannous chloride

was applied, which was described as sufficient to provide an adequate reducing

capacity in the range of 1-10 µg of protein hydrolyzate (Starcher 2001). Additionally,

a boiling water bath was used to heat the reaction mixture and it was found that 5

min was enough to provide the heating necessary to develop the color for all

samples. However, the color development in the samples of the acidic hydrolyzate

was less than the enzymatic hydrolyzate, independent of the duration of enzymatic

hydrolysis. This result could be caused by insufficient buffer capacity since the pH of

the acidic and enzymatic hydrolyzates was 5 and 5.5, respectively.

Interestingly, the intensity of MeSO decreased in the samples of the acidic

hydrolyzate compared to the enzymatic hydrolyzate. A previous study reported that

up to 59 % of methionine can be destroyed during acidic hydrolysis (Jennings. D. M

1969), while others reported that MeSO is reduced to methionine during acidic

hydrolysis (Guy and Fenaille 2006; Brock, Ames et al. 2007).

In the current study, the decrease of MeSO during acidic hydrolysis was observed

when NaBH4 was used. On the other hand, a considerable increase of MeSO was

found in the samples, which were subjected to acidic hydrolysis without NaBH4. This


refers to a possible influence of NaBH4 on MeSO intensity. However, NaBH4 mainly

reduces aldehyde and ketone to yield alcohols (da Costa, Pais et al. 2006).

Moreover, the model mixture of MeSO, NaBH4 and HCl (6 N), which was heated at

110 °C, did not show the conversion of MeSO into me thionin despite the decrease of

MeSO signal intensity. This result suggests that MeSO was not reduced to

methionine, but has undergone further reaction. No effect of NaBH4 on methionine

sulfoxide was reported in literature.

It was decided for several reasons to use enzymatic hydrolysis without the reduction

step to release the target analytes. 1)- The analysis by LC-MS-MS revealed

overestimation of CML as well as underestimation of MeSO in the acidic hydrolyzate

compared to the enzymatic hydrolyzate. 2)- The decrease of CML in the enzymatic

hydrolyzate, which was caused by the reduction of LacLys, was not observed in all

reduced samples and was always lower than 25%. Thus, the effect of NaBH4 on the

artificial formation of CML is still doubtful. 3)- The reduction of LacLys by NaBH4

required the use of SPE as a cleanup tool, which resulted in the adsorption of the

analytes on the stationary phase of the SPE column. 4)- The reduction step would

prevent the detection and quantification of LacLys in parallel with CML and MeSO.

On the basis of these experimental data, it was decided for further investigation to

adopt enzymatic hydrolysis for 72 hours without applying a reduction step.

Additionally, it was decided to use ultrafiltration as a cleanup tool. Finally, the use of

ZIC-HILIC column is also considered an improvement, because it resulted in a better

sensitivity for the detection of CML.


2.7 Method validation for the quantification of

LacLys, CML and MeSO in milk proteins

2.7.1 Introduction

Is the last chapters, a method was established to detect some glycation and

oxidation products in milk proteins by LC-ESI-MS-MS. Additionally, the conditions of

protein hydrolysis were optimized to release the target analytes as complete as

possible as well as to avoid artificial formation of the analytes during sample

preparation. In the present chapter, the validation of the method will be described.

The method was validated for each analyte by evaluation the within-day repeatability

(intraday repeatability), the between-days repeatability (inter-day repeatability), the

recovery as well as the limit of detection (LOD) and the limit of quantitation (LOQ).

The selectivity of the method was achieved by monitoring two mass transitions of

each compound. The most intense transition was used as the quantifier fragment

and the second one as the qualifier fragment.

analyte quantifier fragment qualifier fragment

LacLys m/z 471.2→ 225.1 m/z 471.2→ 128.1 MeSO m/z 166.1→ 74 m/z 166.1→ 56

CML m/z 205.1 → 84.1 m/z 205.1→ 130.1

Table 8: The quantifier and qualifier fragments of LacLys, MeSO and CML

UHT milk was chosen to investigate the repeatability of the method in mildly heated

milk proteins, while UHT milk heated for 1 hour at 120 °C was chosen as an example

for severely heated milk proteins.

To investigate the within-day repeatability, 7 to 8 samples of UHT milk or heated

UHT milk protein were enzymatically hydrolyzed for 3 days, worked up at the same

day, and then analyzed by LC-MS-MS. Since the standard addition method was

used to quantify the three analytes, which is rather time consuming, it was difficult to

analyze all samples at one day. Therefore, each group of samples, UHT milk or

heated UHT milk, required two days for anaylsis.


To investigate the between-day repeatability, 4 to 6 samples of UHT milk or heated

UHT milk were enzymatically hydrolyzed in consecutive days, worked up at the

same day, and were then analyzed by LC-MS-MS in consecutive days.

In order to investigate the recoveries of the analytes, UHT milk proteins were spiked

with three different and known amounts of LacLys, MeSO and CML before

enzymatic hydrolysis.

LOD was defined as the concentration of the analyte for which the signal to noise

ratio of the qualifier fragment is 3, while LOQ was defined as the concentration of the

analyte for which the signal to noise ratio of the quantifier fragment is 10. However,

the noise of the qualifier fragment of MeSO (m/z166.1→ 56) was high, resulting in a

LOD of MeSO similar to the LOQ. In order to avoid this problem, both, LOD and LOQ

of MeSO, were determined based on the signal to noise ratio of the quantifier

fragment. Moreover, since there were no milk samples which did not contain any

MeSO, LOQ and LOD of MeSO were determined by the dilution of three samples of

UHT milk to the extent at which the signal to noise ratio of the quantifier fragment

was 10 and 3, respectively.

LOQ and LOD of LacLys were determined by the dilution of three samples of UHT

milk protein hydrolyzate to the extent at which the signal to noise ratio of the

quantifier (m/z 471.2→ 225.1) and qualifier (m/z 471.2→ 128.1) fragments were 10

and 3, respectively.

Since CML is not detectable in the applied concentration of raw milk, it was used in a

low concentration as a blank matrix to determine LOQ and LOD of CML. Different

amounts of CML were added to this matrix, and then each sample was analyzed four

times by LC-MS-MS. The average of signal to noise ratios of these 4 analyses was

calculated to determine LOQ and LOD.

During the validation of the method, each analyte was quantitatively measured by

standard addition.

2.7.2 Quantitative analysis by standard addition

It is well known that electrospray ionization suffers signal suppression when

polar/ionic compounds coelute with the analyte of interest. The common approaches

to overcome this problem are the use of standard addition or the use of stable

isotope labeled internal standards (Ardrey 2003). Since the isotopic labelled MeSO


and LacLys are not available as internal standards, the method of standard addition

was adopted for quantitative measurement.

For this method, increasing known amounts of each standard compound are added

to the sample which contains an unknown amount of the analyte. The signal of the

analyte of interest is related to the amount of the added standard as well as to the

amount of the analyte originally present in the sample (Ardrey 2003).

The signal intensity was expressed as the peak area under the quantifier fragment of

each analyte in the unknown sample as well as in those to which the standard

compounds were added. The peaks areas (y axis) were then related to the

concentrations of the added analyte (x axis). The following equation is used to

determine the unknown concentration of the analyte:

y= m*x+b

y: the signal of the analyte

m: the slope of the calibration curve

x: the concentration of the analyte

b: y-intercept of calibration curve

In the case of standard addition, y is the signal of the unknown analyte and the

added standard, while the y-intercept is equivalent to the signal of the unknown

analyte. The extension of calibration curve will intercept the x axis at the point

corresponding to the unknown concentration of the analyte. At this point, y is 0,

which means that x=m/b


Figure 64: Schematic illustration shows the method of standard addition. (http://www.chemgapedia.de/vsengine/vlu/vsc/de/ch/3/anc/croma/kalibrierung.vlu/Page/vsc/de/ch/3/anc/croma/datenauswertung/quantitativ/standardaddition/standardadditionm80ht0801.vscml.html, date 9/2/2011)

2.7.3 Results

2.7.3.1 Within-day repeatability

0.59 ± 0.038 µg of MeSO with a coefficient of variation (CV) of 6.4 % was detected in

1 mg of UHT milk protein residue as an average of 8 samples, which were prepared

simultaneously for analysis. The heat treatment of UHT milk resulted in a higher level

of MeSO. 0.69 ± 0.068 µg was detected analysing 7 samples with a CV of 9.96 %.

0.86 ± 0.074 µg and 1.13 ± 0.09 µg of LacLys were found in UHT milk and heated

UHT milk protein, respectively, as an average of 8 samples of each group with

similar coefficients of variation (8.2 and 8.6%). These values of LacLys were

calculated taking into consideration the purity of the synthesized LacLys (6.13%).

The variation within day for CML was good. 0.0279 ± 0.002 µg with a CV of 7.11%

and 0.0486 ± 0.0046 µg with a CV of 9.6% were detected in UHT and heated UHT

milk, respectively.


MeSO

n=8 LacLys

n=8 CML n=7

UHT-milk µg/mg

0.59 ± 0.038 0.86 ± 0.074 0.0279 ±0.002

coefficient of variation % 6.4 8.6 7.1

MeSO

7=8 LacLys

n=8 CML n=8

heated UHT-milk 60min, 120 °C

µg/mg 0.69 ± 0.068 1.13 ± 0.09 0.0486 ±0.0046

coefficient of variation %

9.96 8.2 9.6

Table 9: Within-day repeatability of the method for MeSO, LacLys and CML

2.7.3.2 Between-day repeatability

The between-day repeatability of the method for LacLys and MeSO was calculated

by analysing 6 samples of UHT milk as well as heated UHT milk proteins which were

prepared and analyzed in sequence with a time difference of 24 hours. 4 samples of

each UHT and heated UHT milk proteins were analyzed to investigate the between-

day repeatability for CML. As seen in table 10, 0.56 ± 0.054 to 0.68 ± 0.059 µg of

MeSO with CV of 9.6 and 8.7 % were detected in UHT and heated UHT milk,

respectively. The between-days repeatability of the method for LacLys in UHT was

good (CV 7.9%). In heat treated UHT milk, the CV was slightly higher (9.96%). The

best coefficients of variation were found in for CML, which were between 5.8 and

7.4%.


MeSO n=6

LacLys n=6

CML n=4

UHT-milk µg/mg

0.56 ± 0.054 1.01 ± 0.08 0.019 ± 0.0014

coefficient of variation % 9.6 7.9 7.4

heated UHT-milk 60min, 120 °C

µg/mg 0.68 ± 0.059 1.49 ± 0.15 0.031±0.0017

coefficient of variation %

8.7 9.96 5.8

Table 10: Between-day repeatability of the method for MeSO, LacLys and CML

2.7.3.3 Recovery

Three different amounts of each of the synthesized LacLys, MeSO and CML were

added to 4 samples of UHT milk proteins before applying enzymatic hydrolysis. The

contents of these analytes in the unspiked UHT milk (control) as well as in the spiked

UHT milk samples were determined by standard addition.

The recovery of each analyte was calculated as the ratio of the measured to the

expected concentration. The measured amount is the content of the analyte in the

sample after spiking (spiking 1, spiking 2 or spiking 3). The expected value of the

analyte is theoretically the sum of the spiked standard compound and the analyte

already present in the control. The average of the recoveries of four samples was

calculated and considered as the final recovery of the analyte. As seen in table 11,

the recovery of LacLys ranged between 110.5 and 111.6 % with a tendency for a

higher standard deviation at a higher amount of the spiked standard.

The recoveries of CML were optimal for the first and the second additions (0.012 and

0.024µg/mg) which were almost 100 % ± 5.5. By increasing the amount of spiked

CML to 0.048µg/mg, the standard deviation increased to 9.4, however the recovery

was still very good (96.6 % ± 9.4). In general, MeSO showed a high recovery which

reached 113.1 % as well as a high standard deviation which ranged between 10.7

and 14.3 %.

The first three experiments were simultaneously performed, while a fourth

experiment was carried out after 2 months. The fourth experiment showed generally,


independent of the spiked amounts of the analytes, lower contents of LacLys and

MeSO compared to the other experiments. Such differences in the analyte content

might result from the use of different standard solutions for standard addition.

However, the recoveries of LacLys and MeSO in this experiment were consistent

with the other experiments.

Unspiked sample (control)

Spiking 1 (0.357 µg added to 1 mg protein)

LacLys µg/mg

measured LacLys µg/mg

expected LacLys µg/mg recovery %

Experiment 1 1.55 2.3 1.91 120.4

Experiment 2 1.52 1.84 1.88 97.9

Experiment 3 1.75 2.50 2.1 119

Experiment 4 0.73 1.14 1.09 104.6

mean 110.5±9.6



LacLys µg/mg


expected LacLys µg/mg recovery %

Experiment 1 1.55 2.66 2.27 117.4

Experiment 2 1.52 2.38 2.23 106.5

Experiment 3 1.75 2.4 2.46 97.4

Experiment 4 0.73 1.81 1.45 124.3

mean 111.6±10.6



LacLys µg/mg


expected LacLys µg/mg

recovery %

Experiment 1 1.55 3.61 2.98 121.1

Experiment 2 1.52 3.72 2.95 126.1

Experiment 3 1.75 2.79 3.18 87.7

Experiment 4 0.73 2.33 2.16 107.9

mean 110.7±17

Table 11: Recovery of LacLys




MeSO µg/mg

measured MeSO µg/mg

expected MeSO µg/mg recovery %

Experiment 1 1.86 2.3 2.02 113.9

Experiment 2 1.9 1.87 2.06 90.8

Experiment 3 1.7 2 1.86 107.5

Experiment 4 1.06 1.45 1.22 118.9

mean 107.8 ±10.6



MeSO µg/mg


expected MeSO µg/mg recovery %

Experiment 1 1.86 2.25 2.18 103.2

Experiment 2 1.9 2.91 2.22 131.1

Experiment 3 1.7 1.85 2.02 91.6

Experiment 4 1.06 1.51 1.38 109.4

mean 108.8 ±14.3



MeSO µg/mg


expected MeSO µg/mg

recovery %

Experiment 1 1.86 2.96 2.5 118.4

Experiment 2 1.9 3.05 2.54 120.1

Experiment 3 1.7 2.21 2.34 94.4

Experiment 4 1.06 2.03 1.7 119.4

mean 113.1±10.8

Table 12: Recovery of MeSO




CML µg/mg

measured CML µg/mg

expected CML µg/mg

recovery %

Experiment 1 0.076 0.086 0.088 97.7

Experiment 2 0.042 0.049 0.054 90.7

Experiment 3 0.053 0.07 0.065 107.7

Experiment 4 0.047 0.057 0.059 96.6

mean 98.2 ±6.1

Unspiked sample

(control)) Spiking 2 (0.024 µg added to 1 mg protein)

CML

µg/mg measured CML

µg/mg expected CML

µg/mg recovery %

Experiment 1 0.076 0.106 0.10 106.0

Experiment 2 0.042 0.07 0.066 106.2

Experiment 3 0.053 0.076 0.077 98.3

Experiment 4 0.047 0.066 0.071 93.4

mean 101 ±5.4



CML µg/mg

measured CML µg/mg

expected CML µg/mg

recovery %

Experiment 1 0.076 0.132 0.124 106.5

Experiment 2 0.042 0.095 0.09 105.6

Experiment 3 0.053 0.088 0.101 87.1

Experiment 4 0.047 0.083 0.095 87.4

mean 96.6 ±9.4

Table 13: Recovery of CML

2.7.3.4 Limit of detection and limit of quantitatio n

LOQ and LOD of MeSO were determined based on the quantifier fragment (m/z

166.1→ 74). LOQ and LOD of LacLys were determined based on the quantifier

fragment (m/z 471.2→ 225.1) and the qualifier fragment (m/z 471.2→ 128.1). LOQ

and LOD of MeSO and LacLys were dependent on the injection volume. LOQ and


LOD were 2 and 1 pg of MeSO on column, respectively, equivalent to 400 and 200

pg MeSO pro 1 ml of sample solution (injection volume is 5 µl).

For the determination of LOQ of LacLys, the unknown peak, which showed the

quantifier fragment of LacLys (m/z 471.2→ 225.1) and which appeared directly after

LacLys (figure 65), was not considerd as noise. Otherwise, LOQ would increase

more than 20 fold.

Figure 65: An unknown peak appeared at 8.08 min and showed the quantifier fragment of LacLys

LOQ and LOD were 6.5 and 4 pg of LacLys on column, which is equivalent to 1.3

and 0.75 ng LacLys pro 1 ml of sample solution, respectively.

These values were calculated taking into consideration the purity of the synthesized

LacLys (6.13%) and the injection volume (5µl). Additionally, the unknown peak,

which appeared directly after LacLys (8.08 min), was not considerd as noise.

LOQ and LOD of CML were 8 and 4 ng, respectively, spiked in 25 µg of raw milk

proteins. These values are equivalent to 40 and 20 pg of CML on column (injection

volume is 5 µl).

2.7.4 Discussion

The coefficients of variation of within-day and between-day repeatability of the entire

method to detect the analytes LacLys, CML and MeSO were determined. These

values did not exceed 10 % at low as well as at high level of modifications. The

coefficients of variation reflect the variation in both, enzymatic hydrolysis and the

instrumental analysis. The results for CML are consistent with those obtained by

Assar at al. However, in this study the hydrolyzates were analyzed only on two


different days to estimate the between-day repeatability, and only twice on the same

day to exam the intra-day repeatability (Assar, Moloney et al. 2009).

In lactose free milk, UHT milk and raw milk samples, 0.01 µg CML was detected in

65, 210 and 700 µg protein, respectively, with S/N ratio of about 10. Additionally,

0.005 µg CML was detected in 35 µg protein of infant powder formula and HA infant

powder formula with S/N ratio, based on the qualifier fragment (m/z 205.1→ 130.1)

of about 3. The protein content in these five samples was determined by Kjeldahl.

