Studies on the Formation of 3-Deoxyglucosone- and Methylglyoxal

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Studies on the Formation of 3-Deoxyglucosone- and MethylglyoxalDerived Hydroimidazolones of Creatine during Heat Treatment of Meat Stephanie Treibmann, Franz Spengler, Julia Degen, Jürgen Löbner, and Thomas Henle J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b01243 • Publication Date (Web): 03 May 2019 Downloaded from http://pubs.acs.org on May 3, 2019

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Journal of Agricultural and Food Chemistry

Studies on the Formation of 3-Deoxyglucosone- and Methylglyoxal-Derived Hydroimidazolones of Creatine during Heat Treatment of Meat

Stephanie Treibmann, Franz Spengler, Julia Degen, Jürgen Löbner, Thomas Henle

Chair of Food Chemistry, Technische Universität Dresden, D-01062 Dresden, Germany

Corresponding author: T. Henle Tel.: +49-351-463-34647 Fax: +49-351-463-34138 Email: Thomas.Henle@ tu-dresden.de

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ACS Paragon Plus Environment


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Abstract

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Dicarbonyl compounds such as methylglyoxal (MGO) and 3-deoxyglucosone (3-DG) are

3

formed via caramelization and the Maillard reaction in food during heating or in vivo as by-

4

products of glycolysis. Recently, it was shown that creatine, an amino compound linked to the

5

energy metabolism in vertebrate muscle, reacts rapidly with methylglyoxal under

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physiological conditions to form N-(4-methyl-5-oxo-1-imidazolin-2-yl)sarcosine (MG-HCr),

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a methylglyoxal-derived hydroimidazolone of creatine. Based on the observation that heated

8

meat contains only small amounts of MGO and 3-DG when compared to many other

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foodstuffs, the aim of this study was to investigate a possible reaction of creatine with 3-DG

10

and MGO in meat.

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From incubation mixtures consisting of 3-DG and creatine, a new hydroimidazolone of

12

creatine, namely N-(4-butyl-1,2,3-triol-5-oxo-1-imidazolin-2-yl)sarcosine (3-DG-HCr), was

13

isolated and characterized via spectroscopic means. To quantitate 3-DG-HCr and MG-HCr,

14

meat and fish products were analyzed via HPLC-MS/MS using isotopically labeled standard

15

material. Whereas samples of raw fish and meat contained only trace amounts of the

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hydroimidazolones (below 5 µg/kg), up to 28.3 mg/kg MG-HCr and up to 15.3 mg/kg 3-DG-

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HCr were found in meat and fish products. The concentrations were dependent on the heat

18

treatment and presumably on the smoking process. In comparison to the lysine and arginine

19

derivatives CEL, pyrraline, and MG-H1, the derivatization rate of creatine as MG-HCr and 3-

20

DG-HCr was higher than of lysine and arginine, which clearly demonstrates the 1,2-

21

dicarbonyl scavenging properties of creatine in meat.

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Keywords

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1,2-dicarbonyl compounds; 3-deoxyglucosone; methylglyoxal; Maillard reaction; glycation;

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creatine; meat 2


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Introduction

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Methylglyoxal (MGO) and 3-deoxyglucosone (3-DG) are 1,2-dicarbonyl compounds formed

27

from carbohydrates during caramelization and the Maillard reaction. Under physiological

28

conditions, MGO is also formed as a by-product of glycolysis, and 3-DG from 3-

29

phosphorylated fructose and fructoseamines.1–3 Because of their reactivity, 1,2-dicarbonyl

30

compounds play an important role in food as color and odor precursors and, furthermore, may

31

react with lysine and arginine side chains and N-termini of proteins to form “advanced

32

glycation endproducts” (AGEs).4 Concentrations of 1,2-dicarbonyl compounds were

33

quantitated in a wide range of products like soft drinks, wine, and bakery products.5 In

34

patients with diabetes or uremia, plasma levels of MGO and other 1,2-dicarbonyl compounds

35

were increased.6 This imbalance between production and metabolization was called

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“dicarbonyl stress”.7,8 Due to their reactions with proteins, 1,2-dicarbonyl compounds can

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impair protein functionality and play a role in biological aging and in the pathophysiology of

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several diseases.9

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Based on this, strategies to lower the concentrations of 1,2-dicarbonyl compounds have been

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discussed, which include trapping with molecules like aminoguanidine and quercetin in vivo10

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and in foods.11 For instance, trapping of MGO with a scavenger peptide could reduce diabetic

42

pain in mice.12 Recently it was shown that creatine reacts rapidly with methylglyoxal to form

43

methylglyoxal-derived hydroimidazolone of creatine (MG-HCr, Figure 1) under physiological

44

conditions.13 The compound was also quantitated in urine where higher concentrations were

45

found for omnivores than for vegetarians. The scavenging of MGO with creatine in vivo was

46

shown in a study on a healthy vegetarian, whose MG-HCr excretion increased with creatine

47

supplementation.13

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Creatine is an amino compound, which is linked to the energy metabolism in vertebrate

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muscle. Thus, it is prevalent in fish, meat and respective products with concentrations of 3 to 3



50

10 g/kg.14 Maillard reaction products of creatine such as N-methylacrylamide,15 pentosidine

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like structures16 and heterocyclic amines were found in meat and in vivo.17,18 On the other

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hand, the 1,2-dicarbonyl compounds 3-DG and MGO were not detectable in meat, even when

53

heated with added reducing sugars.19 It was therefore the purpose of this study to investigate

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whether creatine can also react with 3-DG and whether creatine scavenges MGO and 3-DG in

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meat to form hydroimidazolones.

