Article pubs.acs.org/JAFC
Dietary Influence on Urinary Excretion of 3‑Deoxyglucosone and Its Metabolite 3‑Deoxyfructose Julia Degen, Helene Beyer, Björn Heymann, Michael Hellwig, and Thomas Henle* Institute of Food Chemistry, Technische Universität Dresden, D-01062 Dresden, Germany S Supporting Information *
ABSTRACT: 3-Deoxyglucosone (3-DG), a reactive 1,2-dicarbonyl compound derived from D-glucose in food and in vivo, is an important precursor for advanced glycation endproducts (AGEs). At present, virtually no information about the metabolic transit of dietary 3-DG is available. One possible metabolic pathway of 3-DG during digestion is enzymatic transformation to less reactive compounds such as 3-deoxyfructose (3-DF). To study the handling of dietary 1,2-dicarbonyl compounds by the human body, 24 h urinary excretion of 3-DG and its metabolite, 3-deoxyfructose, was investigated. Urinary 3-DG and 3-DF excretion was monitored for nine healthy volunteers following either a diet with no dietary restrictions or a diet avoiding the ingestion of 3DG and other Maillard reaction products (“raw food” diet). During the “raw food” diet, the urinary 3-DG and 3-DF excretion decreased approximately to 50% compared to the excretions during the diet with no restrictions. When subjects received a single dose of wild honey (50 g) naturally containing a defined amount of 3-DG (505 μmol), median excretion of 3-DG and 3-DF increased significantly from 4.6 and 77 to 7.5 and 147 μmol/day, respectively. The obtained experimental data for the first time demonstrate a dietary influence on urinary 3-DG and 3-DF levels in healthy human subjects. KEYWORDS: 3-deoxyglucosone, 3-deoxyfructose, glycation, Maillard reaction, GC-MS, urinary excretion, metabolic transit
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INTRODUCTION 3-Deoxyglucosone (3-DG) is a reactive 1,2-dicarbonyl compound derived from D-glucose. In food, 3-DG is formed nonenzymatically in the course of the Maillard reaction and in caramelization processes.1−3 3-DG in vivo can also originate from fructose-3-phosphate or from the phosphorylated Amadori compound fructosamine-3-phosphate via spontaneous decomposition.4,5 The concentration of 3-DG in the plasma of diabetic subjects ranges between 0.08 and 0.50 μmol/L and is about twice as high as the concentration in the plasma of healthy subjects (0.04−0.16 μmol/L).6−9 The occurrence of 3DG in a variety of foods was recently published.10,11 High contents were measured in balsamic vinegar (up to 16 mmol/ L), honey (10 mmol/kg), and bakery products such as bread and cookies (up to 3.8 mmol/kg). The dietary intake of 3-DG was estimated to range from 0.1 to 1 mmol (20−160 mg) per day.11 3-DG rapidly reacts with lysine and arginine side chains of proteins to form advanced glycation endproducts (AGEs), such as the lysine-derived pyrraline and the arginine derivative “3deoxyglucosone-derived hydroimidazolone” (3-DG-H, Figure 1). Both compounds have already been detected in vivo, for example, in long-lived proteins such as lens crystalline and collagen.12−14 As the plasma concentration of 3-DG is elevated under diabetic conditions, the dicarbonyl compound may play an important role in the development of diabetic complications.15 Elevated levels of 3-DG and other 1,2-dicarbonyl compounds in plasma (and, in consequence, accumulation of AGEs) are also discussed to contribute to long-term complications of uremia.16 In this context, the term “carbonyl stress” was introduced.17 However, both reductive and oxidative mechanisms exist in vivo to metabolize endogenous 3-DG to less reactive © 2014 American Chemical Society
compounds. Aldehyde reductase (EC 1.1.1.2) and aldose reductase (EC 1.1.1.21) both reduce 3-DG NADPH-dependently at the aldehyde group to 3-deoxyfructose (3-DF, Figure 1).18−20 The enzymes are present in all organs, with high expression in the kidneys. Recently, the small intestine aldose reductase (ALKR1B10) was identified as a novel member of the aldose reductase family. ALKR1B10 is primarily expressed in the human colon and small intestine.21,22 Another mechanism for detoxification of 3-DG is oxidation via aldehyde dehydrogenase ALDH1A1 (NAD+-dependent) and 2-oxoaldehyde-dehydrogenase (NADP+-dependent) to 3-deoxy-2-ketogluconic acid. ALDH1A1 was identified as a specific 3-DG oxidizing enzyme in erythrocytes (Figure 1).23 3-Deoxyfructose is the main urinary metabolite of 3-DG, and concentrations between 32 and 92 μmol/g creatinine can be measured in the urine of normoglycemic persons and elevated concentrations (54−196 μmol/g creatinine) in the urine of diabetic patients. In plasma, 3-DF accounts for 85% and in urine for 99% of the summarized amounts of 3-DG and 3-DF,7,24,25 indicating the effective enzymatic transformation of 3-DG to 3-DF. On the basis of recently published contents of 3-DG in commonly consumed foods10,11 and the possible role of 3-DG in diabetic complications, questions may arise concerning eventual health risks resulting from the dicarbonyl compound ingested with the daily diet. Only very limited information is available concerning the bioavailability of 3-DG and its metabolic fate during digestion. No information about the Received: Revised: Accepted: Published: 2449
December 11, 2013 February 28, 2014 March 1, 2014 March 1, 2014 dx.doi.org/10.1021/jf405546q | J. Agric. Food Chem. 2014, 62, 2449−2456
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Figure 1. Degradation reactions of 3-deoxyglucosone: (I) enzymatic transformation of 3-deoxyglucosone to 3-deoxyfructose via aldehyde and aldose reductase and (II) to 3-deoxy-2-ketogluconic acid via 2-oxoaldehyde dehydrogenase and ALDH1A1; (III) nonenzymatic reaction of 3deoxyglucosone with protein-bound arginine to the advanced glycation end product 3-DG-H (3-deoxyglucosone derived hydroimidazolone) and (IV) reaction with protein-bound lysine to form pyrraline.
Figure 2. Synthesis of 3-deoxy-D-erythro-hexulose (3-DF): (I) acetone, H2SO4, 25 °C, 1.5 h; (II) THF, 1,1′-thiocarbonyldiimidazole, 66 °C, 3 h; (III) toluene, tributyltin hydride, 110 °C, 1 h; (IV) acetone/water (1:1, v/v), Lewatit S100, 25 °C, 24 h. Synthesis of 3-Deoxy-D-erythro-hexulose (3-Deoxyfructose). Protocols of synthesis were adopted with minor modifications.27−29 Eighteen grams of D-fructose (1, Figure 2) was suspended in 350 mL of acetone containing 0.5% sulfuric acid and mechanically stirred at room temperature (under exclusion of moisture) until the sugar was dissolved (about 1.5 h). Termination of the reaction was carried out by adding 50 mL of ice-cold sodium hydroxide solution (c = 2.75 mmol/ L) to the mixture while stirring was continued. Acetone was evaporated in vacuo, and the remaining aqueous phase (reddish brown) was extracted with methylene chloride (3 × 100 mL). The combined extracts were washed with water (2 × 50 mL) and dried over sodium sulfate. After evaporation of the organic solvent, the crude reaction product was recrystallized twice from ethanol/water 1:4 (v/v) and from ether followed by pentane. At 6 °C complete crystallization was achieved overnight. After drying, 5.1 g of long white needles of the diacetal sugar 2 (1,2:4,5-di-O-isopropylidene-D-fructopyranose, Figure 2) was obtained (20.2% from compound 1). Solid 1,1′-thiocarbonyldiimidazole (2.2 g) was added to 1.5 g of the partially protected sugar, dissolved in 30 mL of tetrahydrofuran, and heated under reflux (66 °C) until complete conversion to the O-(imidazolylthiocarbonyl) derivative (about 3 h, monitored with thin layer chromtography).28 The target product was isolated from the evaporated reaction mixture by flash chromatography (stationary phase: silica gel 60, mesh 0.063− 0.200 mm) with ethyl acetate/hexane (1:1, v/v) as mobile phase, eluting between 80 and 260 mL. Drying of the combined fractions yielded 1.9 g of the alkoxythiocarbony1imidazolide derivative (85% from 2), which was completely employed for the following reduction step. The product was dissolved in 50 mL of anhydrous toluene, which was added dropwise within 30 min to a boiling tributyltin hydride/toluene solution (2.3 g in 200 mL of anhydrous toluene). After completion of reduction (about 1 h), the cooled solvent was evaporated in vacuo, and the dry residue was extracted with hot acetone (3 × 50 mL); the
metabolic transit of 3-DG in healthy humans is available up to now. The aim of our study, therefore, was to examine the contribution of dietary 3-DG on the “3-DG-load” of the human body. To obtain information about absorption and metabolism of 3-DG, urinary elimination of 3-DG and its major metabolite 3-DF was analyzed in healthy subjects following a diet containing 3-DG and a diet virtually free from 3-DG. During a 3 day intervention study, the influence of a single dose of honey containing a defined amount of 3-DG on the urinary elimination of 3-DG and 3-DF was observed. For this, a sensitive GC-MS for the simultaneous quantification of 3-DG and 3-DF in human urine had to be developed, and both analytes were synthesized as reference material.