This result indicates that the ion suppression is very low up to 1 mg of analyzed milk

proteins. During validation, LOQ and LOD of 8 and 4 ng in 25 µg protein residue,

were determined. These values are equivalent to 40 and 20 pg CML on column,

respectively. Since protein matrix up to 1 mg did not interfere with signal intensity, a

LOQ and LOD of 8 and 4 ng pro 1mg protein were defined.

The value of LOQ and LOD of CML differs from those reported in literature. One

study reported, for example, that LOQ and LOD of CML are 27 and 8 ng per mg

protein, respectively. However, these values were estimated by extrapolation and not

by experimental determination (Delatour, Hegele et al. 2009). Another study reported

that LOQ and LOD of CML were 0.03 and 0.009 mmol per mol lysine, respectively.

The content of lysine in this study was not measured directly, but estimated

depending on milk lysine content reported in literature. Additionally, LOD and LOQ

were calculated based on the quantifier fragment of CML only (Assar, Moloney et al.

2009). In both studies, CML was analyzed by LC-ESI-MS-MS using a stable isotope

labeled standard. It is important to mention that LOD and LOQ of the analytes CML

as well as MeSO and LacLys is dependent on the cleanness of the sprayer needle

and the mass analyzer.

So far, a LC-MS-MS method for the analysis of MeSO and LacLys in milk proteins

has not been reported. The repeatability of the analysis of MeSO and LacLys was

good, since the coefficient of variation did not exceed 10 %. In the current study,

LOQ and LOD were 6.5 and 4 pg of LacLys on column, respectively, and 2 and 1 pg

of MeSO on column, respectively. The detected amount of LacLys and MeSO in

UHT milk, as an example for a mildly heat treated milk product, was much higher

than the LOD and LOQ. Thus, it can be concluded that the method is sensitive

enough to detect these two analytes in all milk products.

The high recovery of LacLys might be caused by the added LacLys standard, which

may contain impurities of lactose. Lactose could then react with the released lysine


during hydrolysis leading to the formation of LacLys. Consequently, the content of

LacLys may enhance the oxidation of methionine leading to a higher recovery of

MeSO.

The validation results indicate that the method is precise and sensitive enough to

quantify lactulosyllysine, carboxymethyllysine and methionine sulfoxide in various

dairy products.


2.8 Quantitation

2.8.1 Introduction

After validation, the method was employed for the analysis of LacLys, CML and

MeSO in several dairy products. The analysis involved raw milk and mildly heat-

treated milk like pasteurized milk, UHT milk as well as lactose free milk and severely

heat-treated milk such as condensed milk and sterilized milk. Additionally, different

infant formulas were analyzed which varied between regular and hydrolyzed

formulas, liquid and powder formulas as well as "from birth" and toddler liquid

formulas. Two samples of each milk product were purchased from two different

manufactures for quantification and then subjected separately to analysis.

The sample preparation involved defatting, dialysis, enzymatic hydrolysis for 72

hours of 1 mg of the lyophilized residue, and then cleanup by ultrafiltration. The

protein content was determined by Kjeldahl which was applied to the milk residues

obtained after defatting and dialysis. The analytes were quantified by standard

addition. The content of CML was expressed as ng per mg protein, while the

contents of both, LacLys and MeSO, were expressed as µg per mg protein.

2.8.2 Results

The contents of MeSO, LacLys and CML in the analyzed samples are summarized in

table 14.


sample MeSO µg per mg protein

LacLys µg per mg protein

CML ng per mg protein

raw milk 1 1.13 0.043 15.10

raw milk 2 0.79 0.087 14.40

pasteurized milk 1 0.91 0.29 17.20

pasteurized milk 2 0.40 0.30 22.10

UHT milk 1 0.85 1.05 60.60

UHT milk 2 0.44 1.03 40.40

lactose free milk 0.49 1.24 142.60

sterilized milk 1 2.38 3.60 454.10

sterilized milk 2 2.14 3.45 547.50

vanilla drink 1 3.05 4.83 322.40

vanilla drink 2 1.97 4.27 451.40

condensed milk 4% , 1 1.52 7.48 532.40

condensed milk 7.5 % 1.79 7.23 538.20

condensed milk 4% , 2 3.09 6.05 899.00

condensed milk 4%, 3 1.06 8.70 586.70

sugared condensed milk 7.5 %, 1 1.41 8.52 624.70

sugared condensed milk 7.5 %, 2 2.20 11.48 832.50

powdered infant formula 1 (from birth) 2.85 4.08 254.40

powdered infant formula 2 (from birth) 7.61 1.50 154.10

powdered infant HA formula 1 (from birth)

3.86 4.26 420.20

powdered infant HA formula 2 (from birth) 11.31 2.23 250.00

liquid toddler formula 1 (from 12 month) 3.46 3.42 321.6

liquid toddler formula 1 (from 12 month) 2.99 2.80 245.74

Table 14: MeSO, LacLys and CML contents in some dairy products determined by LC-ESI-MS-MS


2.8.3 Discussion

The analytes LacLys and CML were quantified in different milk products where they

increased generally with increasing heat treatment. The highest contents were found

in condensed and sterilized milk, while mild heat treatment like pasteurization and

UHT processing induced a low level of modification. Additionally, LacLys and CML

as well as MeSO were detected in important amounts in infant formulas. Some of the

present results are consistent with those reported in literature. For example, Baxter

et al quantified MeSO in milk products by HPLC/UV and found 3 and 11.3 mg in 100

g of pasteurized milk and sugared condensed milk, respectively (Baxter, Lai et al.

2007). These results are in good accordance with the current results, where 0.4-0.91

µg and 1.41-2.2 µg of MeSO per mg proteins were detected in pasteurized milk and

sugared condensed milk proteins (equivalent to 1.29-2.93 mg and 10.7-16.6 mg per

100 g of product, respectively). The latter values were calculated assuming a milk

density of 1.025 g/ml and a protein content of 3.3 % in pasteurized milk. Protein

concentration of sugared condensed milk was taken from the information given by

the supplier (7.6 g protein in 100 g of the product).

However, some results differed completely from literature data. For example, 2.08 ng

of CML per mg protein were detected in raw milk (Ahmed, Mirshekar-Syahkal et al.

2005). In the current study, 14.4-15 ng of CML per mg protein was detected. Such

differences may result from different instrumental methods as well as different

hydrolysis protocols and different methods used for the determination of the protein

content. Additionally, the conditions applied in the production process may vary

between different manufactures.

The content of CML in lactose free milk was 3 folds higher than in UHT milk despite

of the same heat treatment and the same content of LacLys. For the production of

lactose free milk, lactose is enzymaticallly hydrolysed to glucose and galactose prior

to heat treatment. The higher content of CML may result from the participation of

glucose and galactose in glycation. It was found that glucose and galactose can

induce a higher level of glycation in milk proteins compared to lactose (Chevalier,

Chobert et al. 2001). CML levels in hydrolyzed infant formulas were higher than in

the regular formulas from the same manufacture (e.g. powdered infant formula 1

versus powdered infant HA formula 1). This result is consistent with literature data

(Delatour, Hegele et al. 2009). Furthermore, Delatour et al and Birlouez et al


reported that liquid infant formulas show in general higher CML levels than powdered

formulas (Birlouez-Aragon, Pischetsrieder et al. 2004; Delatour, Hegele et al. 2009).

In the current study, toddler liquid formulas showed similar or slightly higher CML

contents compared to powdered regular formulas.

Higher levels of LacLys, CML and MeSO were detected in infant formulas and

toddler formulas compared to processed cow milk (UHT and pasteurized milk). A

combination of factors can lead to this result beside the heat treatment. These

factors include the high content of lactose (8% of lactose) (Assar, Moloney et al.

2009) and the high level of lysine in infant formula caused by the addition of α-LA to

resemble mother milk (Heine, Klein et al. 1991). Additionally, it was shown that the

supplementation of infant formulas with ascorbic acid and iron enhances glycation of

milk proteins and leads to higher levels of CML and furosine (Leclere, Birlouez-

Aragon et al. 2002). This supplementation produces also hydroxyl radicals and thus

promotes oxidation reactions (Almaas, Rootwelt et al. 1997). Furthermore, iron is

chelated by glycated proteins and can generate oxygen species which react with

neighbouring amino acids (Leclere, Birlouez-Aragon et al. 2002). These factors

explain the high level of CML and LacLys as well as the highest level of MeSO in

infant formulas compared to the other analyzed samples. The high content of LacLys

and CML in sterilized milk results from the high temperature applied to ensure

microbiological safety. Subsequently, the degradation of the Amadori product

promotes the generation of hydroxyl radicals (Mossine, Linetsky et al. 1999) and

thus explains the high levels of MeSO in these samples. The detection of MeSO in

raw milk was unexpected, especially since the content was higher than in the mildly

heat-treated milk products. This could result from hydrogen peroxide which is widely

used as bacteriostatic agent to clean milk containers. It has been shown that the

addition of H2O2 to whey protein solutions at bacteriostatic level led to a significant

increase of methionine sulfoxide (Baxter, Lai et al. 2007). High levels of

modifications were found in vanilla drinks which were obtained from two different

suppliers. Unfortunately, additional details were not reported on the product`s label

which might help to interpret the results.


sample MeSO analytical method reference

pasteurized milk 3mg/100g product HPLC/UV Baxter et al (2007)

pasteurized milk 1.29-2.93mg/100g product LC-ESI-MS-MS current study

sugared condensed milk 11.3mg /100g product HPLC/UV Baxter et al (2007)

sugared condensed milk 10.7-16.6mg/100g product LC-ESI-MS-MS current study

Table 15: MeSO content in different milk products reported in literature

sample CML analytical method reference

raw milk 9.3ng/mg protein LC-ESI-MS-MS Assar et al (2009)

raw milk 337nM (2.08ng/mg protein) LC-ESI-MS-MS Ahmad et al (2005)

raw milk 1.19-2.35ng/mg protein LC-ESI-MS-MS Delatour et al (2009)

raw milk 14.4-15.05ng/mg protein LC-ESI-MS-MS current study

pasteurized milk 10.4-16.2ng /mg protein LC-ESI-MS-MS Assar et al (2009)

pasteurized milk 877nM (5.42ng/mg protein) LC-ESI-MS-MS Ahmad et al (2005)

pasteurized milk 0.79-1.81ng/mg protein LC-ESI-MS-MS Delatour et al (2009)

pasteurized milk 16.3mg/mg protein LC-ESI-MS-MS Fenaille et al (2006)

pasteurized milk 17.2-22 ng per mg protein LC-ESI-MS-MS current study

UHT milk 8.88ng/mg protein LC-ESI-MS-MS Delatour et al (2009)

UHT milk 29.2-46.4ng/mg protein LC-ESI-MS-MS Fenaille et al (2006)

UHT milk 40-60.55ng/mg protein LC-ESI-MS-MS current study

sterilized milk 12.77nM

(12.77ng/mg protein) LC-ESI-MS-MS Ahmad et al (2005)

sterilized milk 454-547ng/mg protein LC-ESI-MS-MS current study

condensed milk 205ng/mg protein LC-ESI-MS-MS Delatour et al (2009)

condensed milk 533-899ng/mg protein LC-ESI-MS-MS current study

powdered infant formula 1.2ng /mg protein ELISA Birlouez-Aragon (2004)

powdered infant formula 54ng / mg protein GC-MS Chrissou et al (2007)

powdered infant formula 25-140ng/mg protein LC-ESI-MS-MS Fenaille et al (2006)

powdered infant formula 154-254.44ng /mg protein LC-ESI-MS-MS current study

powdered infant HA formula 184ng/mg protein LC-ESI-MS-MS Delatour et al (2009)

powdered infant HA formula

134-322ng/mg protein LC-ESI-MS-MS Fenaille et al (2006)

powdered infant HA formula 250-420ng/mg protein LC-ESI-MS-MS current study

Table 16: CML content in different milk products reported in literatu

CHAPTER 3. MATERIALS AND METHODS 114

3 Materials and methods

3.1 Materials and apparatus

3.1.1 General materials

Tubes 15 and 50 ml (Sarstedt)

Syringe filter 0.22µm, PVDF (Roth)

Tubes 0.5, 1.5 and 2 ml (Eppendorf)

Pipette tips (Eppendorf)

3.1.2 Apparatus

Analytical balance (Sartorius)

pH-Meter (Metrohm)

Pipette (Brand, Eppendorf)

Ultrasonic bath (Bandelin SONOREX)

Magnetic stirrer (Heidolph)

Thermo mixer (Eppendorf)

Rotary evaporator (Büchi, Heidolph)

Centrifuge (Hettich universal 32R)

Shaking water bath SW22 (Julabo)

Freeze-dryer (Novalyphe-NL150, Savant)

Speed-Vac concentrator (Thermo Electron corporation)

NMR-spectrometry analyzer (Bruker Avance 360 MHz)

High resolution impact mass spectrometry (JMS-GC-Mate JEOL)

Kjeldahl apparatus consists of kjeldahl apparatus IR600, exhauster and water vapour

distillatory (Omnilab)


LC-ESI-MS-MS consists of API 4000 QTRAP (AB SCIEX) coupled to liquid

chromatography Ultimate 3000 RS (Dionex)

Analytic HPLC:

AS-1555, Intelligent Sampler, Jasco

DG-980-50, 3-Line Degasser Jasc

MD-1510, Multiwavelength Detector, Jasco

LG-980-02S, Ternary Gradient Unit; Jasco

PU-1580, Intelligent pump, Jasco

Preparative HPLC:

AS-2057Plus, Intelligent Sampler, Jasco

UV-2077Plus, 4- Intelligent UV/VIS Detector, Jasco

CHF122SB, Fraction Collector, Advantec

PU-2087Plus, Intelligent Prep. Pump, Jasco

CO-200, oven, Jasco

3.1.3 Materials

Synthesis

Nα-FMOC-Lys-HCl (Fluka)

Lactose anhydrous (Fluka)

Triethylamine (Acros Organics)

Morpholine (Fluka)

Dimethylformamide (Sigma-Aldrich)

Diethylether (Fisher scientific)

Dimethylformamide 99.8% (Acros-organics )

Cbz-Lys-OH (Fluka)

Na2HPO4*2H2O (Fluka)

NaH2PO4-2H2O (Fluka)

Iodoacetic acid (Fluka)

Palladium black (Fluka)

Spiritus ethanol (BFB-Nürnberg)

K2HPO4 (Fluka)

KH2PO4 (Fluka)

NaCL (Prolabo)


Urea (Merck)

Methionine (Sigma-Aldrich)

Cysteine (Fluka)

H2O2 30% (Merck)

FeSO4 heptahydrate (Fluka)

Hydrolysis

Pepsin (Roche)

Pronase (Roche)

Leucine aminopeptidase (Sigma-Aldrich)

Ammonium acetate (Acros Organics)

Ammonium carbonate (Acros Organics)

Ammonia (Grüssing)

Sodium tetraborate (Acros Organics)

Sodium hydroxide (Grüssing)

Sodium borohydride (Fluka)

Tris (Acros Organics)

Concentrated hydrochloric acid (Merck)

Ninhydrin reagent

Sodium acetate (Fluka)

Stannous chloride dehydrate (Merck)

Ninhydrin (Sigma-Aldrich)

Ethelyne glycol (Acros Organics)

Materials and equipments for liquid chromatography and mass

spectrometry

RP-thin layer (Merck)

1ml polypropylene column (Qiagen GmbH)

m-Aminophenylboronic acid-agarose (Sigma-Aldrich)

Magnesium chloride hexahydrate (Merck)

D-sorbitol (Fluka)

Concentrated acetic acid (Merck)

ZIC-HILIC column: 150*2.1mm, 3.5 µm (Sequant)


ZIC-HILIC column: 150*4.6 mm, 5 µm (Sequant)

Supelcosil C18 column: 150*2.1mm, 5µm (Supelco)

C18-100 column: 250*4 mm, 5µm (Nucleosil)

C18-100 column: 250*21 mm, 7µm (Nucleosil)

Strata C18-E (phenomenex)

NFPA (Sigma-Aldrich)

Formic acid 99% (Acros Organics)

Acetonitrile MS grade (Fisher scientific)

Ammonium acetate MS grade (Fluka)

Methanol HPLC grade (Prolabo)

Acetonitrile HPLC grade (Prolabo)

5-Hydroxytryptophan (Sigma-Alrich)

Methionine sulfoxide (Sigma-Adrich)

Methionine sulfone (Sigma-Aldrich)

Carboxymethyllysine (NeoMPS GROUPE SNPE)

Oxidation and glycation in milk models

α-lactalbumin typ (Sigma-Aldrich)

Lactose anhydrous (Fluka)

H2O2 30% (Merck)

NaCL (Prolabo)

Na2HPO4*2H2O (Fluka)

NaH2PO4-2H2O (Fluka)

Dialysis membranes MWCO of 4-6 and 8-10kDa (Roth)

Nanosep centrifugal devices 10K (Pall)

3.2 Buffers and solutions

Milk resembling Phosphate Buffer Saline (PBS), pH 6 .8

1000 ml of doubly distilled water

2.26 mM Na2HPO4*2H2O (0.466g)

7.38 mM NaH2PO4-2H2O (1.152g)


8 mM NaCL (0.467g)

Solutions for enzymatic hydrolysis (Hasenkopf et al )

• Pepsin solution: 2 mg pepsin in 1 ml HCl 0.02M

• Pronase solution: 2 mg pronase in 1 ml of ammonium acetate 2M, pH 8.2

buffer.