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Materials and methods

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Chemicals. Creatinine and pronase E (4000 PU/mg protein) were purchased from Merck

58

(Darmstadt, Germany), hydrochloric acid from VWR ProLabo (Leuven, Belgium), creatine

59

monohydrate from AppliChem (Darmstadt, Germany) and acetic acid from Roth (Karslruhe,

60

Germany). Deuterium oxide, nonafluoropentanoic acid (NFPA), n-heptane, silica gel,

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prolidase (581 U/mg protein), pepsin (902 U/mg protein), and leucine aminopeptidase

62

(20 U/mg protein) from Sigma-Aldrich were employed (Steinheim, Germany) and HPLC

63

gradient grade methanol and acetonitrile from Fisher Chemicals (Loughborough, UK).

64

Isotopically labeled D3-creatine and D3-creatinine were obtained from Cambridge Isotope

65

Laboratories (Loughborough, UK) and di sodium hydrogen phosphate and phosphoric acid

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from Grüssing (Filsum, Germany). Water for solutions, buffers and HPLC-MS/MS was

67

obtained from a Bi 18 E double distillation system (QCS, Maintal, Germany). MG-HCr,13 D3-

68

MG-HCr,13 3-DG,20 13C6-3-DG,20 CML,21 CEL,21 Pyrraline,22 and MG-H121 were synthesized

69

and characterized in our laboratory according to the literature stated. The substances met the

70

spectroscopic and chromatographic characteristics published in the respective protocols.

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Model incubation of creatine and 3-DG. Creatine monohydrate (0.149 g, 50 mM) and 3-DG

72

(0.016 g, 5 mM) were incubated in 100 mM phosphate buffer pH 7.4 at 37 °C to simulate

73

plasma conditions and for a comparison with literature.13 The mixtures of both compounds as

74

well as 3-DG without creatine were incubated for up to 48 h. Samples for analysis of 3-DG4


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HCr, creatine, and creatinine were kept at -20 °C until analysis. For analysis of 3-DG, aliquots

76

of 500 µL were instantly derivatized with 150 µL of phosphate buffer (0.5 M, pH 6.5) and

77

150 µL of o-phenylenediamine (0.2%). Analysis was carried out as published previously on a

78

RP-HPLC system with UV detection.5,13

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Isolation of N-(4-butyl-1,2,3-triol-5-oxo-1-imidozolin-2-yl)sarcosine (3-DG-HCr). The

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compound was prepared and isolated according to a literature report for MG-HCr with some

81

modifications.13 Creatine monohydrate (10 mmol) and 3-deoxyglucosone (1 mmol) were

82

heated in 50 mL of 0.1 M sodium phosphate buffer, pH 7.4 at 60 °C for 24 h. After

83

evaporation of the solvent, the residue was dissolved in 5 ml of a mixture of methanol,

84

acetonitrile, and water (5/5/1, v/v/v) and applied to a glass column (3.0 x 20 cm) filled with

85

35 g of silica gel. Elution was performed with 800 mL of a mixture of methanol, acetonitrile,

86

and water (5/5/1, v/v/v) and fractions of 10 mL were collected and characterized by HPLC-

87

MS/MS. Fractions containing the mass to charge ratio [M+H]+ 276.12 were pooled and

88

evaporated. The residue was solved in 10 mL of 0.005 M acetic acid and subjected to anion

89

exchange chromatography using a column (25 x 8 cm) filled with AG Resin 1-X8 in acetate

90

form. Elution was conducted with 150 mL of each 0.005 M, 0.01 M, 0.05 M, and 0.1 M acetic

91

acid. Fractions of 10 mL were collected using a fraction collector (RediFrac, Pharmacia

92

Biotech, Sweden). Again, fractions containing the mass to charge ratio [M+H]+ 276.12 were

93

pooled, dried in vacuo, repeatedly dissolved in water and evaporated, lyophilized, and

94

characterized. This procedure was repeated with

95

creatine monohydrate (2.5 mmol) in 25 mL of 0.1 M sodium phosphate buffer.

