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Creatine is a scavenger for methylglyoxal under physiological conditions via formation of N-(4-methyl-5-oxo-1-imidazolin-2-yl)sarcosine (MG-HCr) Jürgen Löbner, Julia Degen, and Thomas Henle J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf505998z • Publication Date (Web): 06 Feb 2015 Downloaded from http://pubs.acs.org on February 18, 2015

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Journal of Agricultural and Food Chemistry

Creatine is a scavenger for methylglyoxal under physiological conditions via formation of N-(4-methyl-5-oxo-1-imidazolin-2yl)sarcosine (MG-HCr)

Jürgen Löbner*, Julia Degen*, Thomas Henle**

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

*J.L. and J.D. contributed equally to this work

**Corresponding author: T. Henle Tel.: +49-351-463-34647 fax: +49-351-463-34138 email: [email protected]

1 ACS Paragon Plus Environment


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ABSTRACT

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Following incubation of methylglyoxal and creatine under physiological conditions, N-(4-

3

methyl-5-oxo-1-imidazolin-2-yl)sarcosine (MG-HCr), was isolated and identified by NMR

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and mass spectrometry. Due to its rapid formation, MG-HCr represents a specific product

5

following “scavenging” of methylglyoxal by creatine. Using hydrophilic interaction chroma-

6

tography coupled to mass spectrometry, MG-HCr was analyzed in urine samples of healthy

7

volunteers. Daily MG-HCr excretion of non-vegetarians ranged from 0.35 to 3.84 µmol/24 h

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urine (median: 0.90 µmol/24 h urine) and of vegetarians from 0.11 to 0.31 µmol/24 h urine

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(median: 0.19 µmol/24 h urine), indicating that formation of MG-HCr in vivo is influenced by

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the dietary intake of creatine. The trapping of methylglyoxal by creatine may delay the for-

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mation of advanced glycation compounds in vivo and, therefore, could be of special im-

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portance in situations in which the body has to deal with pathophysiologically increased

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amounts of dicarbonyl compounds (“carbonyl stress”), for instance in diabetic patients.

14 15

Keywords:

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Methylglyoxal, creatine, dicarbonyl compounds, glycation, carbonyl stress, diabetes, meat


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INTRODUCTION

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Methylglyoxal (MG, Fig 1) is a highly reactive 1,2-dicarbonyl compound, which is formed in

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vivo mainly via spontaneous degradation of triosephosphates, thus resembling a by-product of

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glycolysis in cellular metabolism.1,2 For healthy subjects, plasma levels of MG ranging from

21

0.06 to 0.13 µmol/L3,4 were reported. Enhanced plasma concentrations of MG and other 1,2-

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dicarbonyl compounds such as glyoxal and 3-deoxyglucosone were measured for diabetic and

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uremic patients.5 Substantial amounts of dicarbonyl compounds are also formed in food dur-

24

ing heating or storage,6 but at present, there is no evidence that dietary MG or

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3-deoxyglucosone, respectively, contribute to the “dicarbonyl load” of the human body.7,8

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1,2-Dicarbonyl compounds are precursors for protein modifications known as advanced gly-

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cation endproducts (AGEs), which are discussed to play a pivotal role in the pathophysiology

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of several diseases as well as biological ageing.9 In this context, Miyata introduced the term

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“carbonyl stress”.10 Within the field of nephrology, 1,2-dicarbonyl compounds are classified

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as “uremic toxins”.11,12 Recent studies point to the fact that increased levels of endogenously

31

formed methylglyoxal are linked to neurological and neurodegenerative disorders such as

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schizophrenia, Morbus Alzheimer or Morbus Parkinson, respectively.

33

was shown for patients with type 2 diabetes mellitus that increased modification by MG ac-

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celerates the degradation of high-density lipoprotein and impairs its functionality in vivo. This

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mechanism could contribute to an increased risk for cardiovascular disease.15

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Considering the pathophysiological role of glycation in vivo, pharmacological strategies to

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inhibit the formation of AGEs in the course of the physiological Maillard-Reaction were in-

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vestigated.16 Aminoguanidine (AG) is able to trap the dicarbonyl compounds through the

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formation of triazines and thus can delay the formation of AGEs on proteins.17 In animal ex-

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periments, the administration of large amounts of aminoguanidine could prevent the late con-


13,14

Very recently, it


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sequences of diabetes, but clinical studies on diabetic patients had to be terminated due to

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adverse side effects.18

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The physiological role of creatine (CR, Fig.1) is mainly linked to energy metabolism. Crea-

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tine kinase (CK) catalyzes the reversible reaction of CR to phosphocreatine (PCr) consuming

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ATP. The CK/PCr system is a buffer for short-term energy supply in tissues and cells with

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high and rapidly changing energy consumption, like skeletal and heart muscles (up to 94% of

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total body CR) and brain cells.19 A male person weighing 70 kg has a total CR pool of ap-

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proximately 120 g, from which 2% per day are non-enzymatically converted to creatinine at a

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constant rate. Creatinine is rapidly excreted via the kidneys into the urine. This daily loss of

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CR of about 2 g/day has to be replaced either endogenously by de novo synthesis or exoge-

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nously with the diet.20

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Meat contains CR in amounts of 4 to 5 g/kg,21 indicating that for non-vegetarians, approxi-

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mately half of the daily requirement of creatine is covered by the diet. Vegetarians solely de-

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pend on the endogenous synthesis to replace excreted creatine. Concentrations of CR in serum

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and erythrocytes of subjects regularly consuming meat were significantly higher when com-

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pared to vegetarians (male subjects, serum 41 ± 19 vs. 25 ± 19 µmol/L, erythrocytes 370 ± 72

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µmol/L vs. 270 ± 41 µmol/L).22

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Studies point to an active transport of CR from plasma into erythrocytes, but the biological

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role of CR in erythrocytes remains enigmatic.23 Dietary supplementation with large doses of

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CR is common, particularly for athletes, but scientific data pointing to ergogenic effects are

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still ambiguous.24,25 In this context it is noteworthy that CR is intensively discussed in recent

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years as a therapeutic supplement for patients suffering from muscle, neuron and brain disor-

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ders.26,27,28 To date, however, no explanations of possible effects of CR on a molecular level

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have been provided.


