Determination of α-Dicarbonyl compounds in traditional Chinese herbal medicines.

α-DCs (α-dicarbonyls) have been proven to be closely related to aging and the onset and development of many chronic diseases. The wide presence of this kind of components in various foods and beverages has been unambiguously determined, but their occurrence in various phytomedicines remains in obscurity. In this study, we established and evaluated an HPLC-UV method and used it to measure the contents of four α-DCs including 3-deoxyglucosone (3-DG), glyoxal (GO), methylglyoxal (MGO), and diacetyl (DA) in 35 Chinese herbs after they have been derivatized with 4-nitro-1,2-phenylenediamine. The results uncover that 3-DG is the major component among the α-DCs, being detectable in all the selected herbs in concentrations ranging from 22.80 µg/g in the seeds of Alpinia katsumadai to 7032.75 µg/g in the fruit of Siraitia grosuenorii. The contents of the other three compounds are much lower than those of 3-DG, with GO being up to 22.65 µg/g, MGO being up to 55.50 µg/g, and DA to 18.75 µg/g, respectively. The data show as well the contents of the total four α-DCs in the herbs are generally in a comparable level to those in various foods, implying that herb medicines may have potential risks on human heath in view of the α-DCs.

Application of tea polyphenols as additives in brown fermented milk: Potential analysis of mitigating Maillard reaction products.

Brown fermented milk (BFM) is favored by consumers in the dairy market for its unique burnt flavor and brown color. However, Maillard reaction products (MRP) from high-temperature baking are also noteworthy. In this study, tea polyphenols (TP) were initially developed as potential inhibitors of MRP formation in BFM. The results showed that the flavor profile of BFM did not change after adding 0.08% (wt/wt) of TP, and its inhibition rates on 5-hydroxymethyl-2-furaldehyde (5-HMF), glyoxal (GO), methylglyoxal (MGO), Nε-carboxymethyl lysine (CML), and Nε-carboxyethyl lysine (CEL) were 60.8%, 27.12%, 23.44%, 57.7%, and 31.28%, respectively. After 21 d of storage, the levels of 5-HMF, GO, MGO, CML, and CEL in BFM with TP were 46.3%, 9.7%, 20.6%, 5.2%, and 24.7% lower than the control group, respectively. Moreover, a smaller change occurred in their color and the browning index was lower than that of the control group. The significance of this study was to develop TP as additives to inhibit the production of MRP in brown fermented yogurt without changing color and flavors, thereby making dairy products safer for consumers.

Phytochemical profiling of Symplocos tanakana Nakai and S. sawafutagi Nagam. leaf and identification of their antioxidant and anti-diabetic potential.

Symplocos sp. contains various phytochemicals and is used as a folk remedy for treatment of diseases such as enteritis, malaria, and leprosy. In this study, we discovered that 70% ethanol extracts of Symplocos sawafutagi Nagam. and S. tanakana Nakai leaves have antioxidant and anti-diabetic effects. The components in the extracts were profiled using high-performance liquid chromatography coupled to electrospray ionization and quadrupole time-of-flight mass spectrometry; quercetin-3-O-(6''-O-galloyl)-ß-d-galactopyranoside (6) and tellimagrandin II (7) were the main phenolic compounds. They acted as strong antioxidants with excellent radical scavenging activity and as inhibitors of non-enzymatic advanced glycation end-products (AGEs) formation. Mass fragmentation analysis demonstrated that compounds 6 and 7 could form mono- or di-methylglyoxal adducts via reaction with methylglyoxal, which is a reactive carbonyl intermediate and an important precursor of AGEs. In addition, compound 7 effectively inhibited the binding between AGE2 and receptor for AGEs as well as the activity of α-glucosidase. Enzyme kinetic study revealed that compound 7 acts as a competitive inhibitor of α-glucosidase, through interaction with the active site of the enzyme. Therefore, compounds 6 and 7, the major constituents of S. sawafutagi and S. tanakana leaves, are promising for developing drugs for preventing or treating diseases caused by aging and excessive sugar consumption.

Antiglycation Effects of Adlay Seed and Its Active Polyphenol Compounds: An In Vitro Study.