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N-(4-butyl-1,2,3-triol-5-oxo-1-imidozolin-2-yl)sarcosine (3-DG-HCr): HPLC-MS/MS: see

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Figure 2 B; elemental analysis: C10H17N3O6 (MW = 275.26), calculated: C 43.82%, H 5.84%,

98

N 15.33%; found: C 33.74%, H 5.63%, N 11.59%; content = 76%, based on nitrogen from

5

13C 6

3-deoxyglucoson (0.25 mmol) and



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elemental analysis and chromatographic purity from LC-MS/MS. Yield = 274 mg (molar

100

yield = 17.5%). NMR data: see Table 1 and supporting information Figure S1–S5.

101

13C

102

analysis:

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found: C 37.56%, H 5.99%, N 14.73%; content = 85%, based on nitrogen from elemental

104

analysis and chromatographic purity from LC-MS/MS. Yield = 140 mg (molar yield =

105

37.5%).

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Characterization of 3-DG-HCr and

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recorded on an Avance III HDX 500 MHz Ascend instrument from Bruker (Rheinstetten,

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Germany) at 500.13 MHz and 125.75 MHz, respectively. 3-DG-HCr (6.9 mg) and 13C6-3-DG-

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HCr (7.1 mg) were each dissolved in 650 µL of deuterium oxide. Assignments of 1H and 13C

110

signals are based on

111

correlation spectroscopy (COSY), 1H-13C heteronuclear single quantum coherence (HSQC),

112

and 1H-13C heteronuclear multiple bond correlation (HMBC) experiments. All chemical shifts

113

are given in parts per million (ppm) relative to external standard tetramethylsilane. Elemental

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analysis was performed on a vario MICRO cube CHNS elemental analyzer (Elementar,

115

Hanau, Germany).

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Preparation of meat samples. Samples were prepared according to a published creatine

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assay with some modifications.23 All samples were obtained from local retail stores and are

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listed in the supporting information (Table S1). Samples were roasted in a normal cooking

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pan in 20 g of sunflower oil. Beef and pork samples (ca. 0.6 cm thickness) were roasted for 2

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minutes, chicken samples (2-3 cm thickness) and sausages were roasted for 6 minutes. Those

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roasting procedures were comparable to typical household methods and resulted in ‘well

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done’ products suitable for consumption. Raw and roasted samples were homogenized with a

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hand blender (Guzzini, Recanati, Italy) after cooling. A portion of homogenized meat (ca. 0.2

6-N-(4-butyl-1,2,3-triol-5-oxo-1-imidozolin-2-yl)sarcosine 12C 13C H N O 4 6 17 3 6

13C

(13C6-3-DG-HCr): elemental

(MW = 281.21), calculated: C 45.01%, H 5.71%, N 15.00%;

13C

6-3-DG-HCr.

1H

and

13C

NMR spectra were

distortionless enhancement by polarization transfer (DEPT), 1H-1H

6


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g) was defatted three times with 1.5 mL of n-heptane. Samples were mixed by vortex,

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centrifuged (2 700 x g) and the n-heptane was discarded. After adding 1.5 mL of 0.05 N

126

hydrochloric acid and 10 µL of an internal standard (0.09 mmol/L

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mmol/L D3-MG-HCr, 11.44 mmol/L D3-creatine, 13.26 mmol/L D3-creatinine), samples were

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mixed and stored for 1 h at 4 °C for extraction. Samples were centrifuged (10 600 x g) and a

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portion (250 µL) was added to 750 µL of acetonitrile for protein precipitation. After 1 h at -18

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°C samples were centrifuged (10 600 x g, 10 min) and subjected to HPLC-MS/MS.

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Hydrolysis. For enzymatic hydrolysis, homogenized meat samples were lyophilized and

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samples containing 2–3 mg of protein (ca. 4 mg of lyophilized meat) were dissolved in 1.05

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mL of 0.02 M hydrochloric acid containing 42 U of pepsin.24 After 24 h incubation in a

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drying chamber at 37°C, 300 µL of 2 M TRIS buffer, pH 8.2 containing 400 PU of pronase E

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was added. Following 24 h of further incubation, 22 µL of 2 M TRIS buffer containing 1 U of

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prolidase and 0.2 U of leucine-aminopeptidase was added. Samples were frozen after 48 h at

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37°C. Centrifugation (10 600 x g, 10 min) of thawed samples was performed prior to HPLC

138

analysis. An aliquot of the hydrolysate (150 µL) was suspended in 450 µL of the loading

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buffer for amino acid analysis (0.12 N lithium citrate, pH 2.20). After membrane filtration

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(0.45 µm), samples were subjected to amino acid analysis.

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For acidic hydrolysis, lyophilized samples containing ca. 2 mg of protein were dissolved in 2

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mL of 6 N hydrochloric acid at 110 °C in a drying oven for 23 h. Aliquots of samples were

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evaporated to dryness in vacuo and suspended in HPLC-MS/MS solvent.