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Creatine shows structural analogy to aminoguanidine and arginine referring to the guanidine

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moiety (Fig. 1). The reaction between MG and aminoguanidine or arginine, respectively, is

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well characterized. MG-H1 (methylglyoxal-derived hydroimidazolone 1, Fig. 1) is a quantita-

68

tively important AGE, resulting from the reaction of MG with the guanidino side chain of

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peptide-bound arginine.2 To the best of our knowledge, however, there are no studies investi-

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gating the reaction between MG and CR. We, therefore, hypothesized that CR should react

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with MG under physiological conditions to CR-specific dicarbonyl adducts similar to peptide-

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bound arginine.

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Following incubation experiments in order to prove the carbonyl-trapping activity of CR, a

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hydromidazolone as a creatine-specific methylglyoxal adduct was isolated and quantified in

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urine samples of healthy volunteers. Considering the relatively high plasma levels of CR in

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comparison to MG, we postulate that CR, formed endogenously and/or applied via dietary

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sources, may constitute a “natural” trapping agent for MG in vivo.

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MATERIALS AND METHODS

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Chemicals and Materials.

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Creatine monohydrate was from Applichem (Darmstadt, Germany), creatinine was from

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Merck (Darmstadt, Germany), ammonium acetate was from Grüssing (Filsum, Germany),

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acetic acid was from Roth (Karlsruhe, Germany), ion exchange material AG Resin 1-X8 was

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from Bio-Rad (Hercules, CA), o-phenylendiamine, silical gel (63−200 µm particle size) and

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methylglyoxal (40% solution in water) were from Sigma-Aldrich (Steinheim, Germany),

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HPLC gradient grade acetonitrile and methanol was from VWR Prolabo (Leuven, Belgium),

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LC-MS Grade acetonitrile was from Fisher (Loughborough, UK). All other chemicals were

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purchased from standard suppliers and were of the highest purity available. Water for prepara-

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tion of buffers and solutions was obtained with a Purelab plus purification system (USFilter,

90

Ransbach-Baumbach, Germany). Water for solutions used for LC-MS/MS analysis was dis-

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tilled twice in the presence of potassium permanganate. Artificial urine was prepared accord-

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ing to literature.29

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Urine Samples.

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Eight healthy, non-vegetarian volunteers (age 21−25 years, 5 women and 3 men) and 7

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healthy, vegetarian volunteers (age 21−23 years, 5 women and 2 men, 5 ovo-lacto-vegetarian

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and 2 vegan) had to collect their 24 h urine (8.00 a.m. until 8.00 a.m. the following day) for

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one day and had to record a nutritional protocol. All participants were normoglycemic (fasting

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blood sugar ≤ 5.5 mmol/L) and were of normal body weigth (body-mass-index ≤ 25.0 kg/m2).

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In an orienting intervention study, one healthy, non-vegetarian volunteer (32 years, female)

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followed a controlled ovo-lacto-vegetarian diet for 7 days (wash-out period). At the last day

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(day 1 of the intervention study), 24 h urine was collected (8.00 a.m. until 8.00 a.m. the fol-

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lowing day). Vegetarian diet was continued for further 7 days (day 2−8), but additionally 2 g

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of creatine (“Creapure”, creatine monohydrate, Olimp Laboratories, Dębica, Poland) were 6 ACS Paragon Plus Environment

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consumed daily after lunch (supplementation period). At the last day of the supplementation

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period (day 8), again 24 h urine was collected.

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Isolation of N-(4-methyl-5-oxo-1-imidazolin-2-yl)sarcosine (MG-HCr).

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12.5 mmol creatine monohydrate and 12.5 mmol methylglyoxal in 125 mL of 0.1 M sodium

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phosphate buffer, pH 7.4, were incubated for 2 d at 60 °C. To remove non reacted creatine via

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flash chromatography, the incubation mixture was dried in vacuo and the residue dissolved in

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5 mL of a mixture of acetonitrile/methanol/water (6/4/2, v/v/v). This was applied to a glass

111

column (dimensions 3.0 x 30 cm) filled with 70 g silica gel with particle size 63-200 µm. Elu-

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tion was performed with 1400 mL of a mixture of acetonitrile/methanol/water (6/4/2, v/v/v).

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The fractions were characterized by HPLC-UV-MS/MS and fractions eluting between 140

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and 460 mL containing a peak with the mass transition 186 -> 87 were pooled. Solvents were

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removed under vacuum. To remove further byproducts, the residue was subjected to anion

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exchange chromatography. A column (2.5 x 8 cm) filled with AG Resin 1-X8 in acetate form

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was used and eluted with 300 mL each of 0.01, 0.05, 0.10, 0.15 and 0.20 M acetic acid. The

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fraction size was 10 mL and a fraction collector (RediFrac, Pharmacia Biotech, Uppsala,

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Sweden) with a flow rate of 0.5 mL/min was used. Fractions eluting with 0.1 M acetic acid

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containing a peak with the mass transition 186 -> 87 were pooled, dried under vacuum,

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washed with destilled water, dried by lyophilization and characterized.

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N-(4-methyl-5-oxo-1-imidazolin-2-yl)sarcosine (MG-HCr): ESI-TOF-MS, positive mode,

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[M+H+] m/z 186.09; UV spectroscopy: λmax (1.0 mM ammonium acetate, pH 5.5, acetoni-

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trile/water, 75/25, v/v) 226 nm (log ε = 4.09); Elemental analysis: C7H11N3O3 (MW =

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185.18), calculated, C 45.40%, H 5.99%, N 22.69%, O 25.92%; found, C 41.65%, H 6.28%,

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N 20.11%; content = 88.6%, based on nitrogen (traces of acetate and methanol were visible in

127

NMR spectra). Yield = 52 mg (molar yield = 2.0%).



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Model Incubations under Physiological Conditions.

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Solutions of MG (10 mM) and CR (100 mM), respectively, in phosphate buffer (100 mM, pH

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7.4) were prepared. 5 mL of the CR solution was mixed with 5 mL of the MG solution in

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glass tubes resulting in a 10 fold excess of CR. The mixtures were incubated at 37 °C and

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aliquots were taken at defined times (0 h, 1 h, 2 h, 4 h and 24 h). For the analysis of CR, cre-

133

atinine and MG-HCr, 200 µL aliquots were immediately frozen until analysis. For analysis of

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MG, aliquots of 500 µL were instantly used for derivatization and pipetted into vials contain-

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ing 150 µL phosphate buffer (500 mM, pH 6.5) and 150 µL o-phenylendiamine solution (1%

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in phosphate buffer). Derivatization was done overnight for 16 h under exclusion of light.30

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The incubations were performed in triplicate. As control, MG was incubated in the absence of

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CR and CR in the absence of MG, respectively, to monitor stability of the compounds during

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the incubation.