• Ammonia 5%

• Ammonium acetate buffer 2M, pH 8.2


15.616 g of ammonium acetate

The pH was adjusted to 8.2 with ammonia 25%

Solutions for enzymatic hydrolysis (Delatour et al)

• Pepsin solution: 1 mg pepsin in 1 ml HCl 0.02M

• Pronase solution: 1 mg pronase in 1 ml of tris buffer 2M, pH 8.2

• Sodium borate 0.2 M, pH 9.5

10 ml doubly distilled water

0.7627 g of NA2[B4O5(OH)].8H2O

• Sodium hydroxide 0.1M


0.0799 g of NaOH

• Sodium borohydride 1M

10 ml of 0.1 M NaOH

0.3783 g of NaBH4

• Ammonium carbonate 1M, pH 10.5


0.9609 g of (NH4)2CO3

• 25% Ammonia to adjust the pH to 10.5

• Tris buffer 2M, pH 8.2


2.4228 g of (HOCH2)3CNH2

35% HCl to adjust pH to 8.2


Solutions for the ninhydrin assay

• Sodium acetate buffer 4N, pH 5.5

10.8 mg of sodium acetate (water free),

2 ml of glacial acetic acid (acetic acid 99%)

Water to complete the volume to 10 ml

pH was adjusted to 5.5 using NaOH 2M (about 10 ml NaOH)

• Stannous chloride dihydrate (MW 225 g)

1 ml ethylene glycol

118 mg of SnCL2

• Ninhydrin reagent

200 mg ninhydrin

7.5 ml of ethelyne glycol

2.5 ml of sodium acetate buffer 4N

250 µl of SnCL2 suspension

Solutions to isolate lysyl oxidase

• Potassium phosphate buffer 16 mM/NaCL 120 mM


12 mM K2HPO4 (2.090g)

3.8 mM KH2PO4 (0.517g)

7.013 g of NaCl

pH was adjusted to 7.7 by NaOH 2N (80 g NaOH dissolved in 1000 ml of doubly

distilled water)

• Potassium phosphate buffer 16 mM/NaCl 120 mM/urea 4N

250 ml of potassium phosphate buffer 16 mM/NaCL 120 mM

60.06 g of Urea


Standard`s concentration to determine the fragments of

LacLys, CML, MeSO and 5-OH-Trp

4.2 µM of the synthesized LacLys, 1µM of CML, 1.2 µM of MeSO and 0.9 µM of 5-

OH-Trp were dissolved in a mixture of formic acid (0.1%) in a water/acetonitrile

mixture (50/50%).

Aqueous mobile phases for liquid chromatography

• 5 mM CH3COONH4, pH 6.8


0.385 g of CH3COONH4

• 10 mM CH3COONH4, pH 6.8


0.770 g of CH3COONH4

• 5 mM NFPA


0.766 µl of NFPA 97%

Solutions for affinity chromatography

• Washing buffer: CH3COONH4 250 mM/MgCl2 50 mM, pH 8


1.927g of CH3COONH4

1.0165g of MgCL2* H2O

• Elution buffer: D-sorbitol 200 mM

100 ml of washing buffer

3.644g D-sorbitol

• Elution buffer: acetic acid 250 mM

98.569 ml doubly distilled water

1.431 ml of concentrated acetic acid 99%, d=1.049

Ammoniac 25% to adjust the pH to 2.83


3.3 Methods

3.3.1 Lactulosyllysine synthesis

3.3.1.1. Trial to synthesize lactulosyllysine

A solution of Nα-FMOC-Lys-HCl (0.493 mmol, 200 mg) and triethylamine (0.5 mmol,

69 µl) in 8 ml of methanol was stirred at room temperature for 20 min, and then 80

mg of lactose anhydrous was added.

The reaction mixture was refluxed at 64 °C for 3 ho urs under nitrogen atmosphere.

After the removal of solvents by rotary evaporator, the residue was reconstituted in

water and purified on a column (200*20mm) filled with RP-18 silica (LiChroprep 25-

40µm). Five mixtures of water and methanol were run on the column and then

collected. These fractions were: fraction A (50 ml, 100% water), fraction B (70 ml,

80:20%, water/MeOH), fraction C (50 ml, 50:50%, water/MeOH), fraction D (50 ml,

20:80%, water/MeOH) and fraction E (100 ml, 100% MeOH).

In order to determine the fractions which contain the target compound, an aliquot of

each fraction (20 µl) was loaded on a RP-thin layer chromatography. Mobile phases

were a mixture of water and methanol (20:80%). After 3-4 hours the plate was

sprayed by ninhydrin solution (5% in ethanol) to detect the target compound FMOC-

LacLys. Pink spots appeared in the fractions C and D indicating the presence of

FMOC-LacLys. After the removal of methanol by rotation evaporator, the rest of the

fractions C and D were lyophilized.

3.3.1.2 Chromatographic conditions for the separati on of FMOC-

LacLys

The lyophilized residues (from the fractions C and D) were dissolved in acetonitrile

(1mg/ml) and then were loaded on a Nucleusil C18 column, 250*4, 5 µm using

mobile phases of formic acid 0.5% in water (solvent A) and acetonitrile (solvent B).

Acetonitrile was 10 % at the beginning of run, it increased then in 10 min to 50%,

and then to 100% during the next 5 min. Acetonitrile was kept at 100% for 5 min. The

flow rate was 0.8 ml/min.


3.3.1.3 Removal of the blocking group of FMOC-LacLy s

242 mg of the lyophilized residue (from the fractions C and D) were dissolved in a

mixture of DMF/MeOH (9:1, 1.6 ml) followed by the addition of 0.52 ml of

morpholine. The mixture was stirred for 2 hours at 25 °C. After the removal of

volatiles, the residue was suspended in 100 ml diethyl ether and filtered. The

synthesized product was obtained as a yellowish powder on the filter paper.

3.3.1.4 Trial to purifiy LacLys using m-Aminoboroni c acid

m-Aminophenylboronic acid attached to insoluble agarose matrix was suspended in

washing buffer (ammonium acetate 250 mM, pH 8 containing 50 mM MgCL2) and

then packed into polypropylene column (1ml, 85* 7mm). In order to keep the reactive

boronate form [RB(OH)3], the column was equilibrated with washing buffer. 200 µl of

the synthesized LacLys (1mg dissolved in ml of washing buffer) were loaded on the

column and kept at 4 °C to retain LacLys. After 1 h our, 10 ml of the washing buffer

was added to remove the unbounded substances and to maintain the medium an

alkaline pH. Finally, LacLys eluted specifically using the elution buffer D-sorbitol (200

Mm), which competes with LacLys to bind to boronate. Alternatively, LacLys eluted

non specifically by acidification of the boronate complex by 250 mM acetic acid pH

2.8. The eluates were collected in 5 ml fractions, and then lyophilized and

reconstituted in water (15 µg of the lyophilized residue per ml water). As control, a

solution of the synthesized LacLys, which was not subjected to affinity

chromatogaphy, was prepared (15µg/ml). The lyophilized residues and the control

were loaded on a C18 column, 150*2.1mm, particle size 5 µm, followed by MS-MS

analysis under the chromatographic and spectrometric conditions used to separate

LacLys on the C18 column.

3.3.1.5 Synthesis of FMOC-LacLys

A solution of Nα-FMOC-Lys-HCl (0.493 mmol, 200 mg) and triethylamine (0.5mmol,

69 µl) in 20 ml MeOH was stirred at room temperature for 20 min, and then a

solution of lactose anhydrous (0.584 mmol, 200mg) dissolved in 1 ml water was

added. The reaction mixture was stirred at 64 °C fo r 3-5 hours under nitrogen

atmosphere then filtered. After the removal of solvents by rotary evaporator, the

residue was reconstituted in water and then lyophilized. The lyophilized rest was


taken up in MeOH, and then filtered to remove the non-reacted lactose. MeOH was

then removed by rotary evaporator; the residue was taken up in water and

lyophilized.

3.3.1.5.1 Fractionation of FMOC-LacLys on a ZIC-HILIC column

30 mg of the lyophilized residue, which resulted from refluxing lactose and FMOC-

Lys, was dissolved in 1 ml of a water/acetonitrile mixture (50/50%). The solution was

loaded on a ZIC-HILIC column (150*4.6 mm, 5µm) to separate FMOC-LacLys from

other reaction products.

The separation was achieved using acetonitrile (solvent B) and ammonium acetate

buffer (10 mM). The flow rate was 800 µl per min and the injection volume was 20 µl.

At the beginning of run Solvent B was 80 % then decreased in 4 min to 50% and

then in the next 4 min to 20%. The fractions between 4.8 and 5.8 min were collected.

Acetonitrile was removed by rotary evaporator followed by freeze-drying to remove

water. In order to be sure that the other the compounds were removed, 1 mg of the

freeze-dried residue was reconstituted in 1 ml of a water/acetonitrile mixture

(20/80%). 20 µl was then loaded on the ZIC-column under the chromatographic

conditions which were mentioned. Only the peak of FMOC-LacLys appeared. Finally,

the blockage group (FMOC) was removed by the use of morpholine (See 5.3.1.3).

3.3.1.5.2 Determination of the purity of LacLys by 1H-NMR

2.4 mg of synthesized LacLys (5.1µmol) were dissolved in 1.3 ml D2O. 0.393 µl of

DMF was added (5.1µmol). DMF (99.8%) was used as standard to estimate the

purity of LacLys. The mixture was measured quantitatively by 1H-NMR, whereby the

relaxation time (D1) was 20 sec. Acetone has been added as a standard for axis

calibration and has appeared as a single signal (S, 2.22 ppm).

3.3.2 Synthesis of Cbz-CML

280 mg of Nα-carbobezyloxy-L-lysine (Nα-Cbz-L-lysine) and 186 mg of iodoacetic

acid were dissolved in 10 ml of 2M phosphate buffer. The pH of the reaction mixture

was adjusted to 10 by 2N NaOH, and then the solution was stirred at room

temperature for 2 days. 2 millilitres of ammonia (25%) were added and the solution

was stirred overnight at room temperature. The product Nα-Cbz-CML was isolated


by preparative HPLC on a Necleusil column, C18-100, 7µm, 250*21 mm using the

mobile phases methanol and 5 mM ammonium format buffer, pH 7. The flow rate

and the injection volume were 8 ml/min and 1 ml, respectively. The total run time

was 40 min. Methanol increased in 22 min from 0% to 65%, and then in 4 min to

100%. Methanol was kept at 100% till the end of run. The fractions between 17 and

21 min were collected followed by the removal of methanol by rotation evaporator. At

last, the fractions were lyophilized.

Removal of the blocking group of Cbz-CML

200 mg of the lyophilized residue, which was obtained after fractionation of the

reaction mixture of Cbz-Lysine and iodoacetic acid, was dissolved in 10 ml dry

ethanol. Palladium black was added as catalyser and the solution was hydrogenated

for one day. The solution was then filtered on diatomite (celite 535) and washed first

with water and then with spirituous ethanol. The obtaind aqueous solution was

lyophilized. CML was obtained as a white powder.

3.3.3 Synthesis of methionine sulfoxide (MeSO)

200 µl of 0.25 M methionine was mixed with 100 µl of 0.5M HCl and 5 µl of 30%

H2O2. The reaction mixture was incubated for 1 hour at 21°C then lyophilized. The

lyophiliyed residue was dissolved in a water/acetonitrile (20/80%) mixture then

MeSO was isolated by fractionation from the reaction mixture. The separation of

MeSO was performed on a ZIC-HILIC column (150*4.6 mm, 5µm) using mobile

phases of acetonitrile (solvent B) and 10 mM ammonium acetate buffer. The

gradients were (time in min/B %): (0/70) (4/50) (12/50). The fractions between 6.5

and 7.5 min were collected and lyophilized.

3.3.4 Trial to synthesize lysine aldehyde

LOX was isolated as reported in literature with some modifications (Kagan, Sullivan

et al. 1979). Shell membranes of 10 fresh chicken eggs were washed with doubly

distilled water, and then were ground and filtered to remove water. 20 ml of

potassium phosphate buffer (16 mM pH 7.7) containing 0.12M NaCl (buffer A) was

added to 8 g of the wet ground membranes. The solution was stirred at +4 °C for 3

hours followed by centrifugation at +4 °C for 30 mi n and RPM of 5000. The


centrifugation process was repeated. The two salt extracts were collected, filtered,

and then dialysed against buffer A for 24 hours at +4 °C (molecular weight cutoff of

4-6 kDa).

The pellet was further extracted by potassium phosphate buffer/NaCL containing

urea 4M (buffer B). 50 ml of buffer B was added to the pellet, and then the solution

was stirred at +4 °C for 16 hours followed by centr ifugation at +4 °C for 30 min and

RPM of 5000. The urea extract was collected and the pellet was stirred again using

the same buffer for 8 hours followed by centrifugation. The two urea extracts were

filtered and dialysed against buffer A for 24 hours at +4 °C. At last, both salt extract

and urea extract were lyophilized.

After obtaining the lysyl oxidase extracts, 100 mg of the lyophilized extract, which

contained urea, were dissolved in 35 ml of 0.1M phosphate buffer, pH 7.4. Another

solution was prepared containing 100 mg of the lyophilized salt extract (without

urea). 5 g of urea and 350 mg of Cbz-lysine were then added to each solution. And

then, both solutions were incubated in a shaking water bath at 37 °C. The aliquots

were taken after 2, 3, 7, 9 and 14 days followed by lyophilization. The lyophilized

residues were reconstituted in water. The samples were then loaded on a C18

column 250*4 mm, 5 µm. Acetonitrile and formic acid 0.5% in water were used as

mobile phases. The flow rate was 800µl/min and the injection volume was 20 µl. At

run beginning, acetonitrile was 10% then increased in 10 min to 50%, and then

increased in the next 10 min to 100%. Acetonitrile was then kept at 100% for 3 min.

Mass spectrometry and UV (280nm) were used for detection. The unknown product

was detected mainly in the presence of LOX urea extract, while its formation in the

presence of LOX salt extract was negligible.

The isolation of the predicted Cbz-Lys was achieved by fractionation using the same

chromatographic conditions used for detection. The only difference was the

replacement of acetonitrile with methanol. This replacement has led to higher

pressure; therefore the flow rate was reduced to 700µl/min. These changes

increased the retention time of the target compound to 15.9 min. The fractions

between 15 and 18 min were collected to isolate the target compound, evaporated

by rotation evaporator to remove methanol, and then lyophilized. Finally the blocking

group (Cbz) was removed by hydrogenation, as described previously to synthesize

CML (see 5.3.2).


3.3.5 Trial to synthesize cysteine sulfenic acid (C ys-SOH)

Briefely, 60 ml of 1mM cysteine, 0.5 mM of FeSO4 heptahydrate and 0.2 mM H2O2

were incubated at 37 °C.

Aliquots of 19 ml were taken after 10, 20 and 30 min. 1 ml of methionine 20 mM was

added to each aliqouts to stop the oxidation of cysteine. The aliquots were

lyophilized, reconstituted in water and then loaded on a C18 column, 250*4 mm,

particle size 5 µm. The mobile phases were acetonitrile and formic acid 0.5% in

water. At run beginning, acetonitrile was 10%, and then it increased in 10 min to

50%. UV detection and mass spectrometry were used for the detection. Later, many

attemps were carried out to synthesize Cys-SOH, in which FeSO4 was not used.

Additionally, different temperature (25, 37 and 120 °C) and/or different incubation

times (5, 10, 20, 30, 60, 100 and 120 min) were applied. The aliquots were

lyophilized then treated as menthioned previously.

3.3.6 The conditions of liquid chromatography for t he

seperation of CML, MeSO, LacLys and 5-OH-Trp on a C 18

column

The compounds CML, MeSO, LacLys and 5-OH-Trp were separated on a C18

column from Supelco, 150*2.1mm, particle size 5 µm, protected with a pre-column

20*2.1mm. The column was housed in the oven at 20 °C. The flow rate and injection

volume were set at 200µl/min and 5 µl, respectively. Mobile phases were acetonitrle

(solvent B) and 5 mM NFPA in water (solvent A). The gradient was optimized as

possible to achieve the better separation between the four analytes. At run

beginning, acetonitrile was 10% then it increased in 3 min to 40%, and then

increased in the next 3 min to 50% where it continued for 4 min at 50%. Acetonitrile

increased then in two min to 100% and continued for 2 min at 100% (until 14 min).

Acetonitrile decreased then in 6 min to the initial conditions (10%) followed by

equilibration for 5 min before taking the next sample. All samples were dissolved in a

mixture of water/acetonitrile (50/50%). The same MS-MS conditions were used for

these analytes on both C18 and ZIC-HILIC columns.


3.3.7 The conditions of liquid chromatography for t he

seperation of CML, MeSO, LacLys and 5-OH-Trp on a Z IC-

HILIC column

The separation was performed on a ZIC-HILIC column from Sequant, 150*2.1 mm

and 3.5 µm particle sizes. The column was protected with a pre-column (Sequant)

20*2.1mm, particle size 5 µm and housed in oven at 20 °C. The mobile phases were

acetonitrile (solvent B) and 5mM ammonium acetate buffer, pH 6.8. The flow rate

was constant during run time at 200µl/min. The injection volume was 5 µl. The initial

composition of acetonitrile was 70%, and then it decreased gradually in 18 min to

25% and then decreased in the next 2 min to 20%. Acetonitrile was kept for 10 min

at 20 %. Acetonitrile increased then to the initial percentage between 30 and 45 min

(70 %) followed by the equilibration for 7 min before taking the next sample. All

samples were dissolved in a mixture of water/acetonitrile (50/50%).

3.3.8 Stimulation of glycation in a milk model mixt ure

13 mg of α-LA and 493 mg of lactose, equivalent to 92 and 144.4 mM, respectively,

were dissolved in 10 ml of milk-resembling phosphate buffer saline (10mM of PBS

with 8mM NaCL). One aliquot was taken before heating and was used as the first

control. The protein solution was heated in a shaking water bath at 60 °C. Aliquots of

2.5 ml were taken after 3, 7 and 14 days followed by cooling on ice to stop the

reaction, dialysis against water (MWCO 8-10 kDa) and hydrolysis with enzymes

according to Hasenkopf et al. At last, the samples were cleaned up by ultrafiltration

(Nanosep centrifugal devices)

As a second control, a solution of α-LA without lactose in the same buffer was

heated for 3, 7 and 14 days and treated like that one with lactose. The third control

was native α-LA.