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MATERIALS AND METHODS
Materials. Chemicals of the highest purity available were purchased from standard suppliers: 1,1′-thiocarbonyldiimidazole (Sigma-Aldrich, Steinheim, Germany); n-hexane, hydroxylamine hydrochloride (Merck, Darmstadt, Germany); N,O-bis(trimethylsilyl)trifluoroacetamide, D-fructose, methanol (Sigma-Aldrich); methyl-αD-galactopyranoside (Acros, Geel, Belgium); pyridine (anhydrous), tributyltin hydride, urease (type C-3, 12KU) (Sigma-Aldrich). Mixedbed ion exchanger Serdolit MB-2 was from Serva (Heidelberg, Germany), and cation exchanger (Lewatit S100) was from Bayer (Leverkusen, Germany). 3-DG was synthesized as already described26 with some modifications.10 [13C6]3-DG was prepared accordingly starting from [13C6]glucose (99%, Euriso-Top, Saint-Aubin Cedex, France). The water used was obtained with a Purelab plus purification system (USFilter, Ransbach-Baumbach, Germany). 2450
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Table 1. GC-MS Parameters (SIM Mode) for the Determination of 3-Deoxyfructose (3-DF) and 3-Deoxyglucosone (3-DG)
3-deoxyfructose 3deoxyglucosone
internal standard
quantifier ion [m/z]
qualifier ion [m/z]
internal standard quantifier ion [m/z]
internal standard qualifier ion [m/z]
dwell time (ms)
MGP [13C 6]3-DG
191 347
217, 524 373, 537
204 351
217, 133 379, 543
80 (for each measured ion)
combined extracts were washed with hexane (4 × 50 mL) to remove stannous components. Acetone was evaporated, and product isolation was performed by flash chromatography (stationary phase: silica gel 60, mesh 0.063−0.200 mm, mobile phase ethylacetate/hexane 1:1 (v/ v), elution between 80 and 170 ml). After recrystallization from pentane, 0.74 g of the reduced diisopropylidene derivative 3 (Figure 2) was obtained as white needles (71% from 2). 1H nuclear magnetic resonance (NMR) analysis of this product gives a spectrum according to data published in the literature.28 Distinct signals with chemical shifts of δ = 2.1 for the two protons at C3 indicate the successful reduction at C3 (literature δ = 2.00 and 2.06).28 For hydrolysis the complete yield was dissolved in 12.5 mL of acetone and 25 mL of water. Three grams of activated cation exchange resin (Lewatit S100) was added; this mixture was then stirred for 24 h at room temperature. Neutralization was achieved by stirring the solution for another 6 min with the addition of 6 g of a mixed-bed ion exchanger (Serdolit MB-2). The product was purified by flash chromatography (stationary phase as above; mobile phase: methylene chloride/ethanol/water 2:1:0.1 (v/v/v), elution between 80 and 170 mL). The combined fractions were evaporated, and the residue was taken up in water and lyophilized to yield 0.16 g of 3-DF (4) (Figure 2; 16% from 2). After 6 weeks of storage at −18 °C, complete crystallization was accomplished with small, light yellow crystals. The product was characterized by 1H NMR and 13C NMR, and purity was examined via GC-FID. 1H NMR (500 MHz, D2O) β-pyranose: δH 3.43 (s, 2H, H1A/H1B), 1.78 (m, 2H, H3A/H3B), 3.95 (m, 1H, H4), 3.70 (s, 1H, H5), 3.59 (dd, 1H, H6), 3.86 (dd, 1H, H6). 13C NMR (125 MHz, D2O): δC 97.21 (C-2), 67.05 (C-5), 65.21 (C-1), 64.76 (C-4), 63.81 (C-6), 41.29 (C-3). For purity control of 3-DF, the corresponding trimethylsilyl (TMS) oxime of 3-DF was subjected to GC-FID analysis. Analysis was performed on a Varian 3900 gas chromatograph with a CP-8410 automatic liquid sampler with split injector and flame ionization detection (FID). Separation was performed on a DB-5 capillary column (30 m, 0.25 mm i.d., 1.00 μm film thickness, J&W Scientific, Germany). Helium was the carrier gas with a constant flow at 1.0 mL/ min, injector temperature was 250 °C, and injection mode was 1 μL at 1:20 split. The oven temperature program was adopted from the parameters of the GC-MS analysis. Stock solutions each of 3-DF and D-fructose were prepared in water (c = 1 mM), and calibration curves (0.05−1 mM) of the TMS oximes were recorded with GC-FID. The slope of the calibration curve of 3DF was compared to the slope of the calibration curve of D-fructose, and the ratio was assumed to indicate the purity/content of synthesized 3-DF. Intervention Study. Nine healthy, normoglycemic volunteers (ages 24−36 years, 7 women and 2 men, nonsmoking, fasting blood sugar ≤5.5 mmol/L) collected their 24 h urine (8.00 a.m. until 8.00 a.m. the following day) on 2 consecutive days with no dietary restrictions (days 1−2). Furthermore, all nine participants took part in a 3 day intervention study consisting of one wash-out day (day 3), one intervention day (day 4), and one run-out day (day 5). From day 3 to day 5, they followed a “raw food” diet, which implies avoiding heated and fermented foods such as dairy and bakery products, coffee, juices, and beer to ensure a diet virtually free of 3-DG and Maillard reaction products. In the morning of the intervention day (day 4), the participants ingested a single dose of 50 g of honey containing a defined amount of 505 μmol of 3-DG (3-DG concentration of the honey was 1641 mg/kg). The “raw food” diet was continued on the fifth day. Twenty-four hour urine was collected every day of the study as described above. Additionally, a placebo study was performed. Two participants (ages 28 and 31 years, respectively, 1 woman and 1 man,
nonsmoking, fasting blood sugar ≤5.5 mmol/L) followed the same 5 day study design as described above, but ingested 50 g of a honey analogue sugar mixture (46.5% D-fructose, 34.5% D-glucose, 1.5% saccharose, and 17.5% water)30 instead of wild honey in the morning of the intervention day (day 4). Aliquots of collected 24 h urine samples were immediately stored at −18 °C until analysis. 3-DG and 3-DF were analyzed within 2 weeks. Preparation of Urine Samples for GC-MS Analysis. After the urine thawed, an aliquot of 100 μL was spiked with 10 μL of internal standard solution (containing 1.0 mg/mL methyl-α-D-galactopyranoside31and 0.1 mg/mL [13C6]3-DG in water) and was then treated with 30 μL of urease (1 mg/mL in 0.2 M sodium phosphate buffer, pH 7.0) for 10 min at 37 °C in a water bath. Termination of the enzymatic reaction was realized by addition of 900 μL of ice-cold ethanol. After centrifugation (10000g, 15 min), the resulting supernatant was transferred to a new tube and evaporated to dryness under a nitrogen stream. Then the dry residue was incubated with 50 μL of hydroxylamine hydrochloride solution (10 mg/mL in anhydrous pyridine) for 30 min at 80 °C followed by evaporation to dryness. Then 50 μL of N,O-bis(trimethylsilyl)trifluoroacetamide was added to the residue and vigorously mixed by vortex for 0.5 min and incubated at room temperature for another 2 h. Subsequently, 50 μL of n-hexane was added; an aliquot of 80 μL was transferred to a GC vial and was then ready for GC-MS analysis. GC-MS Analysis of 3-Deoxyglucosone and 3-Deoxyfructose. GC-MS analysis was performed using a HP6890 gas chromatograph, coupled with a HP7683 automatic liquid sampler and a HP5973 mass selective detector equipped with a HP-5MS capillary column (30 m, 0.25 mm i.d., 0.25 μm film thickness; all from Agilent, Germany) and a Zebron Z-guard column (deactivated, 5 m, 0.25 mm i.d.; Phenomenex, Germany). The carrier gas was helium with a constant flow at 1.0 mL/ min, the injector temperature was 250 °C, and injection mode was 1 μL pulsed splitless. The oven temperature program started at 80 °C (hold time = 2 min) elevated to 250 °C at 10 °C/min (hold time = 3 min) and finally heated to 300 °C at 30 °C/min (hold time = 3 min), resulting in a complete run time of 25 min. Mass spectrometer was used with electron ionization (70 eV) in scan and SIM modes. In scan mode the recorded m/z area range varied from 50 to 800. For detailed SIM parameters see Table 1. For external calibration, stock solutions of the analytes in water (0.06−0.6 mM) were pipetted (1−50 μL) to yield appropriate concentrations along with internal standards. Each calibration sample was subjected to the derivatization procedure after evaporation to dryness (see above). Quantitative evaluation was carried out by the use of the ratio of the area of the most prominent analyte peak and the internal standard peak of the corresponding quantifier ions, despite the occurrence of peaks of syn- and anti-stereoisomers of the analytes. The limits of detection (LOD) and quantitation (LOQ) in standard solution and urine were calculated as the concentrations of the analyte necessary to show a peak at signal-to-noise ratios of 3 and 10, respectively. For the determination of the interday repeatability, one urine sample was applied to the sample workup and derivatization procedure five times on different days. The recovery of 3-DF and 3DG was calculated from the slope of the recovery function after urine had been spiked with three ascending concentrations of the analytes (3-DF, 6−300 μmol/L; 3-DG, 0.6−30 μmol/L) with subsequent sample workup. NMR Spectrometry. 1H and 13C NMR spectra were recorded on a Bruker DRX 500 instrument (Rheinstetten, Germany) at 500 and 125 MHz, respectively. Samples were dissolved in deuterium oxide. Chemical shifts are given in parts per million (ppm), those of protons 2451