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Analysis of N-(4-methyl-5-oxo-1-imidazolin-2-yl)sarcosine (MG-HCr), Creatine and

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Creatinine in Model Incubations and Urine Samples by HPLC-ESI-DAD-MS/MS.

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The HPLC system consisted of a degasser, a pump, an autosampler and a diode array detector,

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all from Agilent Technologies 1200 Series (Boeblingen, Germany). For MS/MS-

144

measurements, a Triple Quad LC/MS 6410 from Agilent Technologies was used. For separa-

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tion a column SeQuant ZIC-HILIC (3.5 µm, 100 Å, PEEK 150 x 2.1 mm; Merck KGaA,

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Darmstadt, Germany) was used. Elution was performed at 25 °C with a flow rate of 0.2

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mL/min with following starting conditions: 100% solvent A (1.0 mM ammonium acetate in

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water/acetonitrile 25:75, v/v, pH 5.5) and 0% solvent B (4.65 mM ammonium acetate in wa-

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ter/acetonitrile 70:30 v/v, pH 6.8). This solvent composition was held for 11 minutes (isocrat-

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ic elution) followed by an increase of solvent B to 100% within 1 min and held for 8 min,

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followed by an equilibration at starting conditions for 11 min. Detection wavelengths were


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214, 226 and 236 nm for CR, MG-HCr and creatinine, respectively. For analysis of model

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incubations 1 µL and for analysis of urine samples 5 µL were injected into the HPLC system.

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Single ion monitoring (SIM) was performed from 1 to 11 min in positive mode for m/z 186

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with 200 ms dwell time, fragmentor voltage 115 V, gas temperature 350 °C, gas flow 11

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mL/min and nebulizer pressure 35 psi.

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Product ion scans were recorded from 1 to 11 min with the precursor ions m/z 186 for MG-

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HCr, m/z 132 for creatine and m/z 114 for creatinine, applying the conditions as described

159

above. The ranges of product ions were from set m/z 50 to 200 with a scan time of 200 ms, a

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fragmentor voltage of 115 V and collision energy of 25 eV.

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Measurements of MG-HCr were performed using multiple reaction monitoring mode (MRM)

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in positive mode. The mass transition (186 -> 87) was used for quantitation of MG-HCr with

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fragmentor voltage and collision energy of 115 V and 20 eV, respectively and a dwell time of

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150 ms. For qualification purposes the mass transitions (186 -> 140) and (186 -> 69) were

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monitored.

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In model incubations, MG-HCr was analyzed without further sample preparation except filtra-

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tion (0.2 µm) and dilution (1:1000 with solvent A) as described above for the HPLC-ESI-

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DAD-MS/MS method. Identification of MG-HCr was realized via MS/MS detection and

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quantitation via UV detection (226 nm) with an external calibration using authentic MG-HCr

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reference material (see above).

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Urine samples were centrifuged (4 °C, 10.000 rpm, 10 min) and 250 µL were deproteinized

172

with 750 µL acetonitrile at 4 °C for 20 min. After centrifugation (4 °C, 10.000 rpm, 10 min)

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and filtration (0.2 µm) the solution was employed to standard addition. Quantification via

174

standard addition was necessary to avoid interferences due to matrix effects. Urine samples of

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vegetarians were diluted 3+2 and urine samples of non-vegetarians 1+4 with 10 µL of MG-

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HCr standard solution and 10 or 30 µL of solvent A (1.0 mM ammonium acetate in wa-



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ter/acetonitrile 25:75, v/v, pH 5.5), respectively. For standard addition each urine was spiked

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with four ascending concentrations of MG-HCr resulting in 5 samples per urine including the

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non-spiked urine (instead of MG-HCr standard solution 10 µL water was added). MG-HCr

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stock solution (35 mmol/L) was prepared from the above described synthesized reference

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material. This solution was diluted to 0.70, 0.35, 0.18 and 0.07 µmol/L with water, respective-

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ly. Each was added to the urine samples. For analysis 5 µL of the spiked urine samples were

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injected into the HPLC-ESI-DAD-MS/MS. MG-HCr was detected via MS/MS and creatinine

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via UV at 236 nm. The quantitation of creatinine was realized via external calibration with

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commercially available reference material. Analysis of urine samples were done in duplicate.

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The limits of detection (LOD) and quantification (LOQ) in urine were calculated as the con-

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centrations of the analyte necessary to show a peak at a signal-to-noise ratio of 3 and 10, re-

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spectively. For determination of method precision, one urine sample was applied to replicate

189

analysis on different days (n = 5). Recovery of MG-HCr was calculated from the slope of the

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recovery function after spiking a urine with ascending MG-HCr concentrations (resulting in

191

the same MG-HCr levels as in the standard addition method) before sample work-up (n = 5).

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Quantitation of Methylglyoxal in Model Incubations by RP-HPLC with UV-detection.

193

Analysis of MG in the model incubations was carried out as previously published.30 Derivati-

194

zation with o-phenylendiamine to the corresponding quinoxaline was realized immediately

195

after withdrawal from the reaction mixture to scavenge the residual MG.

196

Mass Spectrometry.

197

For mass-spectrometric analyses, a PerSeptive Biosystems Mariner time-of-flight mass spec-

198

trometry instrument equipped with an electrospray ionization source (ESI-TOF-MS, Applied

199

Biosystems, Stafford, USA) working in the positive mode was used. Calibration of the mass

200

scale was established using a mixture of bradykinin, angiotensin I, and neurotensin. After


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appropriate dilution with 1% formic acid in 50% acetonitrile, the sample was injected into the

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ESI source by a syringe pump at a flow rate of 5 L/min. Spray Tip Potential, Nozzle Potential,

203

Quadrupol RF Voltage, and Detector Voltage were adjusted to 4812, 80, 1000, and 2400 V,

204

respectively.

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Ultraviolet (UV) Spectroscopy.

206

Absorption spectra were recorded on a Specord S100 diode array spectrophotometer (Carl

207

Zeiss, Jena, Germany) with a wavelength accuracy of ± 1 nm. The compound was dissolved

208

in 1.0 mM ammonium acetate, pH 5.5, in acetonitrile/water (75/25, v/v) at a concentration of

209

100 µM. The maxima and molar coefficient of extinction were calculated using quartz cu-

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vettes 115-QS (Hellma, Müllheim, Germany) of 10.00 mm path length.