3.3.9 Stimulation of oxidation in a milk model mixt ure

A solution containing 13 mg of α-LA and 8.5 µl of 30% H2O2 (d=1.1) in 10 ml of milk-

resembling PBS was heated at 120 °C (equivalent to 92 µM and 8.4 mM of α-LA and

H2O2 , respectively). Aliquots of 2.5 ml were taken after 10, 20 and 30 min. As a


control, a solution of α-LA without H2O2 in the same buffer was heated for 10, 20 and

30 min at 120 °C. The second control was native α-LA (standard). The aliquots were

cooled immediately on ice and treated like the samples of glycated α-LA (see 5.3.8).

3.3.10 Preparation of milk samples for LC-MS-MS ana lysis

3.3.10.1 Milk defatting

5 ml of liquid milk or reconstituted powderd milk were defatted by centrifugation at 4

°C and 3850 RPM. Centrifugation lasted for 1-3 hour s depending on the fat amount.

Centrifugation lasted 1 hour in low fat UHT milk and 3 hours in condensed milk. Fat

layer was then removed with laboratory spatula.

3.3.10.2 Removal of lactose and minerals from milk and milk model

mixture

Lactose and minerals were removed from milk samples (5ml) as well as from milk

models (2.5ml) by dialysis against doubly-distilled water for 24 hours at +4 °C

(molecular weight cut-off of 8-10 kDa). Water was changed after 12 hours. The

dialysis was followed by freeze-drying.

3.3.10.3 Enzymatic protein hydrolysis (Hasenkopf et al)

1mg of the freeze-dried residue, which was obtained after dialysis, was dissolved in

1 ml HCl 0.02M. An aliquot of 50 µl of pepsin solution (2mg/ml in HCl 0.02M) was

added to the protein solution followed by incubation at 37 °C in a thermo mixer. After

24 hours of incubation, the protein solution was buffered with 250 µl of 2M

ammonium acetate buffer pH 8.2. 50 µl of pronase solution was then added (2mg/ml

in 2M ammonium acetate buffer, pH 8.2). The pH was adjusted to 7.5 by ammonia

solution (5%). The sample was then incubated for 24 hours at 37 °C. The protein

solution was then supplemented with 4.1 µl of aminopeptidase and further incubated

for 24 hours at 37 °C.

3.3.10.4 Purification of protein hydrolyzate by ult rafiltration

After enzymatic hydrolysis, the enzymes were separated from the protein

hydrolyzates by ultrafiltration. 411 µl of the protein hydrolyzate (equivalent to 300 µg


of the residue which was enzymatically hydrolyzed) were put in a centrifugal

concentrator device (Nanosep device with molecular weight cutoff of 10 kDa), and

were then centrifuged for 10 min at 4 °C and RPM of 14400. The centrifugation was

followed by lyophilization. The dry residue was reconstituted in 1ml of a water/

acetonitrile mixture (50/50%), and then diluted with the same mixture to the

concentration to be analyzed.

3.3.10.5 Enzymatic protein hydrolysis (Delatour et al)

0.5 mg of the lyophilized residue, which was obtained after milk dialysis, was

dissolved in 90 µl of water, and then 10 µl of 1M ammonium carbonate pH 10.5 and

100 µl of 1M sodium borohydride were added. The protein solution was incubated for

1 hour at 37 °C, lyophilized and then reconstituted in 1 ml of HCl 0.02 M. An aliquot

of 18 µl of pepsin solution (1mg/ml in HCl 0.02M) was added. The protein solution

was incubated for 1 hour at 37 °C in a thermo mixer . This step was repeated two

times. The protein solution was then buffered with 250 µl of 2M tris buffer; pH 8.2

followed by the addition of 15 µl of pronase (1mg/ml in tris buffer 2M). The protein

solution was then incubated for two times at 37 °C (each incubation period lasted for

30 min). After 1 hour, the sample was further supplemented with 4.1 µl of

aminopeptidase and the incubation continued for 24 hours at 37 °C. Finally, 15 µl of

pronase was added to the sample followed by incubation at 37 °C for one hour. This

step was repeated two times. Samples were cleaned up by solid phase extraction.

Some samples were hydrolyzed according to this method without the addition of

sodium borohydride. Thus, 0.5 mg of protein residue was directly dissolved in 1 ml of

HCl followed by the addition of 18 µl of pepsin.

3.3.10.6 Acidic protein hydrolysis (Delatour et al)

0.5 mg of the lyophilized residue, which was obtained after milk dialysis, was

dissolved in 50 µl water, and then was diluted with 1.5 ml of 0.2 M sodium borate pH

9.5. 1ml of 1M sodium borohydride was added and the solution was incubated for 4

hours at 21 °C. 2.75 ml of HCl 35% was then added a nd the protein sample was

hydrolysed at 110 °C for 24 hours.


3.3.10.7 Purification of protein hydrolyzate by sol id phase

extraction

The acidic hydrolyzates were dried by Speed-Vac followed by reconstitution in 500 µl

of water. This means 0.5 mg of the lyophilized residue, which was obtained after

dialysis and was then acidic hydrolysed, was reconstituted in 500 µl of water.

The cartridges (Sartra C18-E) were inserted into the chamber. Tubes were set under

the cartridge to collect the eluates. A vacuum port with a gauge was used to control

the vacuum applied on the chamber. The column (Sartra C18-E) was first

equilibrated with 100 µl of NFPA in 1 ml water. An aliquot of the enzymatic or acidic

hydrolyzate (equivalent to 200 µg of the residue which was previously hydrolyzed)

was diluted with water till 1000 µl. 100 µl of 100 mM NFPA was added, and then the

sample was loaded on the SPE column. 2 ml of 10mM NFPA in water were run on

the column for two consecutive times (this was fraction A about 5 ml). Then, 2 ml of

methanol/10mM NFPA in water (5/95%) were run on the column for two consecutive

times (this was the fraction B about 4 ml). At last, 2 ml of methanol/10mM NFPA in

water (50/50%) were run on the column for two times (fraction C). The three fractions

were collected and dried. The analytes were detected mainly in the first fraction.

Lyophilization was mainly used for drying; however, during the investigation of the

role of drying method, some samples were dried by Speed-Vac at 35 °C.

Figure 66: The chamber for solid phase extraction with the cartridges


3.3.10.7.1 The standards MeSO, LacLys and CML on a SPE column

A solution of the standards MeSO, LacLys and CML was prepared (25 µg of each

standard per 1ml water) and divided into 2 portions. The first one was loaded on a

Sartra C18-E column and treated like the samples of enzymatic and acidic

hydrolyzate (see 5.3.10.7). However, to avoid any loss of the standards in the

fractions B and C, the three fractions were collected. The collected fractions as well

as the second portion of standards were lyophilized, reconstituted in water and then

analyzed by LC-MS-MS on a C18 column, 150*2.1, 5 µm.

3.3.11 Trial to detect ornithine in milk protein

Milk samples were prepared as mentioned previously for the detection of LacLys,

CML, MeSO and 5-OH-Trp. The preparation involved milk defatting, dialysis,

enzymatic hydrolysis (for 72 hours) and cleanup by ultrafiltration. In order to detect

ornithine in heated milk, some samples of UHT milk were heated at 120 °C after fat

removal. The aliquots were taken after 10, 20, 30 and 60 min, cooled on ice.

Enzymatic hydrolysis as well as the other preparation steps was then carried out

(see 5.3.10.2-5.3.10.4). The concentration of the analyzed sample was 25 µg per 1

ml of a water/acetonitrile mixture (25 µg of the residue which was enzymatically

hydrolysed).

To investigate the artificial formation of ornithine from arginine during enzymatic

hydrolysis, 25 µg of arginine were subjected to the conditions of enzymatic

hydrolysis. As a standard, two solutions of arginine and ornithine (0.25µg/ml) were

prepared. The samples of milk as well as the standards were analyzed by LC-MS-

MS on a C 18 column 150*2.1, 5 µm.

3.3.12 Investigation the effect of sodium borohydri de on

MeSO

1.5 ml of sodium borate 0.2 M pH 9.5, 1 ml of 1M sodium borohydride and 2.75 ml of

HCl 35% were added to 1 mg of MeSO. The reaction mixture was heated at 110 °C

for 24 hours. An aliquot, which was corresponding to 200 µg MeSO, was cleaned up

by SPE (Sartra C18-E). The first fraction was then lyophilized and analyzed by LC-

MS-MS on the C18 column (150*2.1, 5µm). Furthermore, in order to investigate the


conversion of MeSO into methionine, the first fraction was analyzed by HPLC/UV on

the ZIC-HILIC column (150*4.6, 5µm) under the chromatographic conditions which

were used previously for the fractionation of MeSO (see 5.3.3).

3.3.13 Ninhydrin assay

An aliquot of the hydrolyzate was diluted with water till 50 µl. The aliquot was

equivalent either to 3 µg of the residue which was enzymatically hydrolyzed

according to Hasenkopf et al, or was equivalent to 10 µg of the residue which was

acidic or enzymatically hydrolyzed according to Delatour et al. And then, 100 µl of

ninhydrin reagent was added. The pH of the samples was in range 5-5.5. The

samples were heated in a boiling water bath for 3-5 min, and then were left in a

water bath at room temperature for 5 min. 250 µl of n-buthanol was added to each

sample then the samples were left at room temperature for 15 min. Finally, 250µl of

each sample was taken and the absorbance was measured at 570 nm.

3.3.14 Method validation

The amounts of LacLys during method validation were calculated taking into account

the purity of the synthesized standard (6.13%)

3.3.14.1 Within-day repeatability (intra-day variat ion)

A solution of the standards was prepared which contained 2.94, 1.28 and 0.096 µg of

the synthesized LacLys, MeSO and CML, respectively, dissolved in 10 ml of a

water/acetonitrile mixture (50/50%). This standard solution was used as a stock

solution for standard addition during the investigation of within-day repeatability

where it was diluted many times (1 to 2, 1 to 4 and 1 to 8).

8 mg of UHT milk proteins or heated UHT milk proteins (heated for 60 min at 120 °C)

were dissolved in 8 ml of 0.02M HCl. These protein residues were obtained after fat

removal, dialysis and lyophilization. The protein solution was divided into 8 aliquots

(1mg per 1 ml) followed by enzymatic hydrolysis according to Hasenkopf et al.

Enzymatic hydrolysis was applied for eight samples at the same time using the same

enzymes and buffers solutions. After 3 days of hydrolysis, 411 µl of the enzymatic

hydrolyzate (corresponding to 300 µg of residue which was enzymatically

hydrolyzed) were cleaned up, lyophilized then reconstituted in 1 ml of a


water/acetonitrile mixture (50/50%). The protein samples were then diluted with

water/acetonitrile mixture to 100µg/ml. An aliquot of 300 µl was then diluted either

with 300 µl of water/acetonitrile mixture or with 300 µl of one of the standards

solutions. This means the concentration of the unknown sample became 50µg/ml.

The analysis by LC-ESI-MS-MS lasted 2 days to investigate the within-day

repeatability of the method for LacLys and MeSO on the C18 column (150*2.1, 5

µm). 4 known amounts of LacLys and MeSO were added to the unknown sample.

The analysis lasted 2 days to investigate the within-day repeatability of the method

for CML on the ZIC-HILIC column (150*2.1, 3.5 µm). However, 3 known amounts of

CML were added to the unknown sample. The addition 0.0048 µg/ml was abdicated

by the analysis of UHT milk, while the addition 0.0006µg/ml was abdicated by the

analysis of heated UHT milk.

sample concentration

added LacLys added MeSO added CML

50 µg/ml 0 0 0

50 µg/ml 0.0183 µg/ml 0.008 µg/ml 0.0006 µg/ml




Table 17 : Concentrations of LacLys, MeSO and CML which were used for standard addition to investigate the within-day repeatability for UHT milk and heated UHT milk. The concentration of the sample, in which the analytes should be quantified, was 50 µg/ml

3.3.14.2 Between-day repeatability (inter-day varia tion)

The concentrations of LacLys and MeSO in the standard solutions were the same

which were mentioned previously to investigate the within-day repeatability.

Addaitionally, the samples were prepared as mentioned previously (5.3.14.1).

However, higher concentrations of CML were used. Additionally, enzymatic

hydrolysis was carried out in consecutive days where the eight samples were

completely hydrolyzed after 11 days. The concentrations of the samples increased to

75 µg/ml. 6 samples were then analyzed in a consecutive days to investigate the

between-day repeatability for LacLys and MeSO on the C18 column (150*2.1, 5µm).

4 samples were analyzed to investigate the between-day repeatability for CML

because the used ZIC-HILIC column (150*2.1, 3.5µm) was damaged. A new ZIC-


HILIC column (150*2.1, 3.5µm) was purchased; however, the samples for the

between-day repeatability were not further investigated.

sample concentration added LacLys added MeSO added CML

75 µg/ml 0 0 0





Table 18: Concentrations of LacLys, MeSO and CML which were used for standard addition to investigate the between-day repeatability in the samples of UHT milk and heated UHT milk. The concentration of the sample, in which the analytes should be quantified, was 75 µg/ml

3.3.14.3 Recovery

A solution of standards was prepared containing 21.45, 9.6 and 0.72 µg of the

synthesized LacLys, MeSO and CML, respectively. The standard compounds were

dissolved in 450 µl of water (this solution was used for addition 3). Two aliquots of

the standards solution, each one was 150 µl, were diluted with water to 300 µl or to

600 µl. These two standard solutions were used for addition 2 and addition 3,

respectively.

16 mg of UHT milk proteins, which were obtained after fat removal, dialysis and

lyophilization, were dissolved in 16 ml of HCl 0.02M. The protein solution was then

divided into 16 aliquots (1mg per 1 ml). An aliquot of each standards solution (30 µl)

was added to 4 aliquots of the protein solution followed by enzymatic hydrolysis

according to Hasenkopf et al. 4 aliquots of the protein solution were enzymatically

hydrolyzed without spiking any analyte. 425 µl of the enzymatic hydrolyzate

(corresponding to 300 µg of the residue which was enzymatically hydrolyzed) were

cleaned up, lyophilized, and reconstituted in 1 ml of a water/acetonitrile mixture

(50/50%). The samples were then diluted with the latter mixture either to 50µg/ml, to

determine the recovery of MeSO and LacLys, or were diluted to 300µg/ml to

determine the recovery of CML.

70 µl of the diluted samples (50 or 300µg/ml) were mixed either with 70 µl of

water/acetonitrile mixture or with 70 µl of one of the standards solutions. Thus, the


concentrations of the unknown samples as well as the concentration of the spiked

analytes decreased to 50% (table 20).

protein dissolved

in 1 ml HCl

0.02M

volume of added water (µl)

volume of added

standards solution

(µl)

LacLys (µg/30µl)

CML (µg/30µl)

MeSO (µg/30µl)

Protein (mg/µl)

without analyte addition

1 mg 30 0 0 0 0 1/1030

addition 3 1 mg 0 30 1.43 0.048 0.64 1/1030

addition 2 1 mg 0 30 0.715 0.024 0.32 1/1030

addition 1 1 mg 0 30 0.357 0.012 0.16 1/1030

Table 19 : The amounts of LacLys, CML and MeSO in 30 µl of the standards solutions which were spiked to each protein sample before enzymatic hydrolysis

sample concentration added LacLys added MeSO sample

concentration added CML

25 µg/ml 0 0 150 µg/ml 0

25 µg/ml 0.0459 µg/ml 0.025 µg/ml 150 µg/ml 0.003 µg/ml



25 µg/ml 0.3678 µg/ml 0.2 µg/ml

Table 20: The concentrations which were used for standard addition during the determination of the recovery of of LacLys, MeSO and CML. The concentration of the sample in which CML should be quantified was 150µg/ml and it was 25µg/ml to quantify LacLys and MeSO

3.3.14.4 LOD and LOQ

LOD and LOQ of LacLys and MeSO

In order to determine LOD and LOQ of LacLys, the content of LacLys was

determined in three samples of UHT milk by standard addition. Each sample

contained 25 µg per ml of a water/acetonitrile mixture (50/50). The analyte`s

concentrations for standard addition were mentioned previously for the within-day

repeatability (see 5.3.14.1). In order to determine LOQ und LOD, the three samples

were diluted many times (1:20, 1:25, 1:35, 1:40 and 1:50) with the water/acetonitrile

mixture to the extent at which the signal to noise ratio of the quantifier fragment (m/z


471.2→225.1) and qualifier fragment (m/z 471.2→128.1) were 10 and 3,

respectively. LOQ or LOD were then calculated by dividing the content of LacLys in

25µg of the hydrolyzed residue into the dilution factor. The calculated value

represents LOQ or LOD in 1 ml of the sample solution, and then it was divided into

200 to calculate the content in 5µl, which was the injection volume

In order to determine LOQ and LOD of MeSO, similar steps were carried out;

however, the determination of LOQ and LOD of MeSO was based only on the

quantifier fragment (m/z166.1→ 74). Additionally, the samples were diluted in the

range 1:35 to 1:150.

LOD and LOQ of CML

A solution of CML was prepared (5 µg/ml), and then was diluted to 0.312µg/ml. The

latter solution of CML was diluted many times as the following: 1:2.5, 1:5, 1:10, 1:20,

1:40, and 1:80. A water/acetonitrile mixture (50/50%) was used to dissolve and to

dilute the samples.