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advantageous because the oximes exist in open-chain form,35 leading to only two silylated isomers per aldehyde or keto group, respectively. Compared to the published method7 for the simultaneous determination of 3-DG and 3-DF, this optimized GC-MS method has the key advantage of higher selectivity. This is accounted for by different retention times of the two analytes and distinct m/z ratios (compare Figure 2). In the cited method7 the retention times for both analytes are the same and the m/z ratio is only 1. The preparation of the urine samples prior to derivatization consisted of an enzymatic removal of urea for the analysis of carbohydrates in urine.36 The dry matter of urine contains approximately 23−49% urea,37 which can consume large amounts of the silylating agent and therefore hamper the silylation of the analytes.36 The adopted derivatization method33 was slightly modified in this work, resulting in a reduction of the applied derivatization agents. The application of oxime formation in carbohydrate analysis has been already examined in detail with regard to stability, efficiency, and the number of byproducts formed.38 The amount of N,Obis(trimethylsilyl)trifluoroacetamide (BSTFA) was adjusted to 50 μL per derivatization assay, and derivatization was complete after 2 h at room temperature, based on time course studies. The solvent evaporation after oxime formation is timeconsuming, and the silylation of the analytes in the dry residue is suboptimal compared to a silylation of the analytes in solution. The drying procedure, however, is necessary to ensure a salt concentration as low as possible in the injected sample solution to minimize contamination and signal suppression in the mass selective detector. In Figure 4, a selected ion chromatogram of a 3-DF (at m/z 191) and 3-DG (at m/z 347) standard solution after derivatization with hydroxylamine hydrochloride and BSTFA to the corresponding TMS oximes is shown. For the TMS
relative to the internal HOD signal (4.70 ppm, residual proton), and those of carbon atoms relative to the external standard tetramethylsilane. Assignments of 1H and 13C signals are based on heteronuclear single-quantum coherence (HSQC) and distortionless enhancement by polarization transfer (DEPT) experiments. Statistical Treatment. Comparisons of means between the daily urinary excretions of the different days were examined using Student’s t test. P values ≤0.05 were considered significant (two-tailed) using OriginPro 8.6.
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RESULTS AND DISCUSSION GC-MS Analysis of Urinary 3-Deoxyglucosone and 3Deoxyfructose: Method Evaluation. The first aim of our study was to establish a sensitive and selective method for the simultaneous determination of 3-DG and its metabolite 3-DF in urine. As 3-DF is commercially not available, it had to be synthesized as reference material. This was achieved by the preparation of the 1,2:4,5-diacetal of D-fructose, selective reduction at C3 with tri-n-butylstannane, and finally hydrolysis of the 3-deoxysugar, according to literature methods (compare Figure 2).27−29 The 1H NMR spectrum of the final product 3DF showed several signals due to five tautomeric forms of the deoxysugar in aqueous solution. According to the literature, most signals of the β-pyranoide structure could be assigned, as this is the predominating form of 3-DF in aqueous solution at room temperature. Chemical shifts of the signals are in line with those reported.29 The chemical shift of the protons at C3 (δ = 1.78) of 3-DF are characteristic for the reduction at C3 and prove the identity of 3-DF along with the other signals. Purity control of the synthesized 3-DF via GC-FID32 indicated a purity of 99% when compared to D-fructose. The GC-FID chromatogram indicated no impurities of other sugars or sugar derivatives (data not shown). For the GC-MS analysis of the sugar derivatives, a conversion to volatile compounds is inevitable. The first step of the derivatization is the formation of the corresponding oximes, followed by the conversion to the TMS derivatives (Figure 3).33 With regard to the 10 isomeric forms of 3-DG34 and the 5 tautomeric forms of 3-DF,29 the formation of sugar oximes is
Figure 4. Selected ion monitoring chromatogram of (a) a 3deoxyglucosone 3-DG)/3-deoxyfructose (3-DF) standard solution (m/z 347 and 191), (b) a urine sample at m/z 191, and (c) a urine sample at m/z 347. All chromatograms were acquired by GC-MS after derivatization with hydroxylamine hydrochloride and N,O-bis(trimethylsilyl)trifluoroacetamide to the corresponding trimethylsilyl oximes.
Figure 3. Structures of the trimethylsilyl oxime derivatives of (A) 3deoxyglucosone and (B) 3-deoxyfructose with their corresponding nominal masses. 2452
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Figure 5. (A) EI-Mass spectra of the peak eluting at 18.5 min of a 3-deoxyglucosone (3-DG) standard solution and a urine sample (scan mode). (B) EI-Mass spectra of the peak eluting at 17.3 min of a 3-deoxyfructose (3-DF) standard solution and a urine sample (scan mode). All spectra were acquired by GC-MS after derivatization with hydroxylamine hydrochloride and N,O-bis(trimethylsilyl)trifluoroacetamide to the corresponding trimethylsilyl oximes.
Table 2. Performance Parameters of the GC-MS Method for the Determination of 3-Deoxyfructose (3-DF) and 3Deoxyglucosone (3-DG) in Urine 3-deoxyfructose 3-deoxyglucosone
R2
linear range (μmol/L)
interday repeatabilitya (%)
LODb (nmol/L)
LOQb (nmol/L)
recoveryc (%)
0.9998 0.9980
6−300 0.6−30
1.5 1.0
52 ± 4 20 ± 4
155 ± 11 59 ± 13
101.8 ± 3.8 97.4 ± 4.2
a Expressed as relative standard deviation. bLODs (limit of detection) and LOQs (limit of quantification) were calculated on the basis of signal-tonoise-ratio. cRecovery was determined by addition of various concentrations of 3-DF (60−300 μmol/L, n = 3) and 3-DG (0.6−30 μmol/L, n = 3) to 100 μL of urine, applying all steps of sample preparation and analysis. The value was calculated from the slope of the recovery function and is given in percent ± SE.
are displayed. The two spectra show the same distinct signals at the mass-to-charge ratio of the quantifier ion (m/z 347) and the qualifier ion (m/z 537). The mass spectra (scan mode) of the peak eluting at 17.3 min of a 3-DF standard solution and a urine sample after derivatization to their corresponding TMS oximes are presented in Figure 5B, having consistent signals at m/z 191 (quantifier ion) and at m/z 524 (qualifier ion). Comparison of the mass spectra of the peaks of the two corresponding analytes recorded from a derivatized standard solution and a urine sample allows a doubtless and unequivocal identification of 3-DG and 3-DF in urine. In Table 2, validation parameters of the method for the determination of 3-DG and 3-DF in urine are summarized. They indicate an accurate sample preparation, a reproducible derivatization procedure, and a reliable GC-MS analysis. On the basis of the relative interday repeatability being 0.05). 3-DG accounted for 4−8% (mean = 6%) of the summarized 3DG and 3-DF amounts. This is in line with published data,7 which show that 3-DF is the main metabolite in urine accounting for >95% of 3-DG. The daily urinary apparent 3DF excretions are consistent with values published in the literature for healthy subjects (32−92 μmol/g creatinine),7,26 when a daily excretion of 1.1−1.9 g creatinine is taken as a 2454
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In conclusion, it was possible for the first time to reveal differences in urinary 3-DG and 3-DF elimination in healthy human subjects, dependent on the diet. These results are a prerequisite for further studies concerning metabolic pathways of 3-DG in human subjects.