211

Nuclear Magnetic Resonance (NMR) Spectroscopy.

212

1

213

Germany) at 400 and 100 MHz, respectively. Substances were resolved in 750 µL deuterium

214

oxide for analysis. Assignments of 1H and 13C signals are based on 1H-1H COSY (correlation

215

spectroscopy), HSQC (heteronuclear single quantum coherence); HMBC (heteronuclear mul-

216

tiple bond correlation), DEPT (distortionless enhancement by polarization transfer) experi-

217

ments. A NOESY (nuclear Overhauser enhancement spectroscopy) experiment for carbon-

218

proton couplings and a HMBC (heteronuclear multiple bond correlation) experiment for ni-

219

trogen-proton couplings were performed in addition to the above-mentioned 2D experiments

220

on a Bruker Avance III instrument at 600 MHz (1H), 150 MHz (13C) and 61 MHz (15N). All

221

chemical shifts are given in parts per million (ppm), those of protons relative to the internal

222

HOD signal (4.80 ppm), those of carbon atoms relative to external standard tetramethylsilane

223

and those of nitrogen atoms relative to external standard urea.

H and 13C NMR spectra were recorded on an AVANCE III instrument (Bruker, Rheinstetten,



224

Elemental Analysis.

225

To know the purity of the isolated compound used as standard material, elemental analysis

226

data were obtained on an Elementar Vario Micro Cube CHNS elemental analyzer (Hanau,

227

Germany). The purity of the isolated compound was calculated as the percentage of nitrogen

228

in the preparation divided by the theoretical percentage of nitrogen of the target substance and

229

expressed in percent by weight.

230

Statistical Treatment.

231

Comparisons of means between the daily urinary excretions of the different days were exam-

232

ined with Student’s t test. P values ≤0.05 were considered significant (two-tailed) using

233

OriginPro 8.6.

234 235

RESULTS AND DISCUSSION

236

Incubation of methylglyoxal in the presence of a tenfold excess of creatine under physiologi-

237

cal conditions resulted in a rapid decrease of the carbonyl compound (Fig. 2 A). After 1 h of

238

incubation at 37 °C, only 13% of the initial concentration could be detected as free MG, and

239

after 24 h nearly complete derivatization of MG was observed. In control samples, MG and

240

CR incubated individually remained stable (Fig. 2 A). No creatinine formation was detectable

241

using HPLC-DAD within 24 h. After prolonged incubation for 4 d, creatinine formation ac-

242

counted for only 2% of the initial CR concentration (data not shown). Therefore, it can be

243

concluded that reactive MG is quantitatively trapped by the guanidino compound CR.

244

From incubation mixtures containing equimolar amounts of MG and CR, the major reaction

245

product was isolated by flash chromatography and semi-preparative ion-exchange chromatog-

246

raphy. For the isolated compound, a relative molecular mass Mr of 185.08 ([M+H]+

247

m/z = 186.09) was determined via ESI-TOF-MS. This corresponds to the addition of one


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molecule of MG to one molecule of CR with the loss of one molecule water and formation of

249

a methylglyoxal-derived hydroimidazolone of creatine, namely N-(4-Methyl-5-oxo-1-

250

imidazolin-2-yl)sarcosine (Fig. 3). The supposed structure for MG-HCr was unequivocally

251

confirmed with 1H NMR and 13C NMR experiments as well as two-dimensional NMR (Tab. 1

252

and Fig. 3). In the 1H NMR and

253

were doubled, indicating two dominating tautomeric forms in a ratio of 40:60 (A:B) based on

254

proton intensities in the 1H spectrum. The signals were assigned to the two tautomers each

255

using the resonance in 1H,1H COSY and HMBC as well as the intensity relation in the 1H ex-

256

periment.

257

Results from 1H NMR showed 4 different signals. The intensities indicated two methyl-, one

258

methylene and one methine group. The singlet of one methyl- and the methylene group were

259

assigned to C-8 and C-9 of the creatine backbone. The second methyl signal was split into a

260

doublet and showed a vicinal coupling to the quartet of the methine group. The 1H,1H COSY

261

experiment showed only one resonance confirming the connection of the methylgroup at C-6

262

and the methine H at C-2. In the

263

signed to three quarternary, one methylene and three methyl- or methine carbon atoms by

264

DEPT measurement. The HSQC experiment confirmed these results and allowed the interpre-

265

tation of C-6 and C-8 as methyl groups, C-2 as methine group and C-9 as methylene group.

266

To confirm the position of the quarternary carbon atoms, a HMBC experiment was accom-

267

plished, which resulted in the assignment shown in Fig. 3. The C-10 of the carboxyl group

268

and the C-5 of the guanidino group are connected to the creatine backbone. By contrast, C-3

269

showed resonance with the protons of C-2 and C-6. Taking the resonance of C-2 and C-6 pro-

270

tons with C-5 into account, the hydroimidazolone substructure is highly likely. In conse-

271

quence of the inflexible heterocyclic hydroimidazolone structure, the NOESY experiment

272

showed only cross peaks of the protons of C-2 with C-6 and C-8 with C-9. An additional 1H-

13

C NMR spectra (except for C-2 in

13

13

C NMR) all signals

C NMR spectra, seven signals appeared, which were as-



273

15

274

1, N-4 and N-7, also verifying the heterocyclic hydroimidazolone structure.