This study aimed to evaluate the antiglycation effects of adlay on protein glycation using in vitro glycation assays. Adlay seed was divided into the following four parts: the hull (AH), testa (AT), bran (AB), and polished adlay (PA). A solvent extraction technique and column chromatography were utilized to investigate the active fractions and components of adlay. Based on a BSA-glucose assay, the ethanolic extracts of AT (ATE) and AB (ABE) revealed a greater capacity to inhibit protein glycation. ATE was further consecutively partitioned into four solvent fractions with n-hexane, ethyl acetate (ATE-Ea), 1-butanol (ATE-BuOH), and water. ATE-BuOH and -Ea show marked inhibition of glucose-mediated glycation. Medium-high polarity subfractions eluted from ATE-BuOH below 50% methanol with Diaion HP-20, ATE-BuOH-c to -f, exhibited superior antiglycation activity, with a maximum inhibitory percentage of 88%. Two phenolic compounds, chlorogenic acid and ferulic acid, identified in ATE-BuOH with HPLC, exhibited potent inhibition of the individual stage of protein glycation and its subsequent crosslinking, as evaluated by the BSA-glucose assay, BS-methylglyoxal (MGO) assay, and G.K. peptide-ribose assay. In conclusion, this study demonstrated the antiglycation properties of ATE in vitro that suggest a beneficial effect in targeting hyperglycemia-mediated protein modification.

Evaluation of the nephrotoxicity and safety of low-dose aristolochic acid, extending to the use of Xixin (Asurum), by determination of methylglyoxal and d-lactate.

ETHNOPHARMACOLOGICAL RELEVANCE: Most Aristolochiaceae plants are prohibited due to aristolochic acid nephropathy (AAN), except Xixin (Asarum spp.). Xixin contains trace amounts of aristolochic acid (AA) and is widely used in Traditional Chinese Medicine. Methylglyoxal and d-lactate are regarded as biomarkers for nephrotoxicity. AIM OF THE STUDY: The use of Xixin (Asarum spp.) is essential and controversial. This study aimed to evaluate tubulointerstitial injury and interstitial renal fibrosis by determining urinary methylglyoxal and d-lactate after withdrawal of low-dose AA in a chronic mouse model. MATERIALS AND METHODS: C3H/He mice in the AA group (n = 24/group) were given ad libitum access to distilled water containing 3 µg/mL AA (0.5 mg/kg/day) for 56 days and drinking water from days 57 to 84. The severity of tubulointerstitial injury and fibrosis were evaluated using the tubulointerstitial histological score (TIHS) and Masson's trichrome staining. Urinary and serum methylglyoxal were determined by high-performance liquid chromatography (HPLC); urinary d-lactate were determined by column-switching HPLC. RESULTS: After AA withdrawal, serum methylglyoxal in the AA group increased from day 56 (429.4 ± 48.3 µg/L) to 84 (600.2 ± 99.9 µg/L), and peaked on day 70 (878.3 ± 171.8 µg/L; p < 0.05); TIHS and fibrosis exhibited similar patterns. Urinary methylglyoxal was high on day 56 (3.522 ± 1.061 µg), declined by day 70 (1.583 ± 0.437 µg) and increased by day 84 (2.390 ± 0.130 µg). Moreover, urinary d-lactate was elevated on day 56 (82.10 ± 18.80 µg) and higher from day 70 (201.10 ± 90.82 µg) to 84 (193.28 ± 61.32 µg). CONCLUSIONS: Methylglyoxal is induced after AA-induced tubulointerstitial injury, so methylglyoxal excretion and metabolism may be a detoxification and repair strategy. A low cumulative AA dose is the key factor that limits tubulointerstitial injury and helps to repair. Thus, AA-containing herbs, especially Xixin, should be used at low doses for short durations (less than one month).

Analysis of α-dicarbonyl compounds in coffee (Coffea arabica) prepared under various roasting and brewing methods.

This study investigated the correlations of α-dicarbonyl compounds (α-DCs), including glyoxal (GO), methylglyoxal (MGO) and diacetyl (DA), formed in coffee prepared under various roasting and brewing methods. The levels of α-DCs in Brazilian coffee beans (Coffea arabica) ranged from 28.3 to 178 µg/mL. Concentration ranges of GO, MGO and DA were 1.31-6.57, 25.5-159 and 1.50-12.9 µg/mL, respectively. The level of α-DCs increased with high roasting temperature, long roasting time, small coffee bean particles, mineral water and espresso brewing. In correlation analysis, the roasting temperature-time showed strong negative correlations with α-DCs in espresso (-0.886) and cold-brew coffee (-0.957). In espresso coffee, there was a strong negative correlation between the α-DCs and coffee bean particle size (-0.918).