In order to determine LOD and LOQ of CML, an aliquot of raw milk hydrolyzate

(50µg/ml: 50 µg of the residue which was enzymatically hydrolyzed) was mixed

either with the same volume of water/acetonitrile mixture to give the sample 1, or it

was mixed with one of the diluted solutions of CML to give the samples 2, 3, 4, 5 and

6. Each sample was then analyzed 4 times by LC-ESI-MS-MS using the ZIC-HILIC

column (150*2.1, 3.5µm).

sample name

raw milk residue spiked CML

sample 1 25 µg/ml 0 µg/ml

sample 2 25 µg/ml 0.0624 µg/ml






Table 21: Amounts of CML which were spiked into 25 µg of raw milk protein hydrolyzate in order to determine LOD and LOQ


3.3.15 Qantitation

The concentrations of the samples to be analyzed varied between 25 and 900µg/ml,

as seen in table 22. The sample concentrations were 25µg/ml in most milk samples

to quantify MeSO or LacLys. In order to quantify LacLys in raw and pasteurized milk,

the concentrations increased to 200µg/ml. This means 25 or 200 µg of the residue,

which was defatted and dialysed prior to enzymatic hyrolysis, was dissolved in 1 ml

of a water/acetonitril mixture (50/50%). In the case of CML, the concentrations were

higher and reached 900µg/ml. However, one should keep in mind that the sample`s

concentration can be changed according the cleanness of the sprayer needle and

the mass analyzer, since the draggle will decrease the sensitivily and thus, will

require the use of higher concentrations to reach S/N of 10 (LOQ). By standard

addition, the concentrations of the added analytes were chosen depending on the

expected values of the analytes in milk products. For example, the concentrations of

added MeSO were 0.025, 0.05, 0.1 and 0.2µg/ml for all milk samples. The

concentrations of added LacLys were, taking into account the purity of LacLys

(6.13%), 0.046, 0.092, 0.184 and 0.368µg/ml. Only three amounts of CML were

added to milk samples, so that the concentrations were 0.05, 0.1 and 0.2µg/ml. In

the case of raw and pasteurized milk, lower amounts of CML were added, so that the

concentrations were 0.02, 0.04 and 0.08 µg CML per 1 ml.

sample name sample

concentration (CML)


(LacLys)


(MeSO)

sterilized milk 75 µg/ml 25 µg/ml 25 µg/ml

condensed milk 75 µg/ml 25 µg/ml 25 µg/ml

vanilla drink 75 µg/ml 25 µg/ml 25 µg/ml

lactose free milk 100 µg/ml 25 µg/ml 25 µg/ml

infant formulas 125 µg/ml 25 µg/ml 25 µg/ml

UHT milk 300 µg/ml 25 µg/ml 25 µg/ml

pasteurized milk 800 µg/ml 200 µg/ml 25 µg/ml

raw milk 900 µg/ml 200 µg/ml 25 µg/ml

Table 22: Concentrations of milk samples which were quantified. The concentration varied between 25µg/ml to quantifiy both MeSO and LacLys to 900µg/ml to quantify CML in raw milk proteins


3.3.15.1 Determination protein content by Kjeldahl

100 mg of the residue, which was obtained after defatting and dialysis, was put in a

kjeldahl flask, and then 20 ml of concentrated sulfuric acidic (98%) and a tablet of

catalyser were added.

The mixture was heated for 30 min at 80% energy level of the apparatus, and then

heated at 70% energy level till the disappearance of the black colour. The solution

was then heated at 60 % energy level for 1 hour followed by cooling for 30 min. A

blank, which contained sulfuric acid and catalyser without protein, was prepared and

treated like milk protein samples.

80ml of water was carefully added to the flask. The addition of NaOH 30% was

programmed for 5 seconds followed by distillation for 5 min. The distillate was

received in Erlenmeyer flask containing 90 ml of boric acid (2%) pH 4.3 and taschiro

indicator (a mixture of methyl red and methylene blue dissolved in ethanol). After the

end of distillation the colour of boric acid solution converted into light green. HCl

0.1N was then titrated on the previous solution untill the colour converted into pink.

HCl 0.1N, which was needed for the blank sample, was substracted from HCl which

was needed for the samples with proteins. The conversion factor was 6.25. Protein

content (%) was calculated as the following:

0.1N HCl(ml)*6.25*0.0014*100/ sample weight (g)

CHAPTER 4. SUMMARY 139

4 Summary

The Maillard reaction comprises a series of chemical reactions between amino

groups and carbonyl compounds leading to the formation of a variety of Maillard

reaction products (MRPs). Controlled browning is often used to develop desirable

flavor, odor or color in food like coffee, bakery products and roasted meat. However,

on the other hand, the Maillard reaction can lead to alteration of protein structure and

function as well as to the production of toxic compounds. The Maillard reaction takes

place also in the body leading to the formation of advanced glycation end products

(AGEs). The accumulation of AGEs in different tissues has been implicated in the

aging process and in the pathogenesis of complications associated with diabetes

mellitus such as nephropathy, retinopathy and neuropathy. Other diseases like

Alzheimer‘s disease, cataract and atherosclerosis were also related to the formation

of AGEs. Some of the consumed MRPs can survive the digestive process and can

be transported into the circulation resulting in elevation of AGE-serum concentration.

Due to the structural similarities between MRPs and AGEs, it is discussed that the

consumption of MRP-rich diet may participate in the development of many diseases

especially in patients with kidney diseases. Additionally, food-derived Maillard

products which cannot pass into the circulation may affect the intestinal immune

function and participate in the pathogenesis of inflammatory bowel diseases.

The presence of lactose and proteins as well as the neutral pH value render milk

as a suitable medium for the Maillard reaction during processing and storage. This

leads to the formation of various products like lactulosyllysine (LacLys) and

carboxymethyllysine (CML). In parallel to glycation, milk proteins are subjected to

various oxidation reactions where tryptophan, methionine and cysteine are

particularly sensitive to oxidative modifications. Glycation and oxidation lead to the

degradation of essential amino acids as well as to a decrease of the protein

digestibility. Consequently, these processes reduce the nutritional value of milk and


can alter the functional properties of milk whey proteins which are widely used in

food industry.

This work aimed to detect and quantify several glycation and oxidation products in

milk by liquid chromatography connected to electrospray ionization tandem mass

spectrometry (LC-ESI-MS-MS). LC-ESI-MS-MS represents a powerful analytical

technique for the qualitative determination since the analytes can be unequivocally

identified by their product ion spectrum. MS-MS is considerd also as a very selective

and sensitive detector for quantitation, even in complex matrices like food.

In the course of this work, experiments were carried out to synthesize reference

compounds of the major glycation and oxidation products which has been identified

before in milk products: LacLys, CML in addition to methionine sulfoxide (MeSO),

lysine aldehyde and cysteine sulfenic acid. The synthesis of LacLys could be

achieved by the reaction of FMOC-Lys and lactose under nitrogen atmosphere.

FMOC-LacLys was then isolated from the reaction mixture by fractionation on a ZIC-

HILIC column followed by the removal of the blocking group. The purity of the

standard was determined by quantitative 1H-NMR (6.13%). Thus, for the first time, a

standard with a defined content of LacLys could be synthesized. CML and MeSO

were synthesized using an alkylating agent (iodoacetic acid) or an oxidizing agent

(H2O2), respectively. For further analysis however, commercial standards of CML

and MeSO were used. Lysine aldehyde and cysteine sulfenic acid could not be

synthesized in sufficient purity, because their high reactivity led to degradation during

purification. Standards of 5-hydroxytryptophan (5-OH-Trp) and ornithine were

commercially available.

A method was then developed to detect the oxidation and glycation products, for

which standards were available, in α-lactalbumin (α-LA), which was heated in a milk

model as well as in processed and stored milk products. After removal of fat and

lactose, the target analytes were released from proteins by a complete enzymatic or

acidic hydrolysis prior to the analysis by LC-ESI-MS-MS. The tandem mass

spectrometry parameters which are dependent on the liquid chromatography flow

rate as well as the parameters which are dependent on the compounds were

optimized to achieve the highest intensity for LacLys, CML, MeSO, 5-OH-Trp and

ornithine.

LacLys and CML have little retention on C18 columns due to their high polarity. The

high polarity requires therefore the use of nonafluropentanoic acid as ion pair


reagent to increase the retention. Both, 5-OH-Trp and ornithine could neither be

detected in milk resembling model of lactose and α-LA nor in milk samples. Even

after the oxidation of α-LA by hydrogen peroxide at 120 °C, only MeSO was

detected. The analytes LacLys, CML and MeSO were detected clearly in milk

models which were heated at 60 °C for 14 days.

However, no separation could be achieved between CML and LacLys on the C18

column. Additionally, it was difficult to detect CML in milk samples, especially in

mildly heat-treated products like UHT milk, since CML is formed only in small

amounts. In order to improve the separation as well as to increase the sensitivity of

the method and thus, to avoid loading of high amounts of milk protein on the column,

a ZIC-HILIC column was introduced as alternative to the C18 column. The use of this

hydrophilic stationary phase allowed complete separation of CML and LacLys.

Additionally, a 3-5 fold increased signal intensity was achieved. However, the signal

intensity of LacLys in milk samples showed a higher variation on the ZIC-HILIC

column compared to the C18 column. Additionally, MeSO is detected easily in milk

samples and the variation of its signal intensity was similar on C18 and ZIC-HILIC

columns. Thus, the ZIC-HILIC column was chosen for further investigation of CML,

while the C18 column was used for further investigation of LacLys and MeSO.. Next, protein hydrolysis was studied and optimized to achieve maximal release of

MeSO, CML and LacLys from milk protein as well as to avoid at the same time

artificial formation. It was shown by LC-ESI-MS-MS analysis of the target analytes

and by ninhydrin assay that longer enzymatic hydrolysis increased the release of the

analytes (hydrolysis for 72 hours versus 27 hours). At the same time, the reduction

of LacLys to the unreactive alcohol by sodium borohydride did not lead to an

important decrease of the CML values. The result indicated that artificial formation of

CML resulting from the oxidative cleavage of LacLys during enzymatic hydrolysis

can be neglected.

MeSO was detected in the blank of the enzymatic hydrolysis due to autohydrolysis of

the enzymes, especially of pepsin. This blank value was taken later into

consideration for the quantitative measurements to avoid overestimation of MeSO.

The use of solid phase extraction (SPE) as a tool to remove the enzymes and purify

the hydrolyzate after enzymatic hydrolysis resulted in a loss of the analytes,

especially of LacLys. Therefore, ultrafiltration was used instead of SPE.


Prior to acidic hydrolysis, LacLys must be reduced by sodium borohydride to avoid

artificial formation of CML during acid treatment. As a consequence, reduction

prevents the detection and quantification of LacLys in parallel to CML and MeSO.

Additionally, sodium borohydride could not completely convert LacLys to the reduced

form. Consequently, it could not prevent the conversion of LacLys to CML resulting

in an overestimation of CML. Therefore, acidic hydrolysis was not adopted.

Furthermore, a decrease of MeSO was observed when NaBH4 was used prior to

acidic hydrolysis. MeSO might undergo further reaction in the presence of sodium

borohydride.

LacLys, CML and MeSO were quantified based on standard addition, since stable

isotope-labeled standards of LacLys and MeSO are not available to be used as

internal standards. The method was validated by evaluating the within-day and

between-day repeatability at low and high modification levels. In addition, the

recovery as well as detection limit and quantitation limit were determined for each

analyte. A high selectivity of the method was achieved by identifiying each analyte by

two characteristic mass transitions. The LC-ESI-MS-MS method showed good

precision. The coefficients of variation for within-day and between-day repetition of 4-

8 samples containing the analytes LacLys, CML and MeSO did not exceed 10%. In

order to investigate the recovery, three different amounts of each analyte were

spiked into UHT milk proteins prior to enzymatic hydrolysis. Some samples showed

a very good recovery. The recoveries of CML, for example, were optimal and ranged

between 96.6±9.4 % to 101±5.4 %. The recoveries of LacLys ranged between

110.5±9.6 % to 111.6±10.6 %, while the recoveriers of MeSO ranged between

107.8±10.6 % to 113.1±10.8 %. Since the recoveries did not exceed 115 %, they

were considered as acceptable.

The limit of detection (LOD) and the limit of quantitation (LOQ) for LacLys were

calculated by the dilution of UHT milk proteins to the extent at which the signal to

noise ratio of the qualifier and quantifier fragments were 3 and 10, respectively. LOD

and LOQ were 4 and 6.5 pg of LacLys on column and 1 and 2 pg of MeSO on

column. LOD and LOQ for MeSO were calculated based on the quantifier fragment

only (m/z 166.1 →74). LOD and LOQ for CML were determined by spiking different

amounts of CML to 25 µg of raw milk protein. LOD and LOQ of 4 and 8 ng CML per

mg protein were defined. These values were equivalent to 20 and 40 pg of CML on

column. Thus, a selective and validated method was developed to detect the major


glycation and oxidation products LacLys, CML and MeSO in dairy products. This

sensitive method allows the detection and the quantitation even of low levels of

modifications as present in mildly processed milk.

In the last part of this work, the method was employed for the quantitative

measurement of LacLys, CML and MeSO in different dairy products. The analysis

involved raw milk, mildly heat-treated milk like pasteurized milk and severely heat-

treated milk like sterilized milk. Additionally, different infant formulas were analyzed.

The concentrations of the analytes in cow milk were clearly dependent on the

applied heat treatment. The lowest levels of modifications were detected in raw milk

and the highest levels were found in sterilized as well as in condensed milk. One

exception was MeSO because its content in raw milk was higher than in mildly heat-

treated milk. High modification rates, especially of oxidation were found in infant

formulas with a tendency to higher values in hypoallergenic formulas compared to

regular formulas.

High modification rates of methionine and lysine block their physiological use as

essential amino acids and decrease the protein digestibility. Monitoring of LacLys,

CML and MeSO by the newly developed LC-ESI-MS-MS methods in dairy products

thus, allows evaluation of their nutritional value and assessment of the production

parameters.

CHAPTER 5. ZUSAMMENFASSUNG 144

5 Zusammenfassung

Die Maillard Reaktion umfasst eine Reihe chemischer Reaktionen zwischen

Aminogruppen und Carbonylverbindungen, die zur Bildung von vielfältigen Maillard-

Reaktionprodukten (MRPs) führen. Diese so genannte nicht-enzymatische Bräunung

ist häufig erwünscht und verleiht vielen Lebensmitteln, wie Kaffee, Backwaren und

gebratenem Fleisch ihren charakteristischen Geruch und Geschmack sowie eine

braune Farbe. Andererseits kann die Maillard-Reaktion zur Veränderung der

Proteinstruktur und der Proteinfunktion sowie zur Entstehung von toxischen

Verbindungen führen. Die Maillard-Reaktion läuft auch im menschlichen Körper ab

und führt zur Bildung von so gennanten „Advanced Glycation End-products (AGEs)“.

Die Akkumulation von AGEs in verschiedenen Geweben wird mit dem

Alterungsprozess und verschiedenen Komplikationen von Diabetes mellitus wie z.B.

Nephropathie, Retinopathie und Neuropathie in Zusammenhang gebracht.

Auch andere Krankheiten wie Alzheimer, Katarakt und Atherosklerose stehen in

Verbindung mit der Bildung von AGEs. Über die Nahrung aufgenommene MRPs

können während des Verdauungsprozesses resorbiert werden, was zu einer

Erhöhung der Konzentrationen von AGEs im Serum führt. Aufgrund der strukturellen

Ähnlichkeiten zwischen MRPs und AGEs wird diskutiert, dass besonders bei

Patienten mit Nierenkrankheiten der Konsum von MRP-reicher Nahrung an der

Entwicklung von verschiedenen Komplikationen beteiligt sein könnte. Zusätzlich

können die von Lebensmitteln stammenden Maillard-Produkte, die nicht resorbiert

werden, die intestinale Immunreaktionen beeinflussen und an der Pathogenese der

chronisch-entzündlichen Darmerkrankung beteiligt sein.

Durch die Anwesenheit von Laktose und Proteinen sowie den neutralen pH-Wert

stellt Milch bei der Verarbeitung und der Lagerung ein ideales Medium für die

Maillard-Reaktion dar, so dass es zur Bildung von verschiedenen Produkten wie

Laktulosyllysin (LacLys) und Carboxymethyllysin (CML) kommt. Parallel zur


Glykierung, unterliegen Milchproteine auch verschiedenen Oxidationsreaktionen,

wobei die Aminosäuren Tryptophan, Methionin und Cystein besonders betroffen

sind. Glykierung und Oxidation führen zum Abbau der essentiellen Aminosäuren und

zur Reduzierung der Proteinverdaubarkeit, wodurch diese Prozesse den Nährwert

der Milch mindern. Auch die funktionellen Eigenschaften der Molkenproteine, welche

in der Lebensmittelindustrie weit eingesetzt werden, können durch Glykierung und

Oxidation geändert werden.

Das Ziel dieser Arbeit war die Detektion und die Quantifizierung von verschiedenen

Glykierungs- und Oxidationsprodukten in Milch mittels

Hochleistungsflüssigchromatographie gekoppelt mit Elektrospray-Ionisation und

Tandem-Massenspektrometrie (LC-ESI-MS-MS). LC-ESI-MS-MS ist ein

leistungstarkes Analyzenverfahren für die Qualifizierung, da die Analyten über ihre

Produktionenspektren eindeutig identifiziert werden können. Weiterhin, stellt die

Tandem-Massenspektrometrie einen selektiven und empfindlichen Detektor zur

Quantifizierung dar, selbst wenn der Analyt in einer komplexen Matrix, wie

beispielweise einem Lebensmittel, zu untersuchen ist.