275

As mentioned above, doubled signals indicating two tautomeric forms were observed. At pre-

276

sent, the NMR data do not allow a definite assignment to two of five predictable tautomeric

277

forms of the hydroimidazolone moiety (possible structures are shown in the Supporting In-

278

formation). However, participation of all tautomers considering an enol at C-2/C-3 is implau-

279

sible (compare structure IV and V, Supporting Information), because this would be indicated

280

by the disappearance of the proton signal at C-2 due to a quantitative H-D-exchange in the

281

solvent D2O. For the methylglyoxal-derived hydroimidazolone of arginine, two tautomers and

282

the appearance of the keto-imine and the keto-amine structure in a ratio 2:1 were postulated as

283

well, supported by NMR experiments.31 This points to structure I and II (see Supporting In-

284

formation). Although one report on X-ray analysis of the compound shown in Fig. 3 can be

285

found in the literature,32 to the best of our knowledge, formation of MG-HCr as a specific

286

reaction product from MG and creatine has not been described yet, although the reaction of

287

guanidino moieties with oxoaldehydes is well known.33,34

288

To further characterize the scavenging reaction between MG and CR, samples of different

289

time points were analyzed with regard to the possible formation of MG-HCr. With proceeding

290

incubation time, one peak at mass transition 186 -> 87 increased in the chromatograms (Fig. 2

291

B). This peak could be assigned to the compound MG-HCr by comparing retention time and

292

fragmentation pattern of product ion scan of MG-HCr reference material (see Fig. II in the

293

Supporting Information). Quantitative data obtained for MG-HCr in the incubation mixtures

294

via external calibration are shown in Fig. 2 C. After 24 h, the concentration of MG-HCr in the

295

incubation samples was quantitated with 4.2 ± 0.2 mmol/L (Fig. 2 C). Initial MG concentra-

296

tion (t = 0 h) was 5.6 ± 0.4 mmol/L, indicating that 75% of MG had been reacted with CR to

297

MG-HCr. Compared with the decrease in MG (Fig. 2 A), the formation of MG-HCr (Fig. 2 C)

N HMBC experiment resulted in cross peaks of the proton at C-2 with all nitrogen atoms N-


Page 14 of 36

Page 15 of 36


298

is somewhat delayed. This can be explained with the proposed reaction mechanism (Fig. 4),

299

in which the conversion to the hydroimidazolone in the last reaction step should be rate-

300

determining.35

301

The fast and quantitative scavenging of MG by CR can be compared with the scavenging ca-

302

pacity of other guanidino compounds such as aminoguanidine17 and acetyl arginine.35 At pre-

303

sent, no studies have been performed to investigate a possible reaction between CR and MG

304

under physiological conditions. The reaction between creatinine and reducing carbohydrates

305

was studied regarding the taste modulating effects of the formed compounds in meat. From

306

these reaction mixtures, a MG-derived creatinine compound was isolated and quantitated in

307

urine after meat ingestion.36,37

308

Our results show that CR reacts with MG under physiological conditions resulting in MG-

309

HCr as the main reaction product. The excellent trapping capacity of CR in vitro for the reac-

310

tive oxoaldehyde MG points to a possible biological role of CR in vivo. As described in the

311

introduction section, oral administration of aminoguanidine (AG) was found to suppress path-

312

ophysiological consequences of diabetes in animal experiments via dicarbonyl scavenging,

313

but exhibited adverse side effects in human studies.17,18 Plasma concentrations of AG between

314

10 to 50 µmol/L were necessary to induce pharmacologically relevant effects.38 In this context

315

it is noteworthy that normal serum concentration of CR in men (non-vegetarian) is 41 ± 19

316

µmol/L.22 Therefore, it can be hypothesized that naturally occurring CR may have MG trap-

317

ping effects in vivo, leading to continuous formation and excretion of MG-HCr.

318

To support this hypothesis, a method was established for the quantitation of MG-HCr in urine

319

samples of vegetarians and non-vegetarians, based on hydrophilic interaction chromatography

320

(HILIC) to separate polar compounds.21 Sensitive detection was realized via tandem-mass

321

spectrometry. SIM chromatograms were recorded at m/z 186, the pseudo-molecular ion of

322

MG-HCr ([M+H]+). Using HILIC column material it turned out, that the retention time of



323

MG-HCr was considerably dependent on the ionic milieu of the injected sample solution. For

324

identification in urine samples, MG-HCr reference material was therefore dissolved in artifi-

325

cial urine, thus simulating the average composition of urine including salts, urea and organic

326

acids.29 Assignment of the peak in the SIM chromatogram of a urine sample (Fig. 5 B1) to

327

MG-HCr was confirmed first of all chromatographically by comparing the retention times of

328

the target compound in artificial urine and urine (compare Fig. 5 A1 and B1). Retention time

329

of the main peak in the chromatogram of the urine sample with m/z 186 (4.3 min in Fig. 5 B1)

330

differed slightly from that of the peak in the chromatogram of the MG-HCr reference material

331

(3.9 min in Fig. 5 A1). Depending on the ionic milieu (artificial urine, dilution of the urine)

332

retention time for MG-HCr could vary within 0.5 min on the applied HILIC column. The

333

shifts in retention time of the peak in the urine samples were verified by standard addition

334

(data not shown). Unequivocal identification was accomplished based on the corresponding

335

mass spectra. In Fig. 5 A2 and B2, the product ion pattern after fragmentation (product ion

336

scan) of the peak with m/z = 186 of a MG-HCr standard solution in artificial urine and of a

337

urine sample are displayed, respectively. The two fragmentation patterns were identical and

338

showed the same distinct signals (Fig. 5 A2 and B2). Thus, unambiguous identification of

339

MG-HCr in urine was achieved.

340

With the applied chromatographic system, a sufficient retardation of the polar compound

341

MG-HCr as well as separation from creatine and creatinine was possible (see Fig. III, Sup-

342

porting Information). Standard addition curves in the concentration range of 0.07−0.7 µmol/L

343

showed linearity (R2 > 0.99) for MG-HCr. The limit of detection (LOD) and limit of quantita-

344

tion (LOQ) was calculated from the signal-to-noise ratio. LOD in urine was 0.01 µmol/L and

345

LOQ was 0.05 µmol/L. Method precision, expressed as relative standard deviation of repli-

346

cate analysis of a urine sample (n = 5), was 8.6%. Repeated recovery experiments (n = 5),

347

where urine samples were spiked before sample work-up, showed a recovery of 81.6 ± 5.5%,


Page 16 of 36

Page 17 of 36


348

calculated from the slope of the recovery function. The validation parameters of the standard

349

addition method indicate a reliable method for accurate quantitation of MG-HCr in urine. The

350

developed method should be easily transferred to other biological matrices like for example

351

plasma.