the average increase of the urinary 3-DG excretion was 3.0 μmol/day (range = 0.4−3.6 μmol/day) and that of the 3-DF excretion was 70 μmol/day (range = 30−123 μmol/day). This corresponds with approximately 10−15% of the ingested amount of 3-DG being recovered in the urine. Slightly lower excretion rates into urine were observed in rats, but it is obvious that results of animal experiments could be only partly transferred to humans and the study design lasted only 2 h.18 Median 3-DG and 3-DF excretions on the run-out day (day 5) did not differ significantly from those of day 3 (P > 0.05), indicating a rapid elimination of intestinally absorbed 3-DG (compare Figure 6). Despite the intake of a high amount of 3DG on the intervention day (day 4), the median ratios of the urinary 3-DG/3-DF excretion did not vary significantly compared to the other days (see Supporting Information). In further studies it might be of interest to investigate interindividual differences of the excreted 3-DG/3-DF ratios to have further insights into possible variations of the metabolic handling, for example, during diabetes. To rule out that the observed increase in urinary excretion of 3-DF on day 4 is due to the high sugar load and a possible formation of 3-DG from glucose or fructose in vivo, a placebo study was performed. Two volunteers consumed 50 g of a honey analogue sugar mixture virtually free of 3-DG instead of 3-DG-containing wild honey on the intervention day (data and figure shown in the Supporting Information). Urinary excretion of 3-DG and 3-DF remained constant, indicating that precursors such as fructose or glucose do not contribute to a measurable 3-DG formation in vivo or an increased urinary 3DG and 3-DF excretion, respectively. Hence, the elevation of the urinary 3-DF excretion on the intervention day of the intervention study was exclusively due to the 3-DG consumed via the wild honey. These experimental findings for the first time prove an influence of dietary 3-DG on urinary excretion of 3-DG and 3DF. The results imply that dietary 3-DG is to some extent taken up from the ingested food in the intestinal tract, metabolized to 3-DF, and eliminated via urine. This allows the conclusion that detoxification systems for endogenously formed reactive aldehydes, such as aldehyde and aldose reductase,21,22 should also be capable to degrade dietary and therewith exogenous 3-DG. In particular, the recently described small intestine aldose reductase (ALKR1B10), which is described to play a key role in detoxifying dietary and lipid-derived carbonyl compounds,21,22 should be examined in this context. As only 10−15% of the administered 3-DG is recovered in urine as 3DG and 3-DF, the question arises about the fate of the remaining 85−90%. Further studies will have to show whether putative 3-DG-specific metabolites such as 3-deoxy-2-ketogluconic acid or hydroxymethylfurfural (HMF) can be found in the urine. At present, it cannot be excluded that 3-DG is to some extent transformed to HMF due to the low pH value of the stomach. 3-DG may also react with proteins to AGEs in the gastrointestinal tract or in vivo. The analysis of 3-DG and 3-DF in blood plasma plus 3-deoxy-2-keto-gluconic acid and other analytes (i.e., fructoselysine, HMF, pyrraline) of study participants after an oral load of 3-DG would unequivocally reveal if and to what extent 3-DG is absorbed into the systemic circulation and if it is metabolized. On the basis of results of animal experiments, it was concluded that orally ingested 3-DG is absorbed in the intestine to only a minor degree and that the major part remains in the content of the gastrointestinal tract.18 This finding should be examined in a following study.
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ASSOCIATED CONTENT
S Supporting Information *
Figures I and II. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*(T.H.) Phone: +49-351-463-34647. Fax: +49-351-463-34138. E-mail: [email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank Dr. Uwe Schwarzenbolz, Institute of Food Chemistry, Technische Universität Dresden, for his support during the LC-MS measurements. Furthermore, we thank Dr. Margit Gruner and Anett Rudolph, Institute of Organic Chemistry, Technische Universität Dresden, for recording the NMR spectra.
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ABBREVIATIONS USED 3-DF, 3-deoxyfructose; 3-DG, 3-deoxyglucosone; 3-DG-H, 3deoxyglucosone-derived hydroimidazolone; AGE, advanced glycation endproduct; BSTFA, N,O-bis(trimethylsilyl)trifluoroacetamide; FID, flame ionization detector; GC-MS, gas chromatography−mass spectrometry; LOD, limit of detection; LOQ, limit of quantitation; MGP, methyl-α-Dgalactopyranoside; SIM, selected ion monitoring; TMS, trimethylsilyl
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REFERENCES
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(10) Hellwig, M.; Degen, J.; Henle, T. 3-Deoxygalactosone, a “new” 1,2-dicarbonyl compound in milk products. J. Agric. Food Chem. 2010, 58, 10752−10760. (11) Degen, J.; Hellwig, M.; Henle, T. 1,2-Dicarbonyl compounds in commonly consumed foods. J. Agric. Food Chem. 2012, 60, 7071− 7079. (12) Jono, T.; Nagai, R.; Lin, X.; Ahmed, N.; Thornalley, P. J.; Takeya, M.; Horiuchi, S. Nε-(Carboxymethyl)lysine and 3-DGimidazolone are major AGE structures in protein modification by 3deoxyglucosone. J. Biochem. 2004, 136, 351−358. (13) Hayase, F.; Nagaraj, R. H.; Miyata, S.; Njoroge, G.; Monnier, V. M. Aging of proteins: immunological detection of a glucose-derived pyrrole formed during Maillard reaction in vivo. J. Biol. Chem. 1989, 263, 3758−3764. (14) Miyata, S.; Liu, B. F.; Shoda, H.; Ohara, T.; Yamada, H.; Suzuki, K.; Kasuga, M. Accumulation of pyrraline-modified albumin in phagocytes due to reduced degradation by lysosomal enzymes. J. Biol. Chem. 1997, 272, 4037−4042. (15) Ahmed, N. Advanced glycation endproducts − role in pathology of diabetic complications. Diabetes Res. Clin. Pract. 2005, 67, 3−21. (16) Henle, T.; Miyata, T. Advanced glycation end products in uremia. Adv. Renal Replace. Ther. 2003, 10, 321−331. (17) Miyata, T.; Ueda, Y.; Yamada, Y.; Izuhara, Y.; Wada, T.; Jadoul, M.; Saito, A.; Kuro-kawa, K.; Van Ypersele de Strihou, C. Accumulation of carbonyls an advanced stress in uremia accelerates the formation of glycation end product: carbonyl stress in uremia. J. Am. Soc. Nephrol. 1998, 9, 2349−2356. (18) Kato, H.; van Chuyen, N.; Shinoda, T.; Sekiya, F.; Hayase, F. Metabolism of 3-deoxyglucosone, an intermediate compound in the Maillard reaction, administered orally or intravenously to rats. Biochim. Biophys. Acta 1990, 1035, 71−76. (19) Kanazu, T.; Shinoda, M.; Nakayama, T.; Deyashiki, Y.; Hara, A.; Sawada, H. Aldehyde reductase is a major protein associated with 3deoxyglucosone reductase activity in rat, pig and human livers. Biochem. J. 1991, 279, 903−906. (20) Vander Jagt, D. L.; Hunsaker, L. A. Methylglyoxal metabolism and diabetic complications: roles of aldose reductase, glyoxalase-I, betaine aldehyde dehydrogenase and 2-oxoaldehyde dehydrogenase. Chem.−Biol. Interact. 2003, 143/144, 341−451. (21) Cao, D.; Fan, S. T.; Chung, S. S. Identification and characterization of a novel human aldose reductase-like gene. J. Biol. Chem. 1998, 273, 11429−11435. (22) Zhong, L.; Liu, Z.; Yan, R.; Johnson, S. Aldo-keto reductase family 1 B10 protein detoxifies dietary and lipid-derived α,βunsaturated carbonyls at physiological levels. Biochem. Biophys. Res. Commun. 2009, 387, 245−250. (23) Collard, F.; Vertommen, D.; Fortpied, J.; Duester, G.; Van Schaftingen, E. Identification of 3-deoxyglucosone dehydrogenase as aldehyde dehydrogenase 1A1 (retinaldehyde dehydrogenase 1). Biochimie 2007, 89, 369−373. (24) Lal, S.; Szwergold, B. S.; Walker, M. A.; Randall, W. C.; Kappler, F.; Beisswenger, P. J.; Brown, T. R. Production and metabolism of 3deoxyglucosone in humans. The Maillard Reaction in Foods and Medicine; Special Publication 223; Crabbe, J., Nursten, H. E., O’Brien, J., Eds.; Royal Society of Chemistry: Cambridge, UK, 1998; pp 291− 297. (25) Wells-Knecht, K. J.; Lyons, T. J.; McCance, D. R.; Thorpe, S. R.; Feather, M. S.; Baynes, J. W. 3-Deoxyfructose concentrations are increased in human plasma and urine in diabetes. Diabetes 1994, 43, 1152−1156. (26) Henle, T.; Bachmann, A. Synthesis of pyrraline reference material. Z. Lebensm. Unters. Forsch. 1996, 202, 72−74. (27) Brady, R. F. Cyclic acetals of ketoses. 3. Re-investigation of synthesis of isomeric di-O-isopropylidene-β-D-fructopyranoses. Carbohydr. Res. 1970, 15, 35−40. (28) Rasmussen, J. R.; Slinger, C. J.; Kordish, R. J.; Newman-Evans, D. D. Synthesis of deoxy sugars. Deoxygenation by treatment with N,N′-thiocarbonyldiimidazole/tri-n-butyl-stannane. J. Org. Chem. 1981, 46, 4843−4846.