Silicon-regulated antioxidant and osmolyte defense and methylglyoxal detoxification functions co-ordinately in attenuating fluoride toxicity and conferring protection to rice seedlings.

The objective of this manuscript was to demonstrate the efficacy of silicon supplementation in relieving the fluoride-induced damages in rice cultivar, Khitish. The exposure of seedlings to two different concentrations of fluoride, viz., 25 and 50 mg L-1 NaF caused increase in fluoride accumulation, as a result of which the seedlings suffered severe oxidative stress, as evident from growth inhibition, reduction in seed germination, tissue biomass, root and shoot length, decline in chlorophyll content, increases in electrolyte leakage, H2O2 content, lipid peroxidation (malondialdehyde content and lipoxygenase activity), protein carbonylation and protease activity. The extent of damage was more at higher fluoride concentration. Silicon amendment, irrespective of fluoride concentrations, led to large build-up of endogenous silicon level and brought considerable improvement in all the parameters examined with respect to fluoride stress. The fluoride-mediated enhancement in methylglyoxal level was lowered by silicon, because of the prominent activation of glyoxalase I and glyoxalase II. While the stress-mediated induction in antioxidative enzymes like GPOX, APX, SOD, GPX and GR was lowered by silicon, the inhibition in CAT activity was relieved. The antioxidative defense mechanism was also boosted up via enhanced content of total phenolics and carotenoids. However, the fluoride-mediated increase in anthocyanins, flavonoids, xanthophyll, ascorbate and reduced glutathione, and osmolytes like total amino acids, proline and glycine-betaine, were all lowered in presence of silicon, together with reduced PAL and P5CS activity. Overall, silicon reduced oxidative damages to develop fluoride-tolerant rice plants through augmentation of different antioxidant and osmolyte defense and methylglyoxal detoxification system.

Formation of Nε-(carboxymethyl)lysine and Nε-(carboxyethyl)lysine during black tea processing.

This study selected common processing methods for orthodox black tea and investigated changes in the levels of Nε-(carboxymethyl)lysine (CML), Nε-(carboxyethyl)lysine (CEL), lysine, and polyphenols during each processing stage and using different parameters of each processing step. The effects of epicatechin gallate, epigallocatechin, epigallocatechin gallate, and gallic acid on the levels of CML, CEL, fructoselysine, glyoxal, and methylglyoxal were investigated by chemical model systems study under black tea processing conditions. In tea samples, CML and CEL levels significantly increased during drying (could reach 51.8 and 8.7 µg/g tea, respectively), while natural withering and extensive rolling and fermentation times facilitated the formation of CML and CEL by altering the substrate concentrations and the cellular structure of tea leaves to be dried. The results of model systems (containing lysine, glucose, and fructose) indicated that polyphenols were able to enhance the production of CML and CEL, and the levels of CML and CEL increased 1.2-3.2- and 1.6-3.5-fold, respectively. Furthermore, the main pathways responsible for CML and CEL formation during black tea processing likely involve fructoselysine and others but not glyoxal or methylglyoxal.

Effects of Catechins on Nε-(Carboxymethyl)lysine and Nε-(Carboxyethyl)lysine Formation in Green Tea and Model Systems.

The effects of catechins on Nε-(carboxymethyl)lysine (CML) and Nε-(carboxyethyl)lysine (CEL) formation in green tea and related model systems were investigated in this study. Since the first step of green tea processing entails enzyme inactivation, the catechin content was maintained at a high level during processing. However, drying still had a great effect on CML and CEL formation, while other steps also contributed. Hence, model systems were developed to analyze the effects of catechins on CML and CEL formation. Catechins ((-)-epicatechin gallate, (-)-epigallocatechin, and (-)-epigallocatechin gallate) could inhibit CML formation in the model imitating the condition of green tea processing, though the inhibitory efficiency was reduced by transition metals. This suggested that catechins could inhibit CML formation in the real tea system, though the inhibitory efficiency may be reduced by tea components which promote its synthesis. However, CEL formation was not always inhibited by the tested catechins, though catechins could significantly decrease the content of methylglyoxal which is considered an important intermediate. Consequently, the main pathway of CEL formation may not be through methylglyoxal.