352

Using this method, MG-HCr in 24 h urine samples of 15 healthy volunteers (7 vegetarians, 8

353

non-vegetarians) was analyzed. The participants followed their own individual diet and col-

354

lected their 24 h urine for one day. Data for urinary excretion of MG-HCr are shown in Fig. 6

355

A. Daily MG-HCr excretion of non-vegetarians ranged from 0.35 to 3.84 µmol/24 h urine

356

(median: 0.90 µmol/24 h urine) and of vegetarians from 0.11 to 0.31 µmol/24 h urine (medi-

357

an: 0.19 µmol/24 h urine). The urinary excretion between non-vegetarians and vegetarians

358

differed significantly (P < 0.01). Based on the dietary recordings and taking literature data of

359

CR in food into account,39,40,41 it turned out that the estimated intake of CR with the daily diet

360

of the vegetarians was significantly lower when compared with the non-vegetarians (0−0.3

361

mmol/day vs. 1.3−12.2 mmol/day). Therefore, MG-HCr detectable in urine of vegetarians

362

must be due to a formation in vivo. The higher amount of creatine taken up with the daily diet

363

of non-vegetarians should lead to higher plasma concentrations of the guanidino compound

364

which may trap more MG and thus can induce an increased excretion of MG-HCr in urine

365

samples of non-vegetarians.

366

To further strengthen this hypothesis, urinary excretion of one healthy volunteer (non-

367

vegetarian) was measured after being for 7 days on a vegetarian diet (day 1, Fig. 6 B). This

368

wash-out period was followed by a supplementation period, in which for 7 days 2 g of CR per

369

day were consumed after lunch. This amount of CR corresponds to an ingestion of about 500

370

g meat.21 After 7 days on a vegetarian diet, MG-HCr increased from 0.18 ± 0.03 µmol/24 h

371

urine (day 1, Fig. 6 B), which is in the range of the vegetarians shown in Fig. 6 A, to 0.33 ±

372

0.01 µmol/24 h urine (day 8, Fig. 6 B). This significant increase of MG-HCr excretion follow-



373

ing an oral load with creatine clearly demonstrates that trapping of MG in vivo can be in-

374

creased by dietary means. On the other hand, however, it cannot be excluded that excretion of

375

MG-HCr at least partly can result from an oral uptake of the reaction product with heated

376

meat and subsequent renal excretion. At present, no data about the occurrence of MG-HCr in

377

meat are available. Corresponding studies are currently underway in our laboratory.

378

In conclusion, with the identification of MG-HCr, the specific reaction product formed from

379

MG and CR under physiological conditions and its unambiguous quantitation in urine, we

380

provide evidence for a new physiological role of creatine. Our results suggest that dietary in-

381

take of CR and resulting increased plasma concentrations may provide a natural “safety

382

mechanism” for the trapping of highly reactive dicarbonyl compounds. This mechanism could

383

be of special importance in situations in which the body has to deal with pathophysiologically

384

increased amounts of dicarbonyl compounds (“carbonyl stress”), for instance in diabetic pa-

385

tients. Furthermore, our findings may shed light on the question why erythrocytes accumulate

386

CR, but do not use the guanidino compound for their energy metabolism. As mentioned in the

387

introduction section, concentrations of CR in erythrocytes are about tenfold higher compared

388

to blood serum, and subjects consuming meat have higher concentrations of CR in erythro-

389

cytes and serum compared to vegetarians.22,23 Taking into account that erythrocytes produce

390

energy solely by the glycolysis of glucose and lactic acid fermentation of the resulting py-

391

ruvate,42 significant amounts of MG are continuously formed as by-product, which have to be

392

metabolized, in particular by the glyoxalase system. Additionally, the arginine residues of

393

hemoglobin non-enzymatically trap MG via formation of MG-H1.2 It is conceivable that CR

394

may significantly contribute to the detoxification of MG in particular in erythrocytes. Acting

395

this way, CR could retard the formation of advanced glycation end products (AGEs) and thus

396

delay those long-term consequences of diabetes and ageing, which are commonly attributed to

397

AGE formation in vivo. As another aspect, CR may positively contribute to MG-linked neuro-


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398

logical disorders.13,14 If this hypothesis can be confirmed, dietary intake of CR via meat or

399

supplements may constitute a so far overlooked nutritional benefit.

400 401

Abbreviations Used: AGE, advanced glycation endproduct; AG, aminoguanidine; CK,

402

creatine kinase; CR, creatine; LOD, limit of detection; LOQ, limit of quantification; MG,

403

methylglyoxal; MG-HCr, methylglyoxal-derived hydroimidazolone of creatine , namely N-(4-

404

Methyl-5-oxo-1-imidazolin-2-yl)sarcosine; PCr, phosphocreatine; SIM, selected ion monitor-

405

ing; tR, retention time.

406 407

Acknowledgment

408

We thank Dr. Uwe Schwarzenbolz, Institute of Food Chemistry, for the ESI-TOF-MS meas-

409

urements. We appreciate the support of the members of the Chair of Inorganic Coordination

410

Chemistry (Group of Professor Jan Weigand), namely Sivathmeehan Yogendra, Maximilian

411

Donath and Stephen Schulz, who recorded NMR spectra and provided the elemental analysis.

412

We are grateful to the members of the Institute of Organic Chemistry, namely Dr. Margit

413

Gruner and Dr. Tilo Lübken, for recording further NMR spectra.

414 415

Supporting Information

416

Supporting Information Available: Figure I Possible tautomeric forms of N-(4-Methyl-5-oxo-

417

1-imidazolin-2-yl)sarcosine (MG-HCr). Figure II Identification of MG-HCr by HPLC-ESI-

418

MS/MS in the model incubation of CR and MG. Figure III HPLC-ESI-MS/MS und HPLC-

419

UV chromatograms of a MG-HCr standard and a urine sample. This material is available free of

420

charge via the Internet at http://pubs.acs.org.



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Phillips SA, Thornalley PJ. The formation of methylglyoxal from triose phosphates. Investigation using a specific assay for methylglyoxal. Eur. J. Biochem. 1993, 212, 101-15.

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Rabbani N, Thornalley PJ. Methylglyoxal, glyoxalase 1 and the dicarbonyl proteome. Amino Acids, 2012, 42, 1133–42.

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Henning C, Liehr K, Girndt M, Ulrich C, Glomb MA. Extending the spectrum of αdicarbonyl compounds in vivo. J. Biol. Chem., 2014, jbc.M114.563593. [Epub ahead of print].

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Rabbani N, Thornalley PJ. Measurement of methylglyoxal by stable isotopic dilution analysis LC-MS/MS with corroborative prediction in physiological samples. Nat. Protoc., 2014, 9, 1969–79.

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Ahmed N. Advanced glycation endproducts - role in pathology of diabetic complications. Diabetes Res. Clin. Pr., 2005, 67, 3–21.

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Degen J, Hellwig M, Henle T. 1,2-Dicarbonyl compounds in commonly consumed foods. J. Agric Food Chem., 2012, 60, 7071–9.