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Amino Acid Analysis. To compare the glycation derivatization rate of creatine with that of

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lysine and arginine, amino acid analysis was utilized. Amino acids were analyzed on a PEEK

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column filled with the cation exchange resin LCA K07/Li (150 mm × 4.6 mm, 7 µm) on the

147

amino acid analyzer S 433 (Sykam, Fürstenfeldbruck, Germany) with a gradient program

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utilized previously.21,25 Loading and running buffers for this lithium system were obtained 7


13C

6-3-DG-HCr,

0.29


149

from Sykam (Fürstenfeldbruck, Germany). Following a post-column derivatization with

150

ninhydrin, the absorbance of the effluent was recorded with a two-channel photometer

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simultaneously working at 440 nm and 570 nm, respectively. A commercial amino acid

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mixture was purchased (Sigma-Aldrich, Steinheim, Germany) and used for external

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calibration.

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High-Pressure Liquid Chromatography (HPLC) with Tandem Mass Spectrometric

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Detection. Creatine hydroimidazolones were analyzed as described before13 with minor

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modifications on a high pressure gradient system 1200 Series (Agilent Technologies,

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Böblingen, Germany), consisting of a binary pump, an online degasser, an autosampler, a

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column oven, and a diode array detector. Chromatographic separation was performed on a

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SeQuant ZIC-HILIC column (100 x 2.1 mm, 3.5 μm, 100 Å, Merck), with 2.5 mM

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ammonium acetate in water/acetonitrile 25:75, v/v (solvent A) and 5.56 mM ammonium

161

acetate in water/acetonitrile 84:16, v/v (solvent B) as the mobile phases. The injection volume

162

was 5 µL. Analysis was conducted at 30 °C using a flow rate of 0.2 mL/min with the starting

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conditions of 0% B. This solvent composition was held for 11 minutes, increased to 80% B

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within 1 minute, held at 80% B for 3 minutes, and finally decreased to 0% B within 5

165

minutes. The HPLC was coupled to a mass spectrometer 6410 Triple Quad (Agilent,

166

Böblingen, Germany) working in the positive mode with a source temperature of 350 °C and

167

a capillary voltage of 4000 V. Nitrogen was used as the nebulizing gas (nitrogen generator

168

5183-2003, Agilent) with a flow rate of 11 L/min and a pressure of 35 psi. The multiple-

169

reaction monitoring mode (MRM) was used with the conditions listed in Table 2. Protein-

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bound AGEs were analyzed as described before25–28 on the same system on a Zorbax SB C-18

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column (50 mm x 2.1 mm, 3.5 µm, Agilent), with 10 mM NFPA in double-distilled water

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(solvent A) and 10 mM NFPA in acetonitrile (solvent B) as the mobile phases. The injection

173

volume was 2 µL. Analysis was conducted at 35 °C using a flow rate of 0.25 mL/min. A

174

gradient of 5% B (0 min) to 32% B (15 min), then to 85% B (16–19 min), and finally to 5% B 8


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(20 min) was used. An equilibration time of 8 min before the next run was implemented. The

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MRM transitions are listed in Table 2. Data acquisition and evaluation was conducted with

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the software Mass Hunter B.02.00 (Agilent, Böblingen, Germany).

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While creatine hydroimidazolones were quantitated by stable isotope dilution analysis, AGEs

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were quantitated by the standard addition method with three runs. For each run, 100 µL of

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enzymatic hydrolysate was mixed either with 20 µL of water, or with 10 µL of water and

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10 µL of a standard solution, or with 20 µL of standard solution. The standard solution

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contained pyrraline (1.68 µg/mL), CML (1.96 µg/mL), CEL (1.86 µg/mL), and MG-H1 (3.83

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µg/mL) dissolved in water. Limits of detection (LOD) and quantitation (LOQ) for the

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compounds are shown in Table 3.

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Statistical Treatment. All samples were analyzed in triplicate. Limits of detection (LOD)

186

and quantitation (LOQ) were calculated as the concentrations of compound necessary to show

187

peaks at signal-to-noise ratios of 3 and 10, respectively. Normal distribution was tested with

188

the Shapiro Wilk test. Most groups were not normally distributed with α of 0.05, therefore

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median and range were used for statistical considerations. Comparisons of medians between

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food groups were conducted using the Mann-Whitney U test.

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Results and discussion

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Model incubations and Isolation of 3-Deoxyglucosone-derived Hydroimidazolone of

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Creatine (3-DG-HCr). The aim of this study was to examine the reaction of 3-DG with

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creatine and to quantitate hydroimidazolones of creatine in meat. Therefore, 3-DG was

195

incubated with creatine (10 fold excess) for 48 h at 37 °C and pH 7.4 to simulate plasma

196

conditions and for a comparison with literature.13 As shown in Figure 3, the concentration of

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3-DG decreased, while a new peak at the mass 275.11 ([M+H]+ = 276.12) emerged. The

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reaction of 3-DG was significantly slower than the reaction of MGO.13 While 14% of 3-DG

199

were still detectable after 24 h of incubation, approximately the same amount of MGO was

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present under the same conditions after 1 h.13

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The major reaction product of a similar incubation mixture of creatine and 3-DG heated at 60

202

°C for 24 h was isolated via flash chromatography and semi preparative ion exchange

203

chromatography. The characterization was conducted with HPLC-MS/MS and 1H and

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NMR experiments. A 3-DG derived hydroimidazolone equivalent to MG-HCr, namely N-(4-

205

butyl-1,2,3-triol-5-oxo-1-imidozolin-2-yl)sarcosine (3-DG-HCr), was found.