Zunächst wurde versucht, die Referenzesubstanzen von den wichtigsten

Glykierungs- und Oxidationprodukten zu synthesiziern, die in Milchprodukten vorher

identifiziert worden waren: LacLys, CML, Methioninsulfoxid (MeSO), Lysin-Aldehyd

und Cysteinsulfensäure (CysSOH). Die Synthese von LacLys erfolgte durch die

Reaktion von FMOC-Lys und Lactose unter Stickstoffatmosphäre. Anschließend

wurde FMOC-LacLys mit Hilfe einer ZIC-HILIC Säule aus der Reaktionsmischung

isoliert und die Schutzgruppe entfernt. Die Reinheit von LacLys wurde durch

qualitative 1H-NMR (6.13 %) bestimmt. Auf diese Weise konnte zum ersten Mal ein

Standard mit einem definierten Gehalt von LacLys synthetisiert werden. CML und

MeSO wurden mittels Alkylierungs- (Iodessigsäure) und Oxidationsreagenzien

(H2O2) synthetisiert. Allerdings wurden für die weiteren Untersuchungen

kommerzielle Standards von CML und MeSO verwendet. Lysin-Aldehyd und

CysSOH konnten nicht in ausreichender Reinheit synthetisiert werden, weil ihre

hohe Reaktivität zum Abbau während die Aufreinigung geführt hat. Die Standards

von 5-Hydroxytryptophan (5-OH-Trp) und Ornithin waren kommerziell erhältlich.

Anschließend wurde eine Methode entwickelt, um Glykierungs- und

Oxidationsprodukte, für die ein Standard zur Verfügung stand, zu detektieren. Der

Nachweis erfolgte in α-Lactalbumin, das in einem Milchmodell erhitzt worden war


und in kommerziell erhältlichen Milchprodukten. Nach der Entfettung und Entfernung

von Laktose wurden die Analyten aus den Milchproteinen durch eine komplette

enzymatische oder saure Hydrolyse freigesetzt und anschließend mittels LC-ESI-

MS-MS bestimmt. Die Parameter des Massenspektrometers, welche vom Fluss bei

der Flüssigchromatographie abhängen und diejenigen, welche von den

Verbindungen abhängig sind, wurden optimiert, um die maximale Intensitäten für

LacLys, CML, MeSO, 5-OH-Trp und Ornithin zu erreichen.

Aufgrund der hohen Polarität von LacLys and CML, weisen beide Verbindungen

kaum Retention an C18 Säulen auf. Deshalb wurde Nonafluropentansäure als

Ionenpaar-Reagenz verwendet, um die Retention der beiden Substanzen zu

erhöhen.

5-OH-Trp und Ornithin konnten weder im Milchmodell noch in Milchproben detektiert

werden. Auch nach der Oxidation von α-LA mittels Wasserstoffperoxid bei 120 °C

wurde nur MeSO detektiert. Die Analyten LacLys, CML and MeSO wurden in den

Milchmodellen detektiert, die bei 60 °C für 14 Tage n erhitzt wurden.

Allerdings konnten CML und LacLys auf einer C18 Säule nicht getrennt werden.

Auch die Detektion von CML in Milchproben, die mild erhitzt wurden, wie

beispielweise UHT-Milch, gestaltete sich als schwierig, da CML nur in geringen

Mengen gebildet wird. Um die Trennung zu verbessern und die Sensitivität zu

steigern, wurde statt einer C18 Säule eine ZIC-HILIC Säule benutzt. Durch eine

verbesserte Sensitivität kann weiterhin die Menge an Milchproteinen, die auf die

Säule geladen wird, reduziert werden. Die Anwendung dieser hydrophilen

stationären Phase hat eine vollständige Trennung zwischen CML und LacLys

ermöglicht. Weiterhin wurde die Signalintensität um das 3-5-fache gesteigert.

Allerdings zeigte die Signalintensität von LacLys in den Milchproben hohe Variation

an der ZIC-HILIC Säule im Vergleich zu der C18 Säule. Weiterhin wurde MeSO

lediglich in den Milchproben detektiert und die Variation ihre Signalintensität war an

der C18 und ZIC-HILIC Säule gleichartig. Aus diesem Grund wurde die ZIC-HILIC

Säule für weitere Untersuchungen von CML und die C18 Säule für weitere

Untersuchungen von LacLys and MeSO gewählt.

Als nächstes wurde die Proteinhydrolyse untersucht und optimiert, um die

maximale Freisetzung der Analyten MeSO, CML und LacLys von Milchproteinen zu

erreichen und, gleichzeitig, eine artifizielle Bildung zu vermeiden. Die Analyze der

Analyten mittels LC-ESI-MS-MS und Ninhydrin-Assay haben gezeigt, dass die


Zunahme der Hydrolysedauer die Freisetzung der Analyten erhöht (72 h gegenüber

27 h Hydrolyse). Gleichzeitig führte die Reduktion von LacLys mit Natriumborhydrid

zum unreaktiven Alkohol zu keiner bedeutenden Abnahme des CML-Werts. Diese

Ergebnisse zeigen, dass die artifizielle Bildung von CML durch oxidative Spaltung

des LacLys während der enzymatischen Hydrolyse vernachlässigt werden kann.

Bedingt durch die Autohydrolyse der Enzyme, insbesonders von Pepsin, wurde

MeSO in der Blindprobe der enzymatischen Hydrolyse detektiert. Deshalb wurde

dieser Blindwert bei der späteren Quantifizierung berücksichtigt. Die Verwendung

einer Festphasenextraktion (SPE) zur Entfernung der Enzyme und zur Aufreinigung

der Proben hatte einen Verlust der Analyten, besonders von LacLys zur Folge.

Deshalb wurde die Ultrafiltration einer SPE vorgezogen.

Vor der sauren Hydrolyse musste LacLys mittels Natriumborhydrid reduziert werden,

um die artifizielle Bildung des CML während der sauren Behandlung zu vermeiden.

Als Folge davon verhindert die Reduzierung den Nachweis und die Quantifizierung

von LacLys paralell zu CML und MeSO. Weiterhin konnte LacLys von

Natriumborhydrid nicht vollständig reduziert werden. Folglich konnte die Bildung von

CML aus LacLys nicht vollständig verhindert werden, was eine Überschätzung von

CML nach sich ziehen würde. Deshalb wurde auf die saure Hydrolyse verzichtet.

Weiterhin wurde eine deutliche Minderung des MeSO-Signals bemerkt, wenn

Natriumborhydrid vor der sauren Hydrolyse verwendet wurde. MeSO könnte in

Gegenwart von Natriumborhydrid weitere Reaktionen eingehen.

Die Analyten LacLys, CML und MeSO wurden durch das

Standardadditionverfahren quantifiziert, da für LacLys und MeSO keine

isotopenmarkierten Standards verfügbar sind, die als interne Standards benutzt

werden können. Für die Validierung wurde die Wiederholbarkeit von Messungen von

Proben mit hohen und niedrigen Modifikationsraten innerhalb eines Tages und

zwischen verschiedenen Tagen bestimmt. Außerdem wurden die Wiederfindung

sowie die Nachweis- und Bestimmungsgrenze für jeden Analyten bestimmt. Durch

die Identifizierung jedes Analyten über zwei charakteristische Massenübergänge

wurde eine hohe Selektivität der Methode erreicht. Die LC-ESI-MS-MS Methode

zeigte eine gute Präzision. Die Variationskoeffizienten von 4 bis 8 Proben, welche

die Analyten LacLys, CML and MeSO enthielten, lagen innerhalb eines Tages und

zwischen verschiedenen Tagen unter 10 %. Um die Wiederfindung zu untersuchen,

wurden drei unterschiedlich hohe Konzentrationen jedes Analyten unmittelbar vor


der enzymatischen Hydrolyse zu UHT-Milch zudotiert. Die Messungen erzielten

meist eine sehr gute Wiederfindung. Beispielsweise lagen die Wiederfindungen von

CML in einem optimalen Bereich zwischen 96,6±9,4 % und 101±5,4 %. Die

Wiederfindungen von LacLys lagen zwischen 110,5±9,6 % und 111,6±10,6 %,

während die Wiederfindungen von MeSO zwischen 107,8±10,6 % und 113,1±10,8

% lagen. Da die Wiederfindungsraten alle unter 115 % lagen, wurden sie als

akzeptabel angesehen.

Die Nachweisgrenze (LOD) und die Bestimmungsgrenze (LOQ) von LacLys wurden

ermittelt, indem die UHT-Milchproben bis zum Bereich, in welchem das Signal-

Rausch-Verhältnis bei ≤ 3 (qualifier) bzw. ≤ 10 (quantifier) lag, verdünnt wurden.

LOD und LOQ von LacLys lagen bei 4 bzw. 6,5 pg LacLys on column und von MeSO

bei 1 bzw. 2 pg on column, wobei diese Werte nur über den Quantifier von MeSO

(m/z 166,1→ 74) berechnet wurden. LOD and LOQ von CML wurden über ein

Zusatz bestimmter Mengen von CML zu 25 µg Rohmilchprotein bestimmt. Es

wurden LOD und LOQ von 4 und 8 ng pro mg Protein bestimmt, was 20 und 40 pg

CML on column entsprachen. Die auf diese Weise validierte Methode wurde

eingesetzt, um die wichtigsten Glykierungs- und Oxidationsprodukte LacLys, CML

und MeSO in Milchprodukten nachweisen zu können. Durch die hohe Selektivität der

Methode können auch geringe Mengen an Modifikationen in schonend verarbeiteter

Milch detekiert und quantifiziert werden.

Im letzten Teil der Arbeit wurde die Methode angewendet, um die Analyten

LacLys, CML und MeSO in Milchprodukten zu quantifizieren. Dazu wurden

verschiedene Proben wie z.B. Rohmilch, schonend erhitzte Milch wie pasteurisierte

Milch sowie stark erhitzte Milch wie Sterilmilch analysiert. Zusätzlich, wurden auch

verschiedene Proben von Babymilchnahrungen analysiert. Die

Analytenkonzentrationen in den Proben waren eindeutig von der angewandten

Prozessierung abhängig. Der niedrigeste Gehalt an Modifikationen wurde in

Rohmilch detektiert und die höchsten in Sterilmilch und Kondensmilch. Die einzige

Ausnahme stellt MeSO dar. Hier war der Gehalt in Rohmilch höher als der Gehalt in

schonend erhitzter Milch. Hohe Modifizierungsraten, vor allem durch Oxidation,

wurden in Babymilchnahrung gefunden. Dabei wurden tendenzielle höhere Gehalte

in hypoallergener als in normaler Babymilchnahrung gefunden.

Hohe Modifizierungsraten von Methionin und Lysin verhindern ihre physiologische

Verwertung als essentielle Aminosäure und reduzieren die Proteinverdaubarkeit.


Durch die Überwachung der LacLys-, CML- und MeSO-Gehalte mit Hilfe der neu

entwickelten LC-ESI-MS-MS Methode, kann die physiologische Wertigkeit der

Proben evaluiert und die Produktionsparameters überprüft werden.

150

Bibliography

Ahmed, M. U., E. B. Frye, et al. (1997). "N-epsilon-(carboxyethyl)lysine, a product of the chemical modification of proteins by methylglyoxal, increases with age in human lens proteins." Biochemical Journal 324: 565-570.

Ahmed, N., U. Ahmed, et al. (2005). "Protein glycation, oxidation and nitration adduct residues and free adducts of cerebrospinal fluid in Alzheimer's disease and link to cognitive impairment." J Neurochem 92(2): 255-263.

Ahmed, N., O. K. Argirov, et al. (2002). "Assay of advanced glycation endproducts (AGEs): surveying AGEs by chromatographic assay with derivatization by 6-aminoquinolyl-N-hydroxysuccinimidyl-carbamate and application to N-epsilon-carboxymethyl-lysine- and N-epsilon-(1-carboxyethyl)lysine-modified albumin." Biochemical Journal 364: 1-14.

Ahmed, N., B. Mirshekar-Syahkal, et al. (2005). "Assay of advanced glycation endproducts in selected beverages and food by liquid chromatography with tandem mass spectrometric detection." Mol Nutr Food Res 49(7): 691-699.

Akagawa, M., D. Sasaki, et al. (2006). "New method for the quantitative determination of major protein carbonyls, alpha-aminoadipic and gamma-glutamic semialdehydes: investigation of the formation mechanism and chemical nature in vitro and in vivo." Chem Res Toxicol 19(8): 1059-1065.

Akagawa, M., Y. Wako, et al. (1999). "Lysyl oxidase coupled with catalase in egg shell membrane." Biochim Biophys Acta 1434(1): 151-160.

Almaas, R., T. Rootwelt, et al. (1997). "Ascorbic acid enhances hydroxyl radical formation in iron-fortified infant cereals and infant formulas." Eur J Pediatr 156(6): 488-492.

Alpert, A. J. (1990). "Hydrophilic-Interaction Chromatography for the Separation of Peptides, Nucleic-Acids and Other Polar Compounds." Journal of Chromatography 499: 177-196.

Alpert, A. J., M. Shukla, et al. (1994). "Hydrophilic-Interaction Chromatography of Complex Carbohydrates." Journal of Chromatography A 676(1): 191-202.

Ames, J. M., A. Wynne, et al. (1999). "The effect of a model melanoidin mixture on faecal bacterial populations in vitro." Br J Nutr 82(6): 489-495.

Andersen, J. H., H. Jenssen, et al. (2003). "Lactoferrin and lactoferricin inhibit Herpes simplex 1 and 2 infection and exhibit synergy when combined with acyclovir." Antiviral Res 58(3): 209-215.

Aoe, S., Y. Toba, et al. (2001). "Controlled trial of the effects of milk basic protein (MBP) supplementation on bone metabolism in healthy adult women." Biosci Biotechnol Biochem 65(4): 913-918.

Ardrey, B. (2003). "Liquid chromatography mass spectrometry: An Introduction."

151

Argirov, O. K., N. D. Leigh, et al. (2005). "Specific MS/MS Fragmentation of Lysine,Arginine, and Ornithine Glycation Products. Provides an Opportunity for Their Selective Detection in Protein Acid Hydrolysates and Enzymatic Digests." Annals of the New York Academy of Sciences 1043: 903.

Ashby, M. T. and P. Nagy (2007). "Revisiting a proposed kinetic model for the reaction of cysteine and hydrogen peroxide via cysteine sulfenic acid." International Journal of Chemical Kinetics 39(1): 32-38.

Assar, S. H., C. Moloney, et al. (2009). "Determination of Nepsilon-(carboxymethyl)lysine in food systems by ultra performance liquid chromatography-mass spectrometry." Amino Acids 36(2): 317-326.

Badoud, R., L. Fay, et al. (1991). "Gas-Chromatographic Determination of N-Carboxymethyl Amino-Acids, the Periodate-Oxidation Products of Amadori Compounds." Journal of Chromatography 552(1-2): 345-351.

Baptista. Jose, C. C. B. R. (2004). "Indirect determination of Amadori compounds in milk-based products by HPLC/ELSD/UV as an index of protein deterioration." Food Research International 37: 739-747.

Baxter, J. H., C. S. Lai, et al. (2007). "Direct determination of methionine sulfoxide in milk proteins by enzyme hydrolysis/high-performance liquid chromatography." J Chromatogr A 1157(1-2): 10-16.

Belitz. H. D, G. W., Schieberle. P (2009). "Food chemistry."

Birlouez-Aragon, I., M. Pischetsrieder, et al. (2004). "Assessment of protein glycation markers in infant formulas." Food Chemistry 87(2): 253-259.

Brock, J. W., J. M. Ames, et al. (2007). "Formation of methionine sulfoxide during glycoxidation and lipoxidation of ribonuclease A." Arch Biochem Biophys 457(2): 170-176.

Brot, N., J. Werth, et al. (1982). "Reduction of N-acetyl methionine sulfoxide: a simple assay for peptide methionine sulfoxide reductase." Anal Biochem 122(2): 291-294.

Bucala, R., Z. Makita, et al. (1994). "Modification of low density lipoprotein by advanced glycation end products contributes to the dyslipidemia of diabetes and renal insufficiency." Proc Natl Acad Sci U S A 91(20): 9441-9445.

Calvo, L. d. l. H. (1992). "Flavour of heated milk. A review." Intenational dairy journal 2: 69-81.

Charissou, A., L. Ait-Ameur, et al. (2007). "Evaluation of a gas chromatography/mass spectrometry method for the quantification of carboxymethyllysine in food samples." J Chromatogr A 1140(1-2): 189-194.

Chevalier, F., J. M. Chobert, et al. (2001). "Maillard glycation of beta-lactoglobulin with several sugars: comparative study of the properties of the obtained polymers and of the substituted sites." Lait 81(5): 651-666.

Claiborne, A., H. Miller, et al. (1993). "Protein-sulfenic acid stabilization and function in enzyme catalysis and gene regulation." Faseb Journal 7(15): 1483-1490.

Czerwenka, C., I. Maier, et al. (2006). "Investigation of the lactosylation of whey proteins by liquid chromatography-mass spectrometry." J Agric Food Chem 54(23): 8874-8882.

152

da Costa, J. C. S., K. C. Pais, et al. (2006). "Simple reduction of ethyl, isopropyl and benzyl aromatic esters to alcohols using sodium borohydride-methanol system." Arkivoc: 128-133.

Dean, R. T., S. Fu, et al. (1997). "Biochemistry and pathology of radical-mediated protein oxidation." Biochem J 324 ( Pt 1): 1-18.

Deignan, J. L., J. C. Livesay, et al. (2007). "Polyamine homeostasis in arginase knockout mice." Am J Physiol Cell Physiol 293(4): C1296-1301.

Delatour, T., F. Fenaille, et al. (2006). "Synthesis, tandem MS- and NMR-based characterization, and quantification of the carbon 13-labeled advanced glycation endproduct, 6-N-carboxymethyllysine." Amino Acids 30(1): 25-34.