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Degen J, Vogel M, Richter D, Hellwig M, Henle T. Metabolic transit of methyglyoxal. J. Agric. Food Chem., 2013, 61, 10253–10260.

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Degen J, Beyer H, Heymann B, Hellwig M, Henle T. Dietary influence on urinary excretion of 3-deoxyglucosone and its metabolite 3-deoxyfructose. J. Agric Food. Chem., 2014, 62, 2449–2456.

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Hellwig M, Henle T. Baking, ageing, diabetes: a short history of the Maillard reaction. Angew. Chem Int. Ed. Engl., 2014, 53, 10316–10329.


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Miyata T, Sugiyama S, Saito A, Kurokawa. Reactive carbonyl compounds related uremic toxicity (“carbonyl stress”). Kidney Int., 2001, 78, S25–S31.

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Henle T, Miyata T. Advanced glycation end products in uremia. Adv. Ren. Replace. Ther., 2003, 10, 321–331.

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Mittelmaier S, Niwa T, Pischetsrieder M. Chemical and physiological relevance of glucose degradation products in peritoneal dialysis. J. Ren. Nutr. 2012, 22, 181–185.

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Arai M, Yuzawa H, Nohara I, Ohnishi T, Obata N, Iwayama Y, Haga S, Toyota T, Ujike H, Ichikawa T, Nishida A, Tanaka Y, Furukawa A, Aikawa Y, Kuroda O, Niizato K, Izawa R, Nakamura. Enhanced carbonyl stress in a subpopulation of schizophrenia. Arch. Gen. Psychiat., 2010, 67, 589–597.

14

Srikanth V, Westcott B, Forbes J, Phan TG, Beare R, Venn A, Pearson S, Greenaway T, Parameswaran V, Münch G. Methylglyoxal, cognitive function and cerebral atrophy in older people. J. Gerontol. A Biol. Sci. Med. Sci., 2013, 68, 68–73.

15

Godfrey L, Yamada-Fowler N, Smith J, Thornalley PJ, Rabbani N. Arginine-directed glycation and decreased HDL plasma concentration and functionality. Nutr. Diabetes, 2014, 4, doi:10.1038/nutd.2014.31.

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Engelen L, Stehouwer CD, Schalkwijk CG. Current therapeutic interventions in the glycation pathway: evidence from clinical studies. Diabetes Obes. Metab., 2013, 15, 677–689.

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Thornalley PJ. Kinetics and mechanism of the reaction of aminoguanidine with the alpha-oxoaldehydes glyoxal, methylglyoxal, and 3-deoxyglucosone under physiological conditions. Biochem. Pharmacol., 2000, 60, 55–65.



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Thornalley PJ. Use of aminoguanidine (Pimagedine) to prevent the formation of advanced glycation endproducts. Arch. Biochem. Biophys., 2003, 419, 31–40.

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Wyss M, Kaddurah-Daouk R. Creatine and creatinine metabolism. Physiol. Rev., 2000, 80, 1107–1213.

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Persky AM, Brazeau G, Hochhaus G. Pharmacokinetics of the dietary supplement creatine. Clin. Pharmacokin., 2003, 42, 557–574.

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Mora L, Sentandreu MA, Toldrá F. Hydrophilic chromatographic determination of carnosine, anserine, balenine, creatine, and creatinine. J. Agric. Food Chem., 2007, 55, 4664–4669.

22

Delanghe J, Deslypere JP, Debuyzere M, Robbrecht J, Wieme R, Vermeulen A. Normal reference values for creatine, creatinine and carnitine are lower in vegetarians. Clin. Chem., 1989, 35, 1802–1803.

23

Jiao Y, Okumiya T, Saibara T, Tsubosaki E, Matsumura H, Park K, Sugimoto K, Kageoka T, Sasaki M. An enzymatic assay for erythrocyte creatine as an index of the erythrocyte life time. Clin. Biochem., 1998, 31, 59–65.

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Branch CD. Effect of creatine supplementation on body composition and performance: a meta-analysis. Int. J. Sport Nutr. Exerc. Metab., 2003, 13, 198–226.

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Brosnan JT. Creatine: endogenous metabolite, dietary, and therapeutic supplement. Annu. Rev. Nutr., 2007, 27, 241–261.

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Kley RA, Tarnopolsky MA, Vorgerd M. Creatine for treating muscle disorders. Cochrane Database Syst Rev., 2013, CD004760.

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Pastula DM, Moore DH, Bedlack RS. Creatine for amyotrophic lateral sclerosis/motor neuron disease. Cochrane Database Syst Rev., 2012, CD005225. 22 ACS Paragon Plus Environment

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Allen PJ. Creatine metabolism and psychiatric disorders: Does creatine supplementation have therapeutic value? Neurosci. Biobehav. Rev., 2012, 36, 1442–1462.

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Brooks T, Keevil CW. A simple artificial urine for the growth of urinary pathogens. Lett. Appl. Microbiol., 1997, 24, 203–206.

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Mavric E, Wittmann S, Barth G, Henle T. Identification and quantification of methylglyoxal as the dominant antibacterial constituent of Manuka (Leptospermum scoparium) honeys from New Zealand. Mol. Nutr. Food Res., 2008, 52, 483–489.

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Henle T, Walter AW, Haeßner R, Klostermeyer H. Detection and identification of a protein-bound imidazolone resulting from the reaction of arginine residues and methylglyoxal. Z. Le-bensm. Unters. Forsch., 1994, 199, 55-58.

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Shima K, Ishikawa K, Izawa K, Suzuki E. Crystal Structure of N-(4-Methyl-5-oxo-1imidazolin-2-yl)sarcosine Monohydrate. Anal. Sci., 1998, 14, 1185–1186.

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Sopio R, Lederer M. Reaction of 3-deoxypentosulose with N-methyl- and N,Ndimethylguanidine as model reagents for protein-bound arginine and for creatine. Z. Le-bensm. Unters. Forsch., 1995, 201, 381–386.

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Thornalley PJ, Battah S, Ahmed N, Karachalis N, Agalou S, Babaei-Jadidi R, Dawnay A. Quantitative screening of advanced glycation endproducts in cellular and extracellular proteins by tandem mass spectrometry. Biochem. J., 2003, 375, 581–592.