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spectra were similar to spectra of MG-HCr.13 However, the methyl group signal for C-7 was

207

replaced by a signal for a methylene group. Additionally, three signals for two methine and

208

one methylene group were found, each with chemical shifts for 13C representing carbon atoms

209

with hydroxyl groups. Each carbon had two assigned 13C signals except carbons 6, 9 and 10

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representing two tautomeric forms in a ratio of ca. 60:40, which aligns with the ratio of the

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two MG-HCr tautomers.13 The tautomers are presumably similar to MG-HCr13 and MG-H129

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tautomers with tautomeric double bonds in the hydroimidazolone ring. The most abundant

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tautomer should have a double bond between C-4 and the N between C-4 and C-5 (Figure 1)

214

and the second most abundant tautomer between C-4 and the N between C-4 and C-6. For

215

quantitation, isotopically labeled 3-DG-HCr was prepared from an incubation mixture of 10


1H

and

13C

13C

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13C -3-DG. 6

216

creatine and

Yields and purity of both substances were sufficiently high for

217

chromatographic analysis.

218

3-DG-HCr was also found in the model incubations of 3-DG and creatine under physiological

219

conditions as mentioned above (Figure 3). In comparison to the formation of MG-HCr from

220

MGO and creatine, the formation of 3-DG-HCr from 3-DG and creatine was delayed. After

221

48 h of incubation time, ca. 2.9% of 3-DG had reacted to 3-DG-HCr, while 75% of MGO had

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reacted to MG-HCr after 24 h.13 Therefore, not only the decrease of 3-DG, but also the

223

formation of the hydroimidazolone of 3-DG was slower in comparison to MGO. Thus, MGO

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has a higher reactivity towards creatine than 3-DG, which was also proven for their reactivity

225

towards arginine30 and aminoguanidine.31 We could therefore show that 3-DG and creatine

226

react in model incubations and we isolated the main reaction product 3-DG-HCr.

227

Quantitation of MG-HCr and 3-DG-HCr in Meat, Fish and Meat Products. A method to

228

quantitate MG-HCr and 3-DG-HCr as well as creatine and creatinine in meat and fish

229

products was established, based on LC-MS/MS and the use of isotopically labeled internal

230

standards. A protocol for creatine analysis23 was adjusted for the sample preparation. After

231

homogenizing the meat, samples were extracted with 0.05 N hydrochloric acid and

232

deproteinized with acetonitrile. MG-HCr and 3-DG-HCr analysis was performed via an

233

HILIC-HPLC-MS/MS method used previously for MG-HCr13 with minor changes. Product

234

ion spectra and MRM spectra of meat samples were compared to those of standard solutions

235

(Figure 2 A-D). Transitions that were both selective and abundant were chosen for

236

quantitation (Table 2). Stable isotope dilution analysis was used for quantitation (Figure 2 E,

237

F). LODs and LOQs were sufficiently low to enable quantitation of all analytes in most

238

samples (Table 3). However, LODs and LOQs for 3-DG-HCr and MG-HCr in chicken were

239

much higher than in the other products because of interfering matrix peaks. Thus,

240

quantification of 3-DG-HCr in chicken was only possible in few samples. Recovery 11



241

experiments were carried out in raw meat products because they contained no detectable

242

analytes. After spiking the samples, they were worked up identically to other samples. Good

243

recoveries were achieved with 95.9% for MG-HCr and 93.7% for 3-DG-HCr for different

244

concentrations. Thus, the performance parameters of the method were sufficient to analyze

245

most samples.

246

Using the established method, MG-HCr and 3-DG-HCr were quantitated in different meat,

247

fish and meat product samples. In raw meat, only minor amounts of MG-HCr and 3-DG-HCr

248

were quantifiable, while the amounts of the compounds increased during roasting (Figure 2E,

249

F, Table 4). As shown in Table 4, up to 28.3 mg/kg MG-HCr and up to 15.3 mg/kg 3-DG-HCr

250

were found in different roasted meat types and meat products. The characterization of all

251

samples and the concentrations of the analytes in the samples are listed in Table S1 in the

252

supporting information. Within each meat group, samples were similar in thickness, water

253

content, fat content, and protein content and were roasted equally long. Nevertheless, the

254

amounts of MG-HCr and 3-DG-HCr varied over a range of one order of magnitude within

255

groups (Table 4). Contents of MG-HCr and 3-DG-HCr did not correlate with concentrations