Delatour, T., J. Hegele, et al. (2009). "Analysis of advanced glycation endproducts in dairy products by isotope dilution liquid chromatography-electrospray tandem mass spectrometry. The particular case of carboxymethyllysine." J Chromatogr A 1216(12): 2371-2381.

Delgado-Andrade, C., I. Seiquer, et al. (2007). "Effects of consumption of Maillard reaction products on magnesium digestibility and tissue distribution in rats." Food Science and Technology International 13(2): 109-115.

Dijkstra, G., H. Moshage, et al. (2002). "Blockade of NF-kappaB activation and donation of nitric oxide: new treatment options in inflammatory bowel disease?" Scand J Gastroenterol Suppl(236): 37-41.

Dittrich, R., F. El-Massry, et al. (2003). "Maillard reaction products inhibit oxidation of human low-density lipoproteins in vitro." J Agric Food Chem 51(13): 3900-3904.

Drinda, S., S. Franke, et al. (2002). "Identification of the advanced glycation end products N(epsilon)-carboxymethyllysine in the synovial tissue of patients with rheumatoid arthritis." Ann Rheum Dis 61(6): 488-492.

Einarsson, H., T. Eklund, et al. (1988). "Inhibitory mechanisms of Maillard reaction products." Microbios 53(214): 27-36.

Einarsson, H., B. G. Snygg, et al. (1983). "Inhibition of Bacterial-Growth by Maillard Reaction-Products." Journal of Agricultural and Food Chemistry 31(5): 1043-1047.

Fedorova, M., N. Kuleva, et al. (2010). "Identification, quantification, and functional aspects of skeletal muscle protein-carbonylation in vivo during acute oxidative stress." J Proteome Res 9(5): 2516-2526.

Fenaille, F., V. Parisod, et al. (2005). "Carbonylation of milk powder proteins as a consequence of processing conditions." Proteomics 5(12): 3097-3104.

Fenaille, F., V. Parisod, et al. (2006). "Modifications of milk constituents during processing: A preliminary benchmarking study." International Dairy Journal 16(7): 728-739.

Fernandez-Artigas, P., E. Guerra-Hernandez, et al. (1999). "Browning indicators in model systems and baby cereals." J Agric Food Chem 47(7): 2872-2878.

Ferreira, A. E., A. M. Ponces Freire, et al. (2003). "A quantitative model of the generation of N(epsilon)-(carboxymethyl)lysine in the Maillard reaction between collagen and glucose." Biochem J 376(Pt 1): 109-121.

153

Finot, P. A., Aeschbacher, et al. (1990). Metabolism and physiological effects of Miallard products (MRP). in The Maillard reaction in food processing, human nutrition, and physiology. Basel ; Boston, Birkhäuser Verlag.

Finot, P. A. and E. Magnenat (1981). "Metabolic transit of early and advanced Maillard products." Prog Food Nutr Sci 5(1-6): 193-207.

Fliss, H., H. Weissbach, et al. (1983). "Oxidation of methionine residues in proteins of activated human neutrophils." Proc Natl Acad Sci U S A 80(23): 7160-7164.

Friedman, M. (1992). "Dietary impact of food processing." Annu Rev Nutr 12: 119-137.

Friedman, M. (1996). "Food browning and its prevention: An overview." Journal of Agricultural and Food Chemistry 44(3): 631-653.

Frolov, A. and R. Hoffmann (2008). "Analysis of amadori peptides enriched by boronic acid affinity chromatography." Ann N Y Acad Sci 1126: 253-256.

Gerrard, J. A. (2002). "New Aspects of an AGEing Chemistry-Recent Development Concerning the Maillard Reaction." CSIRO and Australian Academy of Science 55: 299-310.

Giles, G. I., K. M. Tasker, et al. (2001). "Hypothesis: the role of reactive sulfur species in oxidative stress." Free Radic Biol Med 31(10): 1279-1283.

Glomb, M. A. and V. M. Monnier (1995). "Mechanism of protein modification by glyoxal and glycolaldehyde, reactive intermediates of the Maillard reaction." J Biol Chem 270(17): 10017-10026.

Gonzalez-Reche, L. M., A. Kucharczyk, et al. (2006). "Determination of N-(epsilon)(carboxymethyl)lysine in exhaled breath condensate using isotope dilution liquid chromatography/electrospray ionization tandem mass spectrometry." Rapid Communications in Mass Spectrometry 20(18): 2747-2752.

Grumbach, E. S., D. M. Diehl, et al. (2003). "Hydrphilic interaction chromatography for enhanced ESI-MS sensitivity and retention of polar basic analytes." Lc Gc Europe: 30-31.

Grumbach, E. S., D. M. Wagrowski-Diehl, et al. (2004). "Hydrophilic interaction chromatography using silica columns for the retention of polar analytes and enhanced ESI-MS sensitivity." Lc Gc North America 22(10): 1010-+.

Guerra-Hernandez, E., N. Corzo, et al. (1999). "Maillard reaction evaluation by furosine determination during infant cereal processing." Journal of Cereal Science 29(2): 171-176.

Guo, Y. and S. Gaiki (2005). "Retention behavior of small polar compounds on polar stationary phases in hydrophilic interaction chromatography." J Chromatogr A 1074(1-2): 71-80.

Guy, P. A. and F. Fenaille (2006). "Contribution of mass spectrometry to assess quality of milk-based products." Mass Spectrometry Reviews 25(2): 290-326.

Haagsma, N. and P. Slump (1978). "Evaluation of lysinoalanine determinations in food proteins." Z Lebensm Unters Forsch 167(4): 238-240.

154

Hao, Z., C. Y. Lu, et al. (2007). "Separation of amino acids, peptides and corresponding Amadori compounds on a silica column at elevated temperature." J Chromatogr A 1147(2): 165-171.

Hasenkopf, K., B. Ronner, et al. (2002). "Analysis of glycated and ascorbylated proteins by gas chromatography-mass spectrometry." J Agric Food Chem 50(20): 5697-5703.

Haug, A., A. T. Hostmark, et al. (2007). "Bovine milk in human nutrition--a review." Lipids Health Dis 6: 25.

Hegele, J., T. Buetler, et al. (2008). "Comparative LC-MS/MS profiling of free and protein-bound early and advanced glycation-induced lysine modifications in dairy products." Anal Chim Acta 617(1-2): 85-96.

Hegele, J., V. Parisod, et al. (2008). "Evaluating the extent of protein damage in dairy products: simultaneous determination of early and advanced glycation-induced lysine modifications." Ann N Y Acad Sci 1126: 300-306.

Heine, W. E., P. D. Klein, et al. (1991). "The importance of alpha-lactalbumin in infant nutrition." J Nutr 121(3): 277-283.

Henle, T., H. Walter, et al. (1991). "Evaluation of the extent of the early Maillard-reaction in milk products by direct measurement of the Amadori-product lactuloselysine." Z Lebensm Unters Forsch 193(2): 119-122.

Hiramoto, S., K. Itoh, et al. (2004). "Melanoidin, a food protein-derived advanced maillard reaction product, suppresses Helicobacter pylori in vitro and in vivo." Helicobacter 9(5): 429-435.

Huber, B. and M. Pischetsrieder (1998). "Characterization of ascorbylated proteins by immunochemical methods." Journal of Agricultural and Food Chemistry 46(10): 3985-3990.

Jennings. D. M, O. A. M. L. (1969). "Methionine Loss during Protein Hydrolysis of Plant Material." Journal of Agricultural and Food Chemistry 17(3): 668-669.

Jolles, P., S. Levy-Toledano, et al. (1986). "Analogy between fibrinogen and casein. Effect of an undecapeptide isolated from kappa-casein on platelet function." Eur J Biochem 158(2): 379-382.

Kada, T., T. Inoue, et al. (1986). "Antimutagens and their modes of action." Basic Life Sci 39: 181-196.

Kagan, H. M., K. A. Sullivan, et al. (1979). "Purification and properties of four species of lysyl oxidase from bovine aorta." Biochem J 177(1): 203-214.

Kennedy, R. S., G. P. Konok, et al. (1995). "The use of a whey protein concentrate in the treatment of patients with metastatic carcinoma: a phase I-II clinical study." Anticancer Res 15(6B): 2643-2649.

Koschinsky, T., C. J. He, et al. (1997). "Orally absorbed reactive glycation products (glycotoxins): an environmental risk factor in diabetic nephropathy." Proc Natl Acad Sci U S A 94(12): 6474-6479.

Krause, R., K. Knoll, et al. (2003). "Studies on the formation of furosine and pyridosine during acid hydrolysis of different Amadori products of lysine." European Food Research and Technology 216(4): 277-283.

155

Krissansen, G. W. (2007). "Emerging Health Properties of Whey Proteins and Their

Clinical Implications." The American College of Nutrition, 26: 713S-723S.

Leclere, J., I. Birlouez-Aragon, et al. (2002). "Fortification of milk with iron-ascorbate promotes lysine glycation and tryptophan oxidation." Food Chemistry 76(4): 491-499.

Lindmark-Mansson, H. and B. Akesson (2000). "Antioxidative factors in milk." Br J Nutr 84 Suppl 1 : S103-110.

Luzzana, M., D. Agnellini, et al. (2003). "Milk lactose and lactulose determination by the differential pH technique." Lait 83(5): 409-416.

Lyons, T. J., G. Silvestri, et al. (1991). "Role of Glycation in Modification of Lens Crystallins in Diabetic and Nondiabetic Senile Cataracts." Diabetes 40(8): 1010-1015.

Matsuura, N., T. Aradate, et al. (2002). "Screening system for the Maillard reaction inhibitor from natural product extracts." Journal of Health Science 48(6): 520-526.

Mauron, J. (1990). "Influence of processing on protein quality." J Nutr Sci Vitaminol (Tokyo) 36 Suppl 1 : S57-69.

Meltretter, J., C. M. Becker, et al. (2008). "Identification and site-specific relative quantification of beta-lactoglobulin modifications in heated milk and dairy products." J Agric Food Chem 56(13): 5165-5171.

Meltretter, J. and M. Pischetsrieder (2008). "Application of mass spectrometry for the detection of glycation and oxidation products in milk proteins." Ann N Y Acad Sci 1126: 134-140.

Meltretter, J., S. Seeber, et al. (2007). "Site-specific formation of Maillard, oxidation, and condensation products from whey proteins during reaction with lactose." J Agric Food Chem 55(15): 6096-6103.

Monnier, V. M., D. R. Sell, et al. (1992). "Advanced Maillard Reaction-Products as Markers for Tissue-Damage in Diabetes and Uremia - Relevance to Diabetic Nephropathy." Acta Diabetologica 29(3-4): 130-135.

Morales, F. J. and S. Jimenez-Perez (2004). "Peroxyl radical scavenging activity of melanoidins in aqueous systems." European Food Research and Technology 218(6): 515-520.

Mortier L, B. A., Cartuyvels D, Van Renterghem R, De Block J. (2000). "Intrinsic indicators for monitoring heat damage of consumption milk." Biotechnol. Agron. Soc. Environ 4(4): 221-225.

Mossine, V. V., M. Linetsky, et al. (1999). "Superoxide free radical generation by Amadori compounds: the role of acyclic forms and metal ions." Chem Res Toxicol 12(3): 230-236.

Murray, K., P. S. Rasmussen, et al. (1965). "The Hydrolysis of Arginine." J Biol Chem 240: 705-709.

Muscat, S., J. Pelka, et al. (2007). "Coffee and Maillard products activate NF-kappa B in macrophages via H2O2 production." Molecular Nutrition & Food Research 51(5): 525-535.

156

Nagai, R., C. M. Hayashi, et al. (2002). "Identification in human atherosclerotic lesions of GA-pyridine, a novel structure derived from glycolaldehyde-modified proteins." J Biol Chem 277(50): 48905-48912.

Nakamura, Y., N. Yamamoto, et al. (1995). "Purification and characterization of angiotensin I-converting enzyme inhibitors from sour milk." J Dairy Sci 78(4): 777-783.

Nentwich, I., E. Michkova, et al. (2001). "Cow's milk-specific cellular and humoral immune responses and atopy skin symptoms in infants from atopic families fed a partially (pHF) or extensively (eHF) hydrolyzed infant formula." Allergy 56(12): 1144-1156.

Nielsen, H. K., J. Loliger, et al. (1985). "Reactions of Proteins with Oxidizing Lipids .1. Analytical Measurements of Lipid Oxidation and of Amino-Acid Losses in a Whey-Protein Methyl Linolenate Model System." British Journal of Nutrition 53(1): 61-73.

Nilsson, M., J. J. Holst, et al. (2007). "Metabolic effects of amino acid mixtures and whey protein in healthy subjects: studies using glucose-equivalent drinks." Am J Clin Nutr 85(4): 996-1004.

Niwa, T., T. Katsuzaki, et al. (1997). "Immunohistochemical detection of imidazolone, a novel advanced glycation end product, in kidneys and aortas of diabetic patients." J Clin Invest 99(6): 1272-1280.

Olano, A., M. M. Calvo, et al. (1989). "Changes in the Carbohydrate Fraction of Milk during Heating Processes." Food Chemistry 31(4): 259-265.

Penndorf, I., D. Biedermann, et al. (2007). "Studies on N-terminal glycation of peptides in hypoallergenic infant formulas: quantification of alpha-N-(2-furoylmethyl) amino acids." J Agric Food Chem 55(3): 723-727.

Powrie, W. D., C. H. Wu, et al. (1986). "Browning reaction systems as sources of mutagens and antimutagens." Environ Health Perspect 67: 47-54.

Reddie, K. G., Y. H. Seo, et al. (2008). "A chemical approach for detecting sulfenic acid-modified proteins in living cells." Mol Biosyst 4(6): 521-531.

Requena, J. R., C. C. Chao, et al. (2001). "Glutamic and aminoadipic semialdehydes are the main carbonyl products of metal-catalyzed oxidation of proteins." Proc Natl Acad Sci U S A 98(1): 69-74.

Ronner, B., H. Lerche, et al. (2000). "Formation of tetrahydro-beta-carbolines and beta-carbolines during the reaction of L-tryptophan with D-glucose." J Agric Food Chem 48(6): 2111-2116.

Rosaneli, C. F., A. E. Bighetti, et al. (2002). "Efficacy of a whey protein concentrate on the inhibition of stomach ulcerative lesions caused by ethanol ingestion." J Med Food 5(4): 221-228.

Saurin, A. T., H. Neubert, et al. (2004). "Widespread sulfenic acid formation in tissues in response to hydrogen peroxide." Proc Natl Acad Sci U S A 101(52): 17982-17987.

Schettgen, T., A. Tings, et al. (2007). "Simultaneous determination of the advanced glycation end product N (epsilon)-carboxymethyllysine and its precursor, lysine, in exhaled breath condensate using isotope-dilution-hydrophilic-

157

interaction liquid chromatography coupled to tandem mass spectrometry." Anal Bioanal Chem 387(8): 2783-2791.

Schleicher, E. (1991). "Die Bedeutung der Maillard-Reaktion in der menschlichen Physiologie." Ernflhrungswissenschaft 30: 18-28.

Schlichtherle-Cerny, H., M. Affolter, et al. (2003). "Hydrophilic interaction liquid chromatography coupled to electrospray mass spectrometry of small polar compounds in food analysis." Analytical Chemistry 75(10): 2349-2354.

Sebekova, K., M. Krajcoviova-Kudlackova, et al. (2001). "Plasma levels of advanced glycation end products in healthy, long-term vegetarians and subjects on a western mixed diet." Eur J Nutr 40(6): 275-281.

Sell, D., R. Monnier, V, M. (2004). "Conversion of Arginine into Ornithine by Advanced Glycation in Senescent Human Collagen and Lens Crystallins." THE JOURNAL OF BIOLOGICAL CHEMISTRY 279(Issue of December 24): 54173–54184.

Sell, D. R., C. M. Strauch, et al. (2007). "2-aminoadipic acid is a marker of protein carbonyl oxidation in the aging human skin: effects of diabetes, renal failure and sepsis." Biochem J 404(2): 269-277.

Shetty, V., D. S. Spellman, et al. (2007). "Characterization by tandem mass spectrometry of stable cysteine sulfenic acid in a cysteine switch peptide of matrix metalloproteinases." J Am Soc Mass Spectrom 18(8): 1544-1551.

Skoog, D. H., J. Nieman, T. (2005). "Principles of Instrumental Analysis, fifth edition."

Smith, M. A., S. Taneda, et al. (1994). "Advanced Maillard Reaction End-Products Are Associated with Alzheimer-Disease Pathology." Proceedings of the National Academy of Sciences of the United States of America 91(12): 5710-5714.

Smith, P. R. and P. J. Thornalley (1992). "Preparation and characterisation of N epsilon-(1-deoxy-D-fructos-1-yl)hippuryl-lysine." Carbohydr Res 223: 293-298.

Sochaski, M. A., A. J. Jenkins, et al. (2001). "Isotope dilution gas chromatography/mass spectrometry method for the determination of methionine sulfoxide in protein." Anal Chem 73(19): 4662-4667.

Starcher, B. (2001). "A ninhydrin-based assay to quantitate the total protein content of tissue samples." Analytical Biochemistry 292(1): 125-129.

Suyama, K., M. Akagawa, et al. (2002). "Oxidative deamination of lysine residue in plasma protein from diabetic rat: a-dicarbonyl-mediated mechanism." International Congress Series 1245: 243-248.

Teerlink, T., R. Barto, et al. (2004). "Measurement of N-epsilon-(Carboxymethyl)lysine and N-epsilon-(Carboxyethyl)lysine in human plasma protein by stable-isotope-dilution tandem mass spectrometry." Clinical Chemistry 50(7): 1222-1228.

Thornalley, P. J. (1994). "Methylglyoxal, Glyoxalases and the Development of Diabetic Complications." Amino Acids 6(1): 15-23.

Thornalley, P. J., S. Battah, et al. (2003). "Quantitative screening of advanced glycation endproducts in cellular and extracellular proteins by tandem mass spectrometry." Biochem J 375(Pt 3): 581-592.