(29) Szarek, W. A.; Rafka, R. J.; Yang, T. F.; Martin, O. R. Structuresweetness relationships for fructose analogs. Part III. 3-Deoxy-Derythro-hexulose (3-deoxy-D-fructose): composition in solution and evaluation of sweetness. Can. J. Chem. 1995, 73, 1639−1644. (30) Wahdan, H. A. L. Causes of the antimicrobial activity of honey. Infection 1998, 26, 26−31. (31) Troyano, E.; Olano, A.; Fernaindez-Diaz, M.; Sanz, J.; MartinezCastro, I. Gas chromatographic analysis of free monosaccharides in milk treatment of milk samples. Chromatographia 1991, 32, 379−382. (32) Glomb, M. A.; Tschirnich, R. Detection of α-dicarbonyl compounds in Maillard reaction systems and in vivo. J. Agric. Food Chem. 2001, 49, 5543−5550. (33) Rojas-Escudero, E.; Alarcón-Jiménez, A. L.; Elizalde-Galván, P.; Rojo-Callejas, F. Optimization of carbohydrate silylation for gas chromatography. J. Chromatogr., A 2004, 1027, 117−120. (34) Kopper, S.; Freimund, S. The composition of keto aldoses in aqueous solution as determined by NMR spectroscopy. Helv. Chim. Acta 2003, 86, 827−843. (35) Peterson, G. Gas-chromatographic analysis of sugars and related hydroxy acids as acyclic oxime and ester trimethylsilyl derivatives. Carbohydr. Res. 1974, 33, 47−61. (36) Shoemaker, J. D.; Elliott, W. H. Automated screening of urine samples for carbohydrates, organic and amino acids after treatment with urease. J. Chromatogr. 1991, 562, 125−138. (37) Lentner, C. Units of Measurement, Body Fluids, Composition of the Body, Nutrition; CIBA-Geigy AG: Basel, Switzerland, 1991. (38) Molnár-Perl, I.; Horváth, K. Simultaneous quantitation of mono-, di- and trisaccharides as their TMS ether oxime derivatives by GC-MS: I. In model solutions. Chromatographia 1997, 45, 321−327. (39) Boeniger, M. F.; Lowry, L.; Rosenberg, J. Interpretation of urine results used to assess chemical-exposure with emphasis on creatinine adjustment − a review. Am. Ind. Hyg. Assoc. J. 1993, 54, 615−627. (40) Delpierre, G.; Veiga-da-Cunha, M.; Vertommen, D.; Buysschaert, M.; Van Schaftingen, E. Variability in erythrocyte fructosamine 3-kinase activity in humans correlates with polymorphisms in the FN3K gene and impacts on haemoglobin glycation at specific sites. Diabetes Metab. 2006, 32, 31−39. (41) Krause, R.; Oehme, A.; Wolf, K.; Henle, T. A convenient HPLC assay for the determination of fructosamine-3-kinase activity in erythrocytes. Anal. Bioanal. Chem. 2006, 386, 2019−2025. (42) Hellwig, M.; Geissler, S.; Matthes, R.; Peto, A.; Silow, C.; Brandsch, M.; Henle, T. Transport of free and peptide-bound glycated amino acids: synthesis, transepithelial flux at Caco-2 cell monolayers, and interaction with apical membrane transport proteins. ChemBioChem 2011, 12, 1270−1279.
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Dietary Influence on Urinary Excretion of 3‑Deoxyglucosone and Its Metabolite 3‑Deoxyfructose Julia Degen, Helene Beyer, Björn Heymann, Michael Hellwig, and Thomas Henle* Institute of Food Chemistry, Technische Universität Dresden, D-01062 Dresden, Germany S Supporting Information *
ABSTRACT: 3-Deoxyglucosone (3-DG), a reactive 1,2-dicarbonyl compound derived from D-glucose in food and in vivo, is an important precursor for advanced glycation endproducts (AGEs). At present, virtually no information about the metabolic transit of dietary 3-DG is available. One possible metabolic pathway of 3-DG during digestion is enzymatic transformation to less reactive compounds such as 3-deoxyfructose (3-DF). To study the handling of dietary 1,2-dicarbonyl compounds by the human body, 24 h urinary excretion of 3-DG and its metabolite, 3-deoxyfructose, was investigated. Urinary 3-DG and 3-DF excretion was monitored for nine healthy volunteers following either a diet with no dietary restrictions or a diet avoiding the ingestion of 3DG and other Maillard reaction products (“raw food” diet). During the “raw food” diet, the urinary 3-DG and 3-DF excretion decreased approximately to 50% compared to the excretions during the diet with no restrictions. When subjects received a single dose of wild honey (50 g) naturally containing a defined amount of 3-DG (505 μmol), median excretion of 3-DG and 3-DF increased significantly from 4.6 and 77 to 7.5 and 147 μmol/day, respectively. The obtained experimental data for the first time demonstrate a dietary influence on urinary 3-DG and 3-DF levels in healthy human subjects. KEYWORDS: 3-deoxyglucosone, 3-deoxyfructose, glycation, Maillard reaction, GC-MS, urinary excretion, metabolic transit
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INTRODUCTION 3-Deoxyglucosone (3-DG) is a reactive 1,2-dicarbonyl compound derived from D-glucose. In food, 3-DG is formed nonenzymatically in the course of the Maillard reaction and in caramelization processes.1−3 3-DG in vivo can also originate from fructose-3-phosphate or from the phosphorylated Amadori compound fructosamine-3-phosphate via spontaneous decomposition.4,5 The concentration of 3-DG in the plasma of diabetic subjects ranges between 0.08 and 0.50 μmol/L and is about twice as high as the concentration in the plasma of healthy subjects (0.04−0.16 μmol/L).6−9 The occurrence of 3DG in a variety of foods was recently published.10,11 High contents were measured in balsamic vinegar (up to 16 mmol/ L), honey (10 mmol/kg), and bakery products such as bread and cookies (up to 3.8 mmol/kg). The dietary intake of 3-DG was estimated to range from 0.1 to 1 mmol (20−160 mg) per day.11 3-DG rapidly reacts with lysine and arginine side chains of proteins to form advanced glycation endproducts (AGEs), such as the lysine-derived pyrraline and the arginine derivative “3deoxyglucosone-derived hydroimidazolone” (3-DG-H, Figure 1). Both compounds have already been detected in vivo, for example, in long-lived proteins such as lens crystalline and collagen.12−14 As the plasma concentration of 3-DG is elevated under diabetic conditions, the dicarbonyl compound may play an important role in the development of diabetic complications.15 Elevated levels of 3-DG and other 1,2-dicarbonyl compounds in plasma (and, in consequence, accumulation of AGEs) are also discussed to contribute to long-term complications of uremia.16 In this context, the term “carbonyl stress” was introduced.17 However, both reductive and oxidative mechanisms exist in vivo to metabolize endogenous 3-DG to less reactive © 2014 American Chemical Society
compounds. Aldehyde reductase (EC 1.1.1.2) and aldose reductase (EC 1.1.1.21) both reduce 3-DG NADPH-dependently at the aldehyde group to 3-deoxyfructose (3-DF, Figure 1).18−20 The enzymes are present in all organs, with high expression in the kidneys. Recently, the small intestine aldose reductase (ALKR1B10) was identified as a novel member of the aldose reductase family. ALKR1B10 is primarily expressed in the human colon and small intestine.21,22 Another mechanism for detoxification of 3-DG is oxidation via aldehyde dehydrogenase ALDH1A1 (NAD+-dependent) and 2-oxoaldehyde-dehydrogenase (NADP+-dependent) to 3-deoxy-2-ketogluconic acid. ALDH1A1 was identified as a specific 3-DG oxidizing enzyme in erythrocytes (Figure 1).23 3-Deoxyfructose is the main urinary metabolite of 3-DG, and concentrations between 32 and 92 μmol/g creatinine can be measured in the urine of normoglycemic persons and elevated concentrations (54−196 μmol/g creatinine) in the urine of diabetic patients. In plasma, 3-DF accounts for 85% and in urine for 99% of the summarized amounts of 3-DG and 3-DF,7,24,25 indicating the effective enzymatic transformation of 3-DG to 3-DF. On the basis of recently published contents of 3-DG in commonly consumed foods10,11 and the possible role of 3-DG in diabetic complications, questions may arise concerning eventual health risks resulting from the dicarbonyl compound ingested with the daily diet. Only very limited information is available concerning the bioavailability of 3-DG and its metabolic fate during digestion. No information about the Received: Revised: Accepted: Published: 2449
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Figure 1. Degradation reactions of 3-deoxyglucosone: (I) enzymatic transformation of 3-deoxyglucosone to 3-deoxyfructose via aldehyde and aldose reductase and (II) to 3-deoxy-2-ketogluconic acid via 2-oxoaldehyde dehydrogenase and ALDH1A1; (III) nonenzymatic reaction of 3deoxyglucosone with protein-bound arginine to the advanced glycation end product 3-DG-H (3-deoxyglucosone derived hydroimidazolone) and (IV) reaction with protein-bound lysine to form pyrraline.