256

of creatine, creatinine, fat, or protein of the raw meat. To analyze the influence of different

257

roasting levels, pork steaks from one packet were roasted separately for two or six minutes,

258

resulting in medium and well done steaks, respectively. In pork roasted for 2 min, 2.4 ± 0.5

259

mg/kg of MG-HCr and trace amounts of 3-DG-HCr were formed, while pork roasted for 6

260

min contained 8.0 ± 1.3 mg/kg MG-HCr and 5.0 ± 0.2 mg/kg 3-DG-HCr. Thus, the degree of

261

heat treatment influences the contents of MG-HCr and 3-DG-HCr. Slight variations in

262

thickness, heat transmission, fat distribution and other parameters could therefore explain

263

differences in the concentrations of the substances within one meat group. Different

264

concentrations of substances which may form 1,2-dicarbonyl compounds, like glucose,

265

glycolysis intermediates and glycogen, could also contribute to the varying amounts of 3-DG-

12


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266

HCr and MG-HCr. Because of the high intragroup variations, MG-HCr and 3-DG-HCr

267

concentrations between groups, namely beef, pork, fish, chicken and sausages were not

268

significantly different.

269

While the contents of MG-HCr and 3-DG-HCr did not differ significantly between groups,

270

the ratio of the two substances varied between different food groups. Concentrations of MG-

271

HCr were significantly higher than 3-DG-HCr concentrations in the samples. Higher

272

concentrations of MG-HCr could result from the higher reactivity towards creatine of MGO

273

in comparison to 3-DG as shown above and from higher formation of MGO than of 3-DG. As

274

shown in Figure 4, the molar ratio of MG-HCr to 3-DG-HCr in roasted beef and pork samples

275

was around 3 to 1 (ranging between 1.7 to 1 and 6.4 to 1). Contrary to the small range of the

276

ratios in pork and beef, in sausage products the molar ratio was between 0.08 to 1 and 12.6 to

277

1 (Figure 4A). The analyzed sausages were produced using different heat treatments and also

278

microbial maturing. Additionally, reducing sugars which are precursors of MGO and 3-DG

279

were added to the recipe of some sausages, and many products were smoked. All of these

280

parameters could lead to a higher variation of the ratio between MG-HCr and 3-DG-HCr

281

(Figure 4A). To further analyze the influence of smoking, we examined smoked and non-

282

smoked fish. Smoked fish samples had a large surplus of MG-HCr, with molar ratios of MG-

283

HCr to 3-DG-HCr ranging from 4.3 to 1 up to 167 to 1 (Figure 4B). In comparison, non-

284

smoked fish samples had significantly lower molar ratios between 0.2 to 1 and 15 to 1. In

285

wood smoke MGO was detectable32 and MGO from smoke could explain high concentrations

286

of MG-HCr in smoked fish. Therefore, the ratio of MG-HCr to 3-DG-HCr depends on the

287

production of the product, such as a possible smoking process.

288

Based on this quantitative data, a rough estimate of the daily intake of MG-HCr and 3-DG-

289

HCr was made. With an assumed uptake of 200 g meat per day (average in Germany, 2016),33

290

the intake of MG-HCr is ca. 6.4 µmol (1.2 mg) and ca. 1.4 µmol (0.39 mg) of 3-DG-HCr. In 13



Page 14 of 32

291

comparison, the daily urinary excretion of MG-HCr was found to be 0.1 to 3.8 µmol MG-

292

HCr.

293

which extent MG-HCr is absorbable. This will be a subject of further research.

294

Maillard Reaction Products in Meat, Fish and Meat Products. To evaluate the formation

295

of other protein-bound AGEs, N-ε-carboxymethyllysine (CML), N-ε-carboxyethyllysine

296

(CEL), MGO-derived hydroimidazolone 1 (MG-H1), and pyrraline were quantitated in raw

297

and roasted pork and beef samples. An established method using enzymatic hydrolysis

298

followed by HPLC-MS/MS was utilized.25–28 The rate of release after enzymatic hydrolysis

299

compared to the rate of acid hydrolysis was ca. 80% for CML and CEL. As shown in Figure

300

5, CML contents per kg dry matter were similar in raw and roasted meat samples, while the

301

contents of CEL, MG-H1 and pyrraline increased during roasting. Contents of CML (8.5–

302

20.9 mg/kg), MG-H1 (3.6–11.3 mg/kg) and CEL (2.8–6.3 mg/kg) were in similar ranges as

303

described in the literature.34 To our knowledge, this is the first time that pyrraline was

304

quantitated in roasted meat (3.0–11.8 mg/kg).