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Lo TW, Westwood ME, McLellan AC, Selwood T, Thornalley PJ. Binding and modification of proteins by methylglyoxal under physiological conditions. J. Biol. Chem., 1994, 269, 32299–322305.

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Kunert C, Walker A, Hofmann T. Taste modulating N-(1-methyl-4-oxoimidazolidin2-ylidene) α-amino acids formed from creatinine and reducing carbohydrates. J. Agric. Food Chem., 2011, 59, 8366–8374.



37

Kunert C, Skurk T, Frank O, Lang R, Hauner H, Hofmann T. Development and application of a stable isotope dilution analysis for the quantitation of advanced glycation end products of creatinine in biofluids of type 2 diabetic patients and healthy volunteers. Anal. Chem., 2013, 85, 2961–2969.

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Nyengaard JR, Chang K, Berhorst S, Reiser KM, Williamson JR, Tilton RG, Discordant effect of guanidines on renal structure and function and on regional vascular dysfunction and collagen changes in diabetic rats. Diabetes, 1997, 46, 94–106.

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Balsom PD, Söderlund K, Ekblom B. Creatine in Humans with Special Reference to Creatine Supplementation. Sports Med., 1994, 18, 268–80.

40

Harris RC, Lowe JA, Warnes K, Orme CE (1997). The concentration of creatine in meat, offal and commercial dog food. Res. Vet. Sci., 1997, 62, 58–62.

41

Del Campo G, Gallego B, Berregi I, Casado JA (1998). Creatinine, creatine and protein in cooked meat products. Food Chem., 1998, 63, 187–190.

42

van Wijk R, van Solinge, WW. The energy-less red blood cell is lost: erythrocyte enzyme abnormalities of glycolysis. Blood, 2005, 106, 4034–4042.


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421

FIGURE CAPTIONS

422

Fig. 1. Structures of 1,2-dicarbonyl compound methylglyoxal (MG), of the advanced gly-

423

cation end product methylglyoxal-derived hydroimidazolone (MG-H1) and the guanidino

424

compounds aminoguanidine (AG) and creatine (CR).

425 426

Fig. 2. (A) Physiological model incubations of methylglyoxal in the presence of creatine and

427

MG control sample. MG was analyzed after derivatization with o-phenylendiamine via RP-

428

HPLC-UV.30 Incubations were done in triplicate. (B) Formation of a new peak in the incuba-

429

tion of MG with CR with the transition 186 -> 87 analyzed by HPLC-ESI-DAD-MS/MS. (C)

430

MG-HCr concentration in the incubation of MG and CR. MG-HCr was quantitated via

431

HPLC-ESI-DAD-MS/MS at UV = 226 nm.

432 433

Fig. 3. Structural formula of N-(4-methyl-5-oxo-1-imidazolin-2-yl)sarcosine (MG-HCr). Ar-

434

rows indicate the relevant (A) carbon-proton or (B) nitrogen-proton long-range connectivities

435

from HMBC spectra.

436 437

Fig. 4. Proposed reaction mechanism for the reaction of creatine (CR) with methylglyoxal

438

(MG) to N-(4-Methyl-5-oxo-1-imidazolin-2-yl)sarcosine (MG-HCr) via the schiff base and

439

the dihydroxyimidazolidine. Formation of the hydroimidazolone of creatine should be rate

440

determining.35Error! Bookmark not defined.

441 442

Fig. 5 Identification of MG-HCr by HPLC-ESI-MS/MS. SIM chromatograms of the synthe-

443

sized MG-HCr reference material in artificial urine29 (A1) and a urine sample (B1) at

444

m/z = 186, respectively. Product ion patterns after fragmentation of the peak at 3.9 min of the

445

MG-HCr standard (A2) and the peak at 4.3 min of the urine sample (B2) with m/z = 186. 25 ACS Paragon Plus Environment


446 447

Fig. 6 (A) 24 hour urinary excretion of MG-HCr of 8 non-vegetarians and 7 vegetarians

448

healthy volunteers. Comparisons of means were examined using Student’s t test. **, P < 0.01

449

(significant). (B) Urinary excretion of MG-HCr into 24 h urine of 1 person (non-vegetarian)

450

during a creatine intervention study. Day 1: after 7 days of vegetarian diet (wash-out period),

451

day 8: after 7 days of creatine supplementation (2 g daily after lunch). Error bars give the

452

standard error of the mean (n = 2)


Page 26 of 36

Page 27 of 36


TABLES Tab. 1 15N, 13C and 1H NMR Spectroscopic Data and Results of 2D NMR Experiments of N(4-methyl-5-oxo-1-imidazolin-2-yl)sarcosine (MG-HCr), A and B Represent Tautomers. Assignment of

δN/ppm or

N or C-atom

δC/ppmb

δH/ppm

(Fig. 3) 1a

1

H-1H

1

H-13C

1

H-15N

1

H-13C

COSY

HMBC

HMBC

NOESY

coupling

couplingd

couplingd

couplingd

6

3; 5; 6

1; 4; 7

6

A -278.6; B -277.2

2

A+Bc

pseudo

56.09

quintet:

(CH)

A 4.47 (q, 1H, J = 7.2 Hz); B 4.49 (q, 1H, J = 7.2 Hz)

3

A 179.11 (Ci); B 179.51 (Ci)

4a

A+Bc 303.3

5

A 157.75 (Ci);



Page 28 of 36

B 158.18 (Ci) 6

A+Bc

A 1.48

15.63 +

(d, 3H, J

15.67

= 7.2

(CH3)

Hz);

2

2; 3

1

2

5; 9

1; 7

9

5; 8; 10

1; 7

8

B 1.50 (d, 3H, J = 7.2 Hz) 7a

A+Bc 297.0

8

A 38.35

A 3.22

(CH3);

(s);

B 37.10

B 3.18 (s)

(CH3) 9

A 53.62

A 4.08

(CH2);

(s);

B 54.71

B 4.11 (s)

(CH2) 10

A 172.41 (Ci); B 172.90 (Ci)

a 15N signal dedicated from HMBC coupling. b Type of signal deduced from 13C DEPT measurement is given in parentheses.


Page 29 of 36


c These signals are not resolved d Couplings from the corresponding hydrogen atoms are denoted.



FIGURES Figure 1 (one-column) 453


Page 30 of 36

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Figure 2 (two-column)



Figure 3 (one-column)


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Figure 4 (two-column) 454



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TOC graphic

457


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Creatine Is a Scavenger for Methylglyoxal under ... - ACS Publications

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