Figure 2. Synthesis of 3-deoxy-D-erythro-hexulose (3-DF): (I) acetone, H2SO4, 25 °C, 1.5 h; (II) THF, 1,1′-thiocarbonyldiimidazole, 66 °C, 3 h; (III) toluene, tributyltin hydride, 110 °C, 1 h; (IV) acetone/water (1:1, v/v), Lewatit S100, 25 °C, 24 h. Synthesis of 3-Deoxy-D-erythro-hexulose (3-Deoxyfructose). Protocols of synthesis were adopted with minor modifications.27−29 Eighteen grams of D-fructose (1, Figure 2) was suspended in 350 mL of acetone containing 0.5% sulfuric acid and mechanically stirred at room temperature (under exclusion of moisture) until the sugar was dissolved (about 1.5 h). Termination of the reaction was carried out by adding 50 mL of ice-cold sodium hydroxide solution (c = 2.75 mmol/ L) to the mixture while stirring was continued. Acetone was evaporated in vacuo, and the remaining aqueous phase (reddish brown) was extracted with methylene chloride (3 × 100 mL). The combined extracts were washed with water (2 × 50 mL) and dried over sodium sulfate. After evaporation of the organic solvent, the crude reaction product was recrystallized twice from ethanol/water 1:4 (v/v) and from ether followed by pentane. At 6 °C complete crystallization was achieved overnight. After drying, 5.1 g of long white needles of the diacetal sugar 2 (1,2:4,5-di-O-isopropylidene-D-fructopyranose, Figure 2) was obtained (20.2% from compound 1). Solid 1,1′-thiocarbonyldiimidazole (2.2 g) was added to 1.5 g of the partially protected sugar, dissolved in 30 mL of tetrahydrofuran, and heated under reflux (66 °C) until complete conversion to the O-(imidazolylthiocarbonyl) derivative (about 3 h, monitored with thin layer chromtography).28 The target product was isolated from the evaporated reaction mixture by flash chromatography (stationary phase: silica gel 60, mesh 0.063− 0.200 mm) with ethyl acetate/hexane (1:1, v/v) as mobile phase, eluting between 80 and 260 mL. Drying of the combined fractions yielded 1.9 g of the alkoxythiocarbony1imidazolide derivative (85% from 2), which was completely employed for the following reduction step. The product was dissolved in 50 mL of anhydrous toluene, which was added dropwise within 30 min to a boiling tributyltin hydride/toluene solution (2.3 g in 200 mL of anhydrous toluene). After completion of reduction (about 1 h), the cooled solvent was evaporated in vacuo, and the dry residue was extracted with hot acetone (3 × 50 mL); the
metabolic transit of 3-DG in healthy humans is available up to now. The aim of our study, therefore, was to examine the contribution of dietary 3-DG on the “3-DG-load” of the human body. To obtain information about absorption and metabolism of 3-DG, urinary elimination of 3-DG and its major metabolite 3-DF was analyzed in healthy subjects following a diet containing 3-DG and a diet virtually free from 3-DG. During a 3 day intervention study, the influence of a single dose of honey containing a defined amount of 3-DG on the urinary elimination of 3-DG and 3-DF was observed. For this, a sensitive GC-MS for the simultaneous quantification of 3-DG and 3-DF in human urine had to be developed, and both analytes were synthesized as reference material.
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MATERIALS AND METHODS
Materials. Chemicals of the highest purity available were purchased from standard suppliers: 1,1′-thiocarbonyldiimidazole (Sigma-Aldrich, Steinheim, Germany); n-hexane, hydroxylamine hydrochloride (Merck, Darmstadt, Germany); N,O-bis(trimethylsilyl)trifluoroacetamide, D-fructose, methanol (Sigma-Aldrich); methyl-αD-galactopyranoside (Acros, Geel, Belgium); pyridine (anhydrous), tributyltin hydride, urease (type C-3, 12KU) (Sigma-Aldrich). Mixedbed ion exchanger Serdolit MB-2 was from Serva (Heidelberg, Germany), and cation exchanger (Lewatit S100) was from Bayer (Leverkusen, Germany). 3-DG was synthesized as already described26 with some modifications.10 [13C6]3-DG was prepared accordingly starting from [13C6]glucose (99%, Euriso-Top, Saint-Aubin Cedex, France). The water used was obtained with a Purelab plus purification system (USFilter, Ransbach-Baumbach, Germany). 2450
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Table 1. GC-MS Parameters (SIM Mode) for the Determination of 3-Deoxyfructose (3-DF) and 3-Deoxyglucosone (3-DG)
3-deoxyfructose 3deoxyglucosone
internal standard
quantifier ion [m/z]
qualifier ion [m/z]
internal standard quantifier ion [m/z]
internal standard qualifier ion [m/z]
dwell time (ms)
MGP [13C 6]3-DG
191 347
217, 524 373, 537
204 351
217, 133 379, 543
80 (for each measured ion)
combined extracts were washed with hexane (4 × 50 mL) to remove stannous components. Acetone was evaporated, and product isolation was performed by flash chromatography (stationary phase: silica gel 60, mesh 0.063−0.200 mm, mobile phase ethylacetate/hexane 1:1 (v/ v), elution between 80 and 170 ml). After recrystallization from pentane, 0.74 g of the reduced diisopropylidene derivative 3 (Figure 2) was obtained as white needles (71% from 2). 1H nuclear magnetic resonance (NMR) analysis of this product gives a spectrum according to data published in the literature.28 Distinct signals with chemical shifts of δ = 2.1 for the two protons at C3 indicate the successful reduction at C3 (literature δ = 2.00 and 2.06).28 For hydrolysis the complete yield was dissolved in 12.5 mL of acetone and 25 mL of water. Three grams of activated cation exchange resin (Lewatit S100) was added; this mixture was then stirred for 24 h at room temperature. Neutralization was achieved by stirring the solution for another 6 min with the addition of 6 g of a mixed-bed ion exchanger (Serdolit MB-2). The product was purified by flash chromatography (stationary phase as above; mobile phase: methylene chloride/ethanol/water 2:1:0.1 (v/v/v), elution between 80 and 170 mL). The combined fractions were evaporated, and the residue was taken up in water and lyophilized to yield 0.16 g of 3-DF (4) (Figure 2; 16% from 2). After 6 weeks of storage at −18 °C, complete crystallization was accomplished with small, light yellow crystals. The product was characterized by 1H NMR and 13C NMR, and purity was examined via GC-FID. 1H NMR (500 MHz, D2O) β-pyranose: δH 3.43 (s, 2H, H1A/H1B), 1.78 (m, 2H, H3A/H3B), 3.95 (m, 1H, H4), 3.70 (s, 1H, H5), 3.59 (dd, 1H, H6), 3.86 (dd, 1H, H6). 13C NMR (125 MHz, D2O): δC 97.21 (C-2), 67.05 (C-5), 65.21 (C-1), 64.76 (C-4), 63.81 (C-6), 41.29 (C-3). For purity control of 3-DF, the corresponding trimethylsilyl (TMS) oxime of 3-DF was subjected to GC-FID analysis. Analysis was performed on a Varian 3900 gas chromatograph with a CP-8410 automatic liquid sampler with split injector and flame ionization detection (FID). Separation was performed on a DB-5 capillary column (30 m, 0.25 mm i.d., 1.00 μm film thickness, J&W Scientific, Germany). Helium was the carrier gas with a constant flow at 1.0 mL/ min, injector temperature was 250 °C, and injection mode was 1 μL at 1:20 split. The oven temperature program was adopted from the parameters of the GC-MS analysis. Stock solutions each of 3-DF and D-fructose were prepared in water (c = 1 mM), and calibration curves (0.05−1 mM) of the TMS oximes were recorded with GC-FID. The slope of the calibration curve of 3DF was compared to the slope of the calibration curve of D-fructose, and the ratio was assumed to indicate the purity/content of synthesized 3-DF. Intervention Study. Nine healthy, normoglycemic volunteers (ages 24−36 years, 7 women and 2 men, nonsmoking, fasting blood sugar ≤5.5 mmol/L) collected their 24 h urine (8.00 a.m. until 8.00 a.m. the following day) on 2 consecutive days with no dietary restrictions (days 1−2). Furthermore, all nine participants took part in a 3 day intervention study consisting of one wash-out day (day 3), one intervention day (day 4), and one run-out day (day 5). From day 3 to day 5, they followed a “raw food” diet, which implies avoiding heated and fermented foods such as dairy and bakery products, coffee, juices, and beer to ensure a diet virtually free of 3-DG and Maillard reaction products. In the morning of the intervention day (day 4), the participants ingested a single dose of 50 g of honey containing a defined amount of 505 μmol of 3-DG (3-DG concentration of the honey was 1641 mg/kg). The “raw food” diet was continued on the fifth day. Twenty-four hour urine was collected every day of the study as described above. Additionally, a placebo study was performed. Two participants (ages 28 and 31 years, respectively, 1 woman and 1 man,