305

In the same samples, MG-HCr (3.0–7.8 mg/kg) and 3-DG-HCr (0.7–5.2 mg/kg) were

306

quantitated. To compare the derivatization rate of creatine with lysine and arginine, the

307

concentration rates of the reaction products were calculated per mole of the respective amino

308

compound. About 0.61 mmol MG-HCr per mol creatine were formed (range: 0.08–1.9

309

mmol/mol), while CEL accounted for ca. 0.09 mmol per mol lysine (range: 0.04–0.15

310

mmol/mol) and MG-H1 for 0.37 mmol per mol arginine (range: 0.12–0.73 mmol/mol). Thus,

311

the derivatization rate with MGO of creatine is higher than of lysine and arginine. Equally, 3-

312

DG derivatized creatine to a higher degree (0.27 mmol 3-DG-HCr per mol creatine, range:

313

traces–0.27) than lysine (0.13 mmol pyrraline per mol lysine, range: 0.03–0.26).

314

supports the hypothesis that creatine scavenges 3-DG and MGO and protects protein to some

315

extent from the formation of AGEs.

13

Therefore intake and excretion are in similar ranges, which raises the question to

14


This

Page 15 of 32


316

In conclusion, we could synthesize a new hydroimidazolone of creatine and 3-DG and

317

establish a method to quantitate MG-HCr and 3-DG-HCr in meat, fish, and meat products.

318

We could show, that hydroimidazolones of creatine form during roasting. Up to 28.3 mg/kg

319

MG-HCr and up to 15.3 mg/kg 3-DG-HCr were found in meat, fish, and meat products. The

320

concentrations were dependent on the heat treatment and presumably on the smoking process.

321

In comparison to the lysine and arginine derivatives CEL, pyrraline, and MG-H1, the

322

derivatization rate of creatine with MG-HCr and 3-DG-HCr was higher than of lysine and

323

arginine which demonstrates 1,2-dicarbonyl scavenging properties of creatine in meat.

324

Abbreviations used

325

3-DG, 3-deoxyglucosone; 3-DG-HCr; 3-DG derived hydroimidazolone of creatine; AGE,

326

advanced

327

carboxymethyllysine; HPLC, high pressure liquid chromatography; LOD, limit of detection;

328

LOQ, limit of quantitation; MG-H1, methylglyoxal-derived hydroimidazolone 1; MG-HCr,

329

methylglyoxal-derived hydroimidazolone of creatine; MRM, multiple reaction monitoring;

330

MS, mass spectrometry; NFPA, nonafluoropentanoic acid; RP, reversed-phase

331

Acknowledgments

332

We are grateful to the members of the Chair of Inorganic Molecular Chemistry (Prof. J.J.

333

Weigand), namely Dr. Kai Schwedtmann, for recording the NMR spectra and Phillip Lange

334

for the elemental analysis. We thank Karla Schlosser, Chair of Food Chemistry, for

335

performing the amino acid analysis.

336

Notes

337

The authors declare no competing financial interest.

glycation

end

product;

CEL,

N-ε-carboxyethyllysine;

338 15


CML,

N-ε-


339

References

340

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341

phosphates: investigation using a specific assay for methylglyoxal. Eur. J. Biochem.

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Page 21 of 32


Figures Figure 1. Formation of MG-HCr and 3-DG-HCr from the reaction of creatine with methylglyoxal and 3-deoxyglucosone, respectively.

Figure 2. Product ion spectra of MG-HCr (a, c) and MRM spectra of 3-DG-HCr (b, d) obtained during HPLC-MS/MS analysis of standard solutions (a, b) and a meat sample (c, d). HPLC-MS/MS chromatogram of MG-HCr (e) and 3-DG-HCr (f) and their isotopically labeled standards in raw, medium roasted (2 min) and well done roasted (6 min) pork.

Figure 3. Model incubation of 3-DG in the presence and absence of creatine (10 fold excess) at 37 °C and pH 7.4. (A) 3-DG analyzed after derivatization with o-phenylenediamine via RPHPLC-UV. (B) Formation of a new peak with the mass to charge ratio transition 276  170 analyzed via HPLC-ESI-MS/MS. (C) 3-DG-HCr concentration quantitated with HPLC-ESIMS/MS.

Figure 4. Molar ratio of MG-HCr to 3-DG-HCr in different types of meat and meat products (A) and different fish products (B), contents of 3-DG-HCr between LOD and LOQ were calculated as mean of LOD and LOQ, contents under LOD as LOD/2.

Figure 5. Content of the protein-bound AGEs N-ε-carboxymethyllysine (CML), N-εcarboxyethyllysine (CEL), methylglyoxal-derived hydroimidazolone 1 (MG-H1) and pyrraline (Pyrr) in raw (black diamonds, left) and in roasted meat (gray diamonds, right) after enzymatic hydrolysis, analyzed via HPLC-MS/MS with standard addition, in mg/kg dry matter (DM).