Determination of glyoxal, methylglyoxal, and diacetyl in red ginseng products using dispersive liquid-liquid microextraction coupled with GC-MS.

A simple and rapid dispersive liquid-liquid microextraction method coupled with gas chromatography and mass spectrometry was applied for the determination of glyoxal as quinoxaline, methylglyoxal as 2-methylquinoxaline, and diacetyl as 2,3-dimethylquinoxaline in red ginseng products. The performance of the proposed method was evaluated under optimum extraction conditions (extraction solvent: chloroform 100 µL, disperser solvent: methanol 200 µL, derivatizing agent concentration: 5 g/L, reaction time: 1 h, and no addition of salt). The limit of detection and limit of quantitation were 1.30 and 4.33 µg/L for glyoxal, 1.86 and 6.20 µg/L for methylglyoxal, and 1.45 and 4.82 µg/L for diacetyl. The intra- and interday relative standard deviations were <4.95 and 5.80%, respectively. The relative recoveries were 92.4-103.9% in red ginseng concentrate and 99.4-110.7% in juice samples. Red ginseng concentrates were found to contain 191-4274 µg/kg of glyoxal, 1336-4798 µg/kg of methylglyoxal, and 0-830 µg/kg of diacetyl, whereas for red ginseng juices, the respective concentrations were 72-865, 69-3613, and 6-344 µg/L.

Effect of hydroxytyrosol and olive leaf extract on 1,2-dicarbonyl compounds, hydroxymethylfurfural and advanced glycation endproducts in a biscuit model.

The antiglycative activity of hydroxytyrosol (HT) and olive leaf extract (OLE) was investigated in wheat-flour biscuits. Quercetin (QE) and gallic acid (GA) were used as reference of antiglycative activity of phenolic compounds. HT, OLE, QE and GA were added in the range of 0.25-0.75% (w/w). Samples were compared against a control recipe baked at 180°C/20min. HT biscuit was able to inhibit efficiently the formation of hydroxymethylfurfural (HMF) and 3-deoxyglucosone (3-DG), as well as reduced the formation of overall free fluorescent AGEs and pentosidine. The inhibition of the 3-DG and HMF formation was directly and significantly correlated under controlled baking conditions. However, samples formulated with OLE exerted similar antiglycative capacity against pentosidine and Nε-carboxyethyl-lysine, although the amount of HT in the biscuit was 100-fold lower than the biscuit formulated with HT. Methylglyoxal, 3-DG, and glyoxal were the predominant 1,2-dicarbonyl compounds after baking but only 3-DG was significantly reduced by HT.

Glucitol-core containing gallotannins inhibit the formation of advanced glycation end-products mediated by their antioxidant potential.

Glucitol-core containing gallotannins (GCGs) are polyphenols containing galloyl groups attached to a 1,5-anhydro-d-glucitol core, which is uncommon among naturally occurring plant gallotannins. GCGs have only been isolated from maple (Acer) species, including the red maple (Acer rubrum), a medicinal plant which along with the sugar maple (Acer saccharum), are the major sources of the natural sweetener, maple syrup. GCGs are reported to show antioxidant, α-glucosidase inhibitory, and antidiabetic effects, but their antiglycating potential is unknown. Herein, the inhibitory effects of five GCGs (containing 1-4 galloyls) on the formation of advanced glycation end-products (AGEs) were evaluated by MALDI-TOF mass spectroscopy, and BSA-fructose, and G.K. peptide-ribose assays. The GCGs showed superior activities compared to the synthetic antiglycating agent, aminoguanidine (IC50 15.8-151.3 vs. >300 µM) at the early, middle, and late stages of glycation. Circular dichroism data revealed that the GCGs were able to protect the secondary structure of BSA protein from glycation. The GCGs did not inhibit AGE formation by the trapping of reactive carbonyl species, namely, methylglyoxal, but showed free radical scavenging activities in the DPPH assay. The free radical quenching properties of the GCGs were further confirmed by electron paramagnetic resonance spectroscopy using ginnalin A (contains 2 galloyls) as a representative GCG. In addition, this GCG chelated ferrous iron, an oxidative catalyst of AGE formation, supported a potential antioxidant mechanism of antiglycating activity for these polyphenols. Therefore, GCGs should be further investigated for their antidiabetic potential given their antioxidant, α-glucosidase inhibitory, and antiglycating properties.