nonsmoking, fasting blood sugar ≤5.5 mmol/L) followed the same 5 day study design as described above, but ingested 50 g of a honey analogue sugar mixture (46.5% D-fructose, 34.5% D-glucose, 1.5% saccharose, and 17.5% water)30 instead of wild honey in the morning of the intervention day (day 4). Aliquots of collected 24 h urine samples were immediately stored at −18 °C until analysis. 3-DG and 3-DF were analyzed within 2 weeks. Preparation of Urine Samples for GC-MS Analysis. After the urine thawed, an aliquot of 100 μL was spiked with 10 μL of internal standard solution (containing 1.0 mg/mL methyl-α-D-galactopyranoside31and 0.1 mg/mL [13C6]3-DG in water) and was then treated with 30 μL of urease (1 mg/mL in 0.2 M sodium phosphate buffer, pH 7.0) for 10 min at 37 °C in a water bath. Termination of the enzymatic reaction was realized by addition of 900 μL of ice-cold ethanol. After centrifugation (10000g, 15 min), the resulting supernatant was transferred to a new tube and evaporated to dryness under a nitrogen stream. Then the dry residue was incubated with 50 μL of hydroxylamine hydrochloride solution (10 mg/mL in anhydrous pyridine) for 30 min at 80 °C followed by evaporation to dryness. Then 50 μL of N,O-bis(trimethylsilyl)trifluoroacetamide was added to the residue and vigorously mixed by vortex for 0.5 min and incubated at room temperature for another 2 h. Subsequently, 50 μL of n-hexane was added; an aliquot of 80 μL was transferred to a GC vial and was then ready for GC-MS analysis. GC-MS Analysis of 3-Deoxyglucosone and 3-Deoxyfructose. GC-MS analysis was performed using a HP6890 gas chromatograph, coupled with a HP7683 automatic liquid sampler and a HP5973 mass selective detector equipped with a HP-5MS capillary column (30 m, 0.25 mm i.d., 0.25 μm film thickness; all from Agilent, Germany) and a Zebron Z-guard column (deactivated, 5 m, 0.25 mm i.d.; Phenomenex, Germany). The carrier gas was helium with a constant flow at 1.0 mL/ min, the injector temperature was 250 °C, and injection mode was 1 μL pulsed splitless. The oven temperature program started at 80 °C (hold time = 2 min) elevated to 250 °C at 10 °C/min (hold time = 3 min) and finally heated to 300 °C at 30 °C/min (hold time = 3 min), resulting in a complete run time of 25 min. Mass spectrometer was used with electron ionization (70 eV) in scan and SIM modes. In scan mode the recorded m/z area range varied from 50 to 800. For detailed SIM parameters see Table 1. For external calibration, stock solutions of the analytes in water (0.06−0.6 mM) were pipetted (1−50 μL) to yield appropriate concentrations along with internal standards. Each calibration sample was subjected to the derivatization procedure after evaporation to dryness (see above). Quantitative evaluation was carried out by the use of the ratio of the area of the most prominent analyte peak and the internal standard peak of the corresponding quantifier ions, despite the occurrence of peaks of syn- and anti-stereoisomers of the analytes. The limits of detection (LOD) and quantitation (LOQ) in standard solution and urine were calculated as the concentrations of the analyte necessary to show a peak at signal-to-noise ratios of 3 and 10, respectively. For the determination of the interday repeatability, one urine sample was applied to the sample workup and derivatization procedure five times on different days. The recovery of 3-DF and 3DG was calculated from the slope of the recovery function after urine had been spiked with three ascending concentrations of the analytes (3-DF, 6−300 μmol/L; 3-DG, 0.6−30 μmol/L) with subsequent sample workup. NMR Spectrometry. 1H and 13C NMR spectra were recorded on a Bruker DRX 500 instrument (Rheinstetten, Germany) at 500 and 125 MHz, respectively. Samples were dissolved in deuterium oxide. Chemical shifts are given in parts per million (ppm), those of protons 2451
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advantageous because the oximes exist in open-chain form,35 leading to only two silylated isomers per aldehyde or keto group, respectively. Compared to the published method7 for the simultaneous determination of 3-DG and 3-DF, this optimized GC-MS method has the key advantage of higher selectivity. This is accounted for by different retention times of the two analytes and distinct m/z ratios (compare Figure 2). In the cited method7 the retention times for both analytes are the same and the m/z ratio is only 1. The preparation of the urine samples prior to derivatization consisted of an enzymatic removal of urea for the analysis of carbohydrates in urine.36 The dry matter of urine contains approximately 23−49% urea,37 which can consume large amounts of the silylating agent and therefore hamper the silylation of the analytes.36 The adopted derivatization method33 was slightly modified in this work, resulting in a reduction of the applied derivatization agents. The application of oxime formation in carbohydrate analysis has been already examined in detail with regard to stability, efficiency, and the number of byproducts formed.38 The amount of N,Obis(trimethylsilyl)trifluoroacetamide (BSTFA) was adjusted to 50 μL per derivatization assay, and derivatization was complete after 2 h at room temperature, based on time course studies. The solvent evaporation after oxime formation is timeconsuming, and the silylation of the analytes in the dry residue is suboptimal compared to a silylation of the analytes in solution. The drying procedure, however, is necessary to ensure a salt concentration as low as possible in the injected sample solution to minimize contamination and signal suppression in the mass selective detector. In Figure 4, a selected ion chromatogram of a 3-DF (at m/z 191) and 3-DG (at m/z 347) standard solution after derivatization with hydroxylamine hydrochloride and BSTFA to the corresponding TMS oximes is shown. For the TMS
relative to the internal HOD signal (4.70 ppm, residual proton), and those of carbon atoms relative to the external standard tetramethylsilane. Assignments of 1H and 13C signals are based on heteronuclear single-quantum coherence (HSQC) and distortionless enhancement by polarization transfer (DEPT) experiments. Statistical Treatment. Comparisons of means between the daily urinary excretions of the different days were examined using Student’s t test. P values ≤0.05 were considered significant (two-tailed) using OriginPro 8.6.