21



Page 22 of 32

Table 1.13C and 1H NMR Spectroscopic Data of N-(4-butyl-1,2,3-triol-5-oxo-1-imidozolin-2yl)sarcosine (3-DG-HCr) C Atom δC [ppm]

δH [ppm]

1H-1H

1H-13C

COSY

HMBC

coupling

coupling

-

-

-

-

-

1; 3; 4

-

-

2; 4

-

J(x,y) [Hz]

(figure 1)

1

A 173.12 (Ci);

-

B 172.81 (Ci)a 2

A 54.84 (CH2); A 4.01 (s); B 53.74 (CH2

3

4

B 3.99(s)

A 37.00 (CH3); A 3.11(s); B 38.39 (CH3)

B 3.13 (s)

A 158.96 (Ci);

-

-

B 158.43 (Ci) 5

A 179.63 (Ci);

-

-

6

A+B

A + B 4.50 (t)b

6.4

7

4; 5; 7; 8

7.1/6.4

6

5; 6; 8; 9

3.8/7.1

9

7; 9; 10

8

7; 8; 10

57.66(CH)b 7

8

9

A 32.77 (CH2); A 2.01(dd); B B 34.01 (CH2)

1.90 (dd)

A 67.34 (CH);

A 3.69 (dt); B

B 68.11 (CH)

3.79(dt)

A + B 74.59

A + B 3.50(m)b

(CH)b 10

A + B 62.37

A + B 3.51 (s)b

(CH2)b [a] A and B represent tautomers [b] signals not resolved

22


9

Page 23 of 32


Table 2. Transitions Recorded during MRM Measurement of AGEs in Meat, Fish and Meat Products transition

fragmentor

collision

energy dwell

voltage [V]

[eV]

[ms]

time Q/q[a]

MRM for creatine and creatine derivatives 3-DG-HCr

13C

6-3-DG-

276 → 170

120

20

70

Q

276 → 132

120

20

70

q

282 → 172

120

20

70

Q

282 → 132

120

30

70

q

186 → 87

90

20

70

Q

186 → 44

90

30

70

q

189 → 90

90

20

70

Q

189 → 143

90

13

70

q

132 → 90

75

10

200

Q

132 → 44

75

20

200

q

135 → 93

90

10

200

Q

135 → 47

90

20

200

q

114 → 86

105

10

70

Q

114 → 44

105

14

70

q

117 → 89

105

10

70

Q

117 → 47

105

14

70

q

HCr

MG-HCr

D3-MG -HCr

Creatine

D3-Creatine

Creatinine

D3-Creatinine

MRM for lysine and arginine derived AGEs CML

205 → 84

100

20

100

Q

205 → 130

100

10

100

q

23



CEL

MG-H1

Pyrraline

Page 24 of 32

219 → 84

100

20

100

Q

219 → 130

100

10

100

q

229 → 114

90

20

100

Q

229 → 166

90

20

100

q

255 → 175

75

10

100

Q

255 → 148

75

10

100

q

[a] Q, transition used for quantitation; q, transition used for the confirmation of the presence of the analyte.

24


Page 25 of 32


Table 3. Performance Parameters for the HPLC-MS/MS Method LOD [mg/kg]a

LOQ [mg/kg]a

recovery [%]b

rel. interday repeatability [%]c

otherd

chicken otherd

chicken

MG-HCr

0.05

0.33

0.17

1.09

3-DG-HCr

0.49

2.84

1.63

9.50

pork

fish

95.9 ± 1.5

6.1

5.8

93.8 ± 7.8

1.5

9.1

[a] Limits of detection (LOD) and limits of quantitation (LOQ) were calculated from the signal-to-noise ratio [b] Recovery was determined by addition of various concentrations of hydroimidazolones (1.0–4.7 µg) to raw pork samples before extraction [c] Coefficient of variation in percent of the relative interday repeatability achieved by working up a roasted pork and a tuna fish sample on four different days and measuring on four different days [d] beef, pork, fish and sausage samples are included in ‘other’.

25



Page 26 of 32

Table 4. Amounts of MG-HCr and 3-DG-HCr in Different Food Items in mg/kg food product

n

MG-HCr

3-DG-HCr

range

median

range

median

Raw meat

25

nd–0.90

tr

nd–tr

nd

Roasted pork

9

2.6–19.5

5.2

tr–13.9

2.8

Roasted beef

9

1.1–13.0

7.7

tr–6.7

3.3

Roasted chicken

7

4.3–28.3

6.2

nd–9.5

nd

Sausages

17

tr–22.4

3.6

nd–15.3

tr

Smoked fish

5

0.7–27.5

6.0

nd

nd

Non-smoked fish

5

tr–10.8

7.8

nd–6.0

tr

nd, not detected (below LOD); tr, traces (between LOD and LOQ)

26


Page 27 of 32


Figures Fig. 1 (one-column figure)

27



Fig. 2 (two-column figure)

28


Page 28 of 32

Page 29 of 32


Fig. 3 (two-column figure)

29



Fig. 4 (one-column figure)

30


Page 30 of 32

Page 31 of 32


Fig. 5 (one-column figure)

31



TOC graphic

32


Page 32 of 32

Studies on the Formation of 3-Deoxyglucosone- and Methylglyoxal

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