158

Valle-Riestra, J. B., R. (1970). "Digestion of Heat-damaged Egg Albumen ' by the Rat2." Nutrition 100: 873-882.

van Boekel, M. A. J. S. (1998). "Effect of heating on Maillard reactions in milk." Food Chemistry 62(4): 403-414.

Villmann, C., B. Sandmeier, et al. (2007). "Myelin proteolipid protein (PLP) as a marker antigen of central nervous system contaminations for routine food control." J Agric Food Chem 55(17): 7114-7123.

Vinale, F., V. Fogliano, et al. (1999). "Development of a stable isotope dilution assay for an accurate quantification of protein-bound N(epsilon)-(1-deoxy-D-fructos-1-yl)-L-lysine using a (13)C-labeled internal standard." J Agric Food Chem 47(12): 5084-5092.

Vlassara, H., W. Cai, et al. (2002). "Inflammatory mediators are induced by dietary glycotoxins, a major risk factor for diabetic angiopathy." Proc Natl Acad Sci U S A 99(24): 15596-15601.

Wagner, K. H., S. Reichhold, et al. (2007). "The potential antimutagenic and antioxidant effects of Maillard reaction products used as "natural antibrowning" agents." Molecular Nutrition & Food Research 51(4): 496-504.

Weigel, K. U. (2004). "α-Dicarbonyle in Lebensmitteln und glucosehaltigen Lösungen der Peritonealdialyse."

Wit, J. N. d. (1998). "Nutritional and Functional Characteristics

of Whey Proteins in Food Products." Diary Science 81: 597-608.

Yalcin, T. and A. G. Harrison (1996). "Ion chemistry of protonated lysine derivatives." J Mass Spectrom 31(11): 1237-1243.

Yue, D. K., S. McLennan, et al. (1982). "The measurement of glycosylated hemoglobin in man and animals by aminophenylboronic acid affinity chromatography." Diabetes 31(8 Pt 1): 701-705.

Zhang, Q., N. Tang, et al. (2007). "Enrichment and analysis of nonenzymatically glycated peptides: boronate affinity chromatography coupled with electron-transfer dissociation mass spectrometry." J Proteome Res 6(6): 2323-2330.

Zill, H., S. Bek, et al. (2003). "RAGE-mediated MAPK activation by food-derived AGE and non-AGE products." Biochem Biophys Res Commun 300(2): 311-315.

Zimmer, D. (2003). "Introduction to quantitative liquid chromatography-tandem mass spectrometry (LC-MS-MS)." Chromatographia 57: S325-S332.

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List of Tables

Table 1: Milk components and their concentrations in whole milk (Haug, Hostmark et al. 2007) ........................................................................................................... 11

Table 2: Composition of casein micelles (Belitz. H. D 2009) .................................... 12

Table 3: Composition, abundance and biological function of whey proteins. Adapted from (Wit 1998) ................................................................................................ 13

Table 4: Amount of methionine (0.25 M), HCl and H2O2 (30%) used to form MeSO 46

Table 5: Optimized MS-parameters dependent on the LC-flow ................................ 54

Table 6: Monitored transitions of LacLys, 5-OH-Trp, CML and MeSO and the optimized MS-parameters dependent on these compounds ............................ 55

Table 7: Mass spectrometric compound-dependent parameters of ornithine ........... 82

Table 8: The quantifier and qualifier fragments of LacLys, MeSO and CML ............ 97

Table 9: Within-day repeatability of the method for MeSO, LacLys and CML ........ 101

Table 10: Between-day repeatability of the method for MeSO, LacLys and CML .. 102

Table 11: Recovery of LacLys ................................................................................ 103

Table 12: Recovery of MeSO ................................................................................. 104

Table 13: Recovery of CML .................................................................................... 105

Table 14: MeSO, LacLys and CML contents in some dairy products determined by LC-ESI-MS-MS .............................................................................................. 110

Table 15: MeSO content in different milk products reported in literature ................ 113

Table 16: CML content in different milk products reported in literature .................. 113

Table 17: Concentrations of LacLys, MeSO and CML which were used for standard addition to investigate the within-day repeatability for UHT milk and heated UHT milk. The concentration of the sample, in which the analytes should be quantified, was 50 µg/ml ................................................................................ 133

Table 18: Concentrations of LacLys, MeSO and CML which were used for standard addition to investigate the between-day repeatability in the samples of UHT milk and heated UHT milk. The concentration of the sample, in which the analytes should be quantified, was 75 µg/ml ................................................................ 134

Table 19 : The amounts of LacLys, CML and MeSO in 30 µl of the standards solutions which were spiked to each protein sample before enzymatic hydrolysis ....................................................................................................... 135

Table 20: The concentrations which were used for standard addition during the determination of the recovery of of LacLys, MeSO and CML. The concentration of the sample in which CML should be quantified was 150µg/ml and it was 25µg/ml to quantify LacLys and MeSO .......................................................... 135

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Table 21: Amounts of CML which were spiked into 25 µg of raw milk protein hydrolyzate in order to determine LOD and LOQ ........................................... 136

Table 22: Concentrations of milk samples which were quantified. The concentration varied between 25µg/ml to quantifiy both MeSO and LacLys to 900µg/ml to quantify CML in raw milk proteins .................................................................. 137

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List of Figures

Figure 1 : The early phase of the Maillard reaction .................................................... 2

Figure 2: Phases of the Maillard reaction ................................................................... 3

Figure 3: Some structures of AGEs ............................................................................ 5

Figure 4: Early stage of the Maillard reaction in milk ................................................ 18

Figure 5: Formation of CML through different pathways (Ferreira, Ponces Freire et al. 2003) ........................................................................................................... 19

Figure 6: Degradation of Amadori product under acidic, neutral or alkaline conditions ......................................................................................................................... 20

Figure 7: Schematic illustration of tandem mass spectrometry in MRM mode [adapted from (Zimmer 2003)] ......................................................................... 25

Figure 8: Degradation of LacLys via oxidative cleavage, acid hydrolysis and heating in the presence of oxalic acid ........................................................................... 28

Figure 9: Products of methionine oxidation .............................................................. 30

Figure 10: Lysine oxidation to form lysine aldehyde ................................................. 30

Figure 11: Some cysteine oxidation products .......................................................... 32

Figure 12: The chromatogram of the reaction mixture of FMOC-lys and lactose shows the formation of a new product with m/z of 693 indicating the formation of FMOC-LacLys .............................................................................................. 34

Figure 13 : MS2 spectrum of the synthesized LacLys .............................................. 34

Figure 14: Fragmentation pathway of lactulosyllysine .............................................. 35

Figure 15: Illustration of affinity chromatography. [Adapted according to the handbook (Affinity Chromatography) from Amersham Biosciences] ................ 37

Figure 16: The interaction of m-aminophenylboronic acid with lactulosyllysine ........ 38

Figure 17: Functional group packed in ZIC-HILIC columns ...................................... 40

Figure 18: Chromatogram of the reaction mixture of FMOC-Lys and lactose separated on a ZIC-HILIC column ................................................................... 40

Figure 19: Chromatogram and mass spectrum of FMOC-LacLys obtained after fractionation of the reaction mixture of FMOC-Lys and lactose on a ZIC-HILIC column. ............................................................................................................ 41

Figure 20: Synthesis of LacLys ................................................................................ 41

Figure 21: 1H-NMR spectrum of the synthesized LacLys shows the signal of α-C and ε-C of lysine as well as the expected signal of the bond between lactose and lysine ................................................................................................................ 43

Figure 22: Signal of the aldehyde proton of DMF which appeared at 7.926 ppm ..... 44

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Figure 23: Chromatogram of the reaction mixture of Cbz-lysine and iodoacetic acid shows the formation of Cbz-CML ..................................................................... 44

Figure 24: Synthesis of CML .................................................................................... 45

Figure 25: Chromatogram of the reaction mixture of methionine and H2O2 which was incubated at 21 °C. The chromatogram shows the form ation of MeSO and MeSOO. ........................................................................................................... 46

Figure 26: Chromatogram and mass spectrum of the reaction mixture of Cbz-lysine and urea extract of ESM shows unreacted Cbz-lysine as well as the formation of unknown compound. The unknown compound showed m/z 280.2 corresponding to the protonated Cbz-lysine aldehyde with a mass difference of -1Da compared to Cbz-lysine........................................................................... 48

Figure 27: Chromatogram of the reaction mixture of cysteine and H2O2 (recorded at 250 nm) shows the scavenger methionine and a new product with m/z 241.2 which was assigned to cystine ......................................................................... 49

Figure 28: Reaction of lysine with lactose during heating of whey protein ............... 51

Figure 29: Some protein oxidation products ............................................................. 52

Figure 30: Ion optic in the vacuum chamber of an MS-MS triple quadrupole system (Manual user API 4000 QTRAP, 2008). Q1: first quadrupole; Q2: collision cell; Q3: third quadrupole; ST: stubby lens between Q0 and Q1; ST2: stubby lens between Q1 and Q2; ST3: stubby lens between Q2 and Q3; RF: radio frequency; CEM: channel electron multiplier .................................................... 54

Figure 31: Product ion mass spectrum of CML and the corresponding fragments ... 56

Figure 32: Product ion mass spectrum of MeSO and the corresponding fragments 56

Figure 33: Product ion mass spectrum of LacLys and the corresponding fragments 57

Figure 34: Product ion mass spectrum of 5-OH-Trp and the corresponding fragments ......................................................................................................................... 57

Figure 35: LC-MS-MS chromatogram of the standards mixture, obtained in MRM mode, on a C18 column. The standard mixture contained MeSO, 5-OH-Trp, LacLys and CML .............................................................................................. 59

Figure 36: A centrifugal concentrator device which was used for the clean-up of the enzymatic hydrolyzate ..................................................................................... 60

Figure 37: Extracted ion chromatogram (XIC) of glycated α-LA shows the formation of CML (right). LC-MS-MS chromatogram of glycated α-LA obtained in MRM mode shows the formation of LacLys (left). α-LA and lactose were heated in a milk matrix at 60 °C for 3 (A), 7 (B) and 14 (C) da ys and subjected to enzymatic hydrolysis prior to LC-MS-MS analysis. CML formation increased during the whole incubation period, while the highest signal of LacLys was obtained after 7 days of incubation. ........................................................................................... 62

Figure 38: LC-MS-MS chromatogram of glycated α-LA obtained in MRM mode shows the formation of MeSO. α-LA and lactose were heated in a milk matrix at 60 °C for 3 (1 a), 7 (2 a) and 14 (3 a) days and subjected to enzymatic hydrolysis prior to LC-MS-MS analysis. MeSO was detected in native α-LA (standard) and in α-LA heated without lactose for 3 (1 b), 7 (2 b) and 14 (3 b) days ................................................................................................................. 63

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Figure 39: LC-MS-MS chromatogram of oxidized α-LA obtained in MRM mode shows the formation of MeSO. 5-OH-Trp was not detected. α-LA and H2O2 were heated in a milk matrix for 10, 20 and 30 min at 120 °C (1a, 2a and 3a, respectively) and subjected to enzymatic hydrolysis prior to LC-MS-MS analysis. LC-MS-MS spectra obtained in MRM mode of native α-LA (standard) and α-LA heated for 10, 20 and 30 min at 120 °C (1b, 2b and 3 b, respectively) which were used as control .............................................................................. 65

Figure 40: LC-MS-MS chromatogram of UHT milk obtained in MRM mode shows the formation of MeSO and LacLys. After the removal of lactose and fat, milk proteins were subjected to enzymatic hydrolysis prior to LC-MS-MS analysis using a C18 column. LacLys and MeSO were identified by their specific mass transitions (LacLys: m/z 471.1→225.1, m/z 471.1→128.1, m/z 471.1→84.1; MeSO: m/z 166.1→74, m/z 166.1→56, m/z 166.1→102.1). ............................ 69

Figure 41: The increase of the MeSO signal and the stability of the unknown peak during heat treatment of milk............................................................................ 70

Figure 42: Relative signal of MeSO and LacLys in different milk samples ............... 71

Figure 43: Formation of MeSO during the different steps of enzymatic protein hydrolysis. A blank without sample protein was subsequently hydrolysed by pepsin, pronase and aminopeptidase. The enzymes remained in the samples throughout hydrolysis (A). Pepsin was removed before further hydrolysis (B). Pepsin and pronase were removed before further hydrolysis (C) .................... 72

Figure 44: The decrease of MeSO and LacLys signal intensities after the removal of pepsin from milk hydrolysate after 24 hours of enzymatic hydrolysis ............... 72

Figure 45: LC-MS-MS chromatogram of sterilized milk obtained in MRM mode shows a clear formation of MeSO and LacLys and a possible formation of CML. After the removal of lactose and fat, milk proteins were subjected to enzymatic hydrolysis prior to LC-MS-MS analysis using a C18 column. MeSO and LacLys were identified by their specific mass transitions. Only one mass transition of CML was detected (m/z 205.1→ 84.1). ............................................................ 73

Figure 46: LC-MS-MS chromatogram obtained in MRM mode of 5-OH-Trp, MeSO, CML and LacLys (standard mixture) on a ZIC-HILIC column 150*2.1mm, 3.5 µm .................................................................................................................... 75

Figure 47: Comparison of the signal intensities of the standards MeSO, CML, 5-OH-Trp and LacLys on C18 and ZIC-HILIC columns ............................................. 76

Figure 48: LC-MS-MS chromatogram of UHT milk obtained in MRM mode shows the formation of CML. Milk proteins were subjected to enzymatic hydrolysis prior to the LC-MS-MS analysis using a ZIC-HILIC column. An unknown peak with the same specific mass transitions (m/z 205.1→ 84.1, m/z 205.1→ 130.1) appeared after 5 min. ....................................................................................... 77

Figure 49: The increase of CML signal and the stability of the signal of the unknown compound during heat treatment of milk .......................................................... 78

Figure 50: Ornithine formation from arginine ............................................................ 81

Figure 51: Some derivatives of imidazolone formed from the reaction of arginine and dicarbonyls ....................................................................................................... 82

164

Figure 52: Product ion scan of ornithine shows the fragments and their proposed structures ......................................................................................................... 83

Figure 53: LC-MS-MS chromatogram of the standard ornithine (A) and UHT milk proteins (B) obtained in MRM mode. Milk proteins were subjected to enzymatic hydrolysis prior to LC-MS-MS analysis using a C18 column. Panel B shows a peak at the retention time of ornithine which showed only one of its characteristic fragments (m/z 133.1→ 70.1). The second fragment, which was characteristic for the standard (m/z 133.1→ 116), was not detected ............... 84

Figure 54: Peak areas obtained for the quantifier fragment (m/z 133.1→ 70.1) of the compound detected at the retention time of ornithine ...................................... 84

Figure 55: Reduction of lactulosyllysine with borohydride to form a product stable during acidic hydrolysis .................................................................................... 87

Figure 56: Protein samples hydrolyzed with a cocktail of enzymes for 72 hours or 27 hours were analyzed by ninhydrin assay. Enzymatic hydrolysis of sterilized milk and UHT milk heated for 10, 20, 30 and 60 min (120 °C) for 72 hours resulted in higher absorbance indicating a higher content of free amino acids .............. 88

Figure 57: LC-MS-MS signal intensity of MeSO, CML and LacLys obtained after the enzymatic hydrolysis of sterilized milk sample for 72 hours or 27 hours .......... 88

Figure 58: Decrease of the signal intensity of the standards MeSO, CML and LacLys after purification by SPE column compared to control. The samples were analyzed by LC-MS-MS. The highest loss was noted for LacLys .................... 89

Figure 59: Signal intensity of MeSO, CML and LacLys detected by LC-MS-MS in UHT milk which was heated for 60 min at 120 °C. The sample was purified either by ultrafiltration or by SPE, and then dried by Speed-Vac ..................... 90

Figure 60: Signal intensity of CML detected by LC-MS-MS in the samples of heated UHT milk which were dried either by lyophilization or Speed-Vac after purification ....................................................................................................... 90

Figure 61: Decrease of the signal intensity of CML in heated UHT milk and sterilized milk in which LacLys was reduced by NaBH4 prior to enzymatic hydrolysis. The samples were analyzed by LC-MS-MS ............................................................ 91

Figure 62: LC-MS-MS signal intensity of the analytes MeSO, CML, and LacLys which were detected in (A) HA-infant powder formula and (B) sterilized milk. The diagrams show an increase of CML in the acidic hydrolyzate compared to both enzymatic hydrolyzates. LacLys was not detected after the reduction by NaBH4 in the acidic nor the enzymatic hydrolyzates. Clear decrease of MeSO signal was found in the acidic hydrolyzate ....................................................... 92

Figure 63: Protein samples of UHT milk heated for 10, 20, 30 and 60 min (120 °C), sterilized milk, HA-infant powder formula and lactalbumin. The samples were analyzed by ninhydrin assay after acidic or enzymatic hydrolysis for 72 hours. The absorbance at 570 nm of the enzymatic hydrolyzate (3µg protein) was higher compared to the acidic hydrolyzate (10µg protein) ............................... 93

Figure 64: Schematic illustration shows the method of standard addition. (http://www.chemgapedia.de/vsengine/vlu/vsc/de/ch/3/anc/croma/kalibrierung.vlu/Page/vsc/de/ch/3/anc/croma/datenauswertung/quantitativ/standardaddition/standardadditionm80ht0801.vscml.html, date 9/2/2011) .................................. 100

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Figure 65: An unknown peak appeared at 8.08 min and showed the quantifier fragment of LacLys ........................................................................................ 106

Figure 66: The chamber for solid phase extraction with the cartridges .................. 130

LC-ESI-MS-MS Analysis of Non-enzymatic Posttranslational ...Der größte Dank geht an meinen Mann...

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