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RESULTS AND DISCUSSION GC-MS Analysis of Urinary 3-Deoxyglucosone and 3Deoxyfructose: Method Evaluation. The first aim of our study was to establish a sensitive and selective method for the simultaneous determination of 3-DG and its metabolite 3-DF in urine. As 3-DF is commercially not available, it had to be synthesized as reference material. This was achieved by the preparation of the 1,2:4,5-diacetal of D-fructose, selective reduction at C3 with tri-n-butylstannane, and finally hydrolysis of the 3-deoxysugar, according to literature methods (compare Figure 2).27−29 The 1H NMR spectrum of the final product 3DF showed several signals due to five tautomeric forms of the deoxysugar in aqueous solution. According to the literature, most signals of the β-pyranoide structure could be assigned, as this is the predominating form of 3-DF in aqueous solution at room temperature. Chemical shifts of the signals are in line with those reported.29 The chemical shift of the protons at C3 (δ = 1.78) of 3-DF are characteristic for the reduction at C3 and prove the identity of 3-DF along with the other signals. Purity control of the synthesized 3-DF via GC-FID32 indicated a purity of 99% when compared to D-fructose. The GC-FID chromatogram indicated no impurities of other sugars or sugar derivatives (data not shown). For the GC-MS analysis of the sugar derivatives, a conversion to volatile compounds is inevitable. The first step of the derivatization is the formation of the corresponding oximes, followed by the conversion to the TMS derivatives (Figure 3).33 With regard to the 10 isomeric forms of 3-DG34 and the 5 tautomeric forms of 3-DF,29 the formation of sugar oximes is
Figure 4. Selected ion monitoring chromatogram of (a) a 3deoxyglucosone 3-DG)/3-deoxyfructose (3-DF) standard solution (m/z 347 and 191), (b) a urine sample at m/z 191, and (c) a urine sample at m/z 347. All chromatograms were acquired by GC-MS after derivatization with hydroxylamine hydrochloride and N,O-bis(trimethylsilyl)trifluoroacetamide to the corresponding trimethylsilyl oximes.
Figure 3. Structures of the trimethylsilyl oxime derivatives of (A) 3deoxyglucosone and (B) 3-deoxyfructose with their corresponding nominal masses. 2452
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Figure 5. (A) EI-Mass spectra of the peak eluting at 18.5 min of a 3-deoxyglucosone (3-DG) standard solution and a urine sample (scan mode). (B) EI-Mass spectra of the peak eluting at 17.3 min of a 3-deoxyfructose (3-DF) standard solution and a urine sample (scan mode). All spectra were acquired by GC-MS after derivatization with hydroxylamine hydrochloride and N,O-bis(trimethylsilyl)trifluoroacetamide to the corresponding trimethylsilyl oximes.
Table 2. Performance Parameters of the GC-MS Method for the Determination of 3-Deoxyfructose (3-DF) and 3Deoxyglucosone (3-DG) in Urine 3-deoxyfructose 3-deoxyglucosone
R2
linear range (μmol/L)
interday repeatabilitya (%)
LODb (nmol/L)
LOQb (nmol/L)
recoveryc (%)
0.9998 0.9980
6−300 0.6−30
1.5 1.0
52 ± 4 20 ± 4
155 ± 11 59 ± 13
101.8 ± 3.8 97.4 ± 4.2
a Expressed as relative standard deviation. bLODs (limit of detection) and LOQs (limit of quantification) were calculated on the basis of signal-tonoise-ratio. cRecovery was determined by addition of various concentrations of 3-DF (60−300 μmol/L, n = 3) and 3-DG (0.6−30 μmol/L, n = 3) to 100 μL of urine, applying all steps of sample preparation and analysis. The value was calculated from the slope of the recovery function and is given in percent ± SE.
are displayed. The two spectra show the same distinct signals at the mass-to-charge ratio of the quantifier ion (m/z 347) and the qualifier ion (m/z 537). The mass spectra (scan mode) of the peak eluting at 17.3 min of a 3-DF standard solution and a urine sample after derivatization to their corresponding TMS oximes are presented in Figure 5B, having consistent signals at m/z 191 (quantifier ion) and at m/z 524 (qualifier ion). Comparison of the mass spectra of the peaks of the two corresponding analytes recorded from a derivatized standard solution and a urine sample allows a doubtless and unequivocal identification of 3-DG and 3-DF in urine. In Table 2, validation parameters of the method for the determination of 3-DG and 3-DF in urine are summarized. They indicate an accurate sample preparation, a reproducible derivatization procedure, and a reliable GC-MS analysis. On the basis of the relative interday repeatability being 0.05). 3-DG accounted for 4−8% (mean = 6%) of the summarized 3DG and 3-DF amounts. This is in line with published data,7 which show that 3-DF is the main metabolite in urine accounting for >95% of 3-DG. The daily urinary apparent 3DF excretions are consistent with values published in the literature for healthy subjects (32−92 μmol/g creatinine),7,26 when a daily excretion of 1.1−1.9 g creatinine is taken as a 2454
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In conclusion, it was possible for the first time to reveal differences in urinary 3-DG and 3-DF elimination in healthy human subjects, dependent on the diet. These results are a prerequisite for further studies concerning metabolic pathways of 3-DG in human subjects.
the average increase of the urinary 3-DG excretion was 3.0 μmol/day (range = 0.4−3.6 μmol/day) and that of the 3-DF excretion was 70 μmol/day (range = 30−123 μmol/day). This corresponds with approximately 10−15% of the ingested amount of 3-DG being recovered in the urine. Slightly lower excretion rates into urine were observed in rats, but it is obvious that results of animal experiments could be only partly transferred to humans and the study design lasted only 2 h.18 Median 3-DG and 3-DF excretions on the run-out day (day 5) did not differ significantly from those of day 3 (P > 0.05), indicating a rapid elimination of intestinally absorbed 3-DG (compare Figure 6). Despite the intake of a high amount of 3DG on the intervention day (day 4), the median ratios of the urinary 3-DG/3-DF excretion did not vary significantly compared to the other days (see Supporting Information). In further studies it might be of interest to investigate interindividual differences of the excreted 3-DG/3-DF ratios to have further insights into possible variations of the metabolic handling, for example, during diabetes. To rule out that the observed increase in urinary excretion of 3-DF on day 4 is due to the high sugar load and a possible formation of 3-DG from glucose or fructose in vivo, a placebo study was performed. Two volunteers consumed 50 g of a honey analogue sugar mixture virtually free of 3-DG instead of 3-DG-containing wild honey on the intervention day (data and figure shown in the Supporting Information). Urinary excretion of 3-DG and 3-DF remained constant, indicating that precursors such as fructose or glucose do not contribute to a measurable 3-DG formation in vivo or an increased urinary 3DG and 3-DF excretion, respectively. Hence, the elevation of the urinary 3-DF excretion on the intervention day of the intervention study was exclusively due to the 3-DG consumed via the wild honey. These experimental findings for the first time prove an influence of dietary 3-DG on urinary excretion of 3-DG and 3DF. The results imply that dietary 3-DG is to some extent taken up from the ingested food in the intestinal tract, metabolized to 3-DF, and eliminated via urine. This allows the conclusion that detoxification systems for endogenously formed reactive aldehydes, such as aldehyde and aldose reductase,21,22 should also be capable to degrade dietary and therewith exogenous 3-DG. In particular, the recently described small intestine aldose reductase (ALKR1B10), which is described to play a key role in detoxifying dietary and lipid-derived carbonyl compounds,21,22 should be examined in this context. As only 10−15% of the administered 3-DG is recovered in urine as 3DG and 3-DF, the question arises about the fate of the remaining 85−90%. Further studies will have to show whether putative 3-DG-specific metabolites such as 3-deoxy-2-ketogluconic acid or hydroxymethylfurfural (HMF) can be found in the urine. At present, it cannot be excluded that 3-DG is to some extent transformed to HMF due to the low pH value of the stomach. 3-DG may also react with proteins to AGEs in the gastrointestinal tract or in vivo. The analysis of 3-DG and 3-DF in blood plasma plus 3-deoxy-2-keto-gluconic acid and other analytes (i.e., fructoselysine, HMF, pyrraline) of study participants after an oral load of 3-DG would unequivocally reveal if and to what extent 3-DG is absorbed into the systemic circulation and if it is metabolized. On the basis of results of animal experiments, it was concluded that orally ingested 3-DG is absorbed in the intestine to only a minor degree and that the major part remains in the content of the gastrointestinal tract.18 This finding should be examined in a following study.
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ASSOCIATED CONTENT
S Supporting Information *
Figures I and II. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*(T.H.) Phone: +49-351-463-34647. Fax: +49-351-463-34138. E-mail: [email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank Dr. Uwe Schwarzenbolz, Institute of Food Chemistry, Technische Universität Dresden, for his support during the LC-MS measurements. Furthermore, we thank Dr. Margit Gruner and Anett Rudolph, Institute of Organic Chemistry, Technische Universität Dresden, for recording the NMR spectra.
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ABBREVIATIONS USED 3-DF, 3-deoxyfructose; 3-DG, 3-deoxyglucosone; 3-DG-H, 3deoxyglucosone-derived hydroimidazolone; AGE, advanced glycation endproduct; BSTFA, N,O-bis(trimethylsilyl)trifluoroacetamide; FID, flame ionization detector; GC-MS, gas chromatography−mass spectrometry; LOD, limit of detection; LOQ, limit of quantitation; MGP, methyl-α-Dgalactopyranoside; SIM, selected ion monitoring; TMS, trimethylsilyl
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