Honey - a potential agent against Porphyromonas gingivalis: an in vitro study.

BACKGROUND: Honey has been discussed as a therapeutic option in wound healing since ancient time. It might be also an alternative to the commonly used antimicrobials in periodontitis treatment. The in-vitro study was aimed to determine the antimicrobial efficacy against Porphyromonas gingivalis as a major periodontopathogen. METHODS: One Manuka and one domestic beekeeper honey have been selected for the study. As a screening, MICs of the honeys against 20 P. gingivalis strains were determined. Contents of methylglyoxal and hydrogen peroxide as the potential antimicrobial compounds were determined. These components (up to 100 mg/l), propolis (up to 200 mg/l) as well as the two honeys (up to 10% w/v) were tested against four P. gingivalis strains in planktonic growth and in a single-species biofilm. RESULTS: 2% of Manuka honey inhibited the growth of 50% of the planktonic P. gingivalis, the respective MIC50 of the German beekeeper honey was 5%. Manuka honey contained 1.87 mg/kg hydrogen peroxide and the domestic honey 3.74 mg/kg. The amount of methylglyoxal was found to be 2 mg/kg in the domestic honey and 982 mg/kg in the Manuka honey. MICs for hydrogen peroxide were 10 mg/l - 100 mg/l, for methylglyoxal 5 - 20 mg/l, and for propolis 20 mg/l - 200 mg/l. 10% of both types of honey inhibited the formation of P. gingivalis biofilms and reduced the numbers of viable bacteria within 42 h-old biofilms. Neither a total prevention of biofilm formation nor a complete eradication of a 42 h-old biofilm by any of the tested compounds and the honeys were found. CONCLUSIONS: Honey acts antibacterial against P. gingivalis. The observed pronounced effects of Manuka honey against planktonic bacteria but not within biofilm can be attributed to methylglyoxal as the characteristic antimicrobial component.

Methylglyoxal may affect hydrogen peroxide accumulation in manuka honey through the inhibition of glucose oxidase.

Although hydrogen peroxide (H2O2) is one of the major antibacterial factors in most honeys, it does not accumulate in medical-grade manuka honey. The goal of this study was to investigate the effect of artificially added methylglyoxal (MGO) on H2O2 accumulation in natural non-manuka honeys. H2O2 concentrations in the honey solutions were determined using a fluorimetric assay. Two, the most potent H2O2 producers honeydew honeys were mixed with MGO at final concentrations of 250, 500, and 1000 mg/kg, and incubated for 4 days at 37°C. Subsequently, H2O2 concentrations were determined in 50% (wt/vol) MGO supplemented honey solutions. In vitro crosslinking of the enzyme glucose oxidase (GOX) after incubation with MGO was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Tested honeys at a concentration of 50% (wt/vol) accumulated up to 495.8±9.1 µM H2O2 in 24 h. The most potent producers were the two honeydew honeys, whose 50% solutions accumulated 306.9±6.8 and 495.8±9.1 µM H2O2, respectively. Levels of H2O2 increased significantly over time in both honey solutions. Contrary to this, the MGO-treated honeys generated significantly lower amounts of H2O2 (P<.001), and this reduction was dose dependent. In addition, MGO-treated GOX formed high molecular weight adducts with increasing time of incubation accompanied by loss of its enzymatic activity. High levels of MGO in manuka honey, by modifying the enzyme GOX, might be responsible for suppressing H2O2 generation. These data highlight the detrimental effect of MGO on significant proteinaceous components of manuka honey.

Pre-column incubation followed by fast liquid chromatography analysis for rapid screening of natural methylglyoxal scavengers directly from herbal medicines: case study of Polygonum cuspidatum.

Methylglyoxal (MGO), a very reactive metabolite of glucose, plays a pivotal role in the pathogenesis of several chronic diseases associated with diabetes, and it has been validated as an attractive target for them. In the present study, a simple and effective method, namely pre-column incubation followed by fast high performance liquid chromatography based on superficially porous particles (shell), coupled with diode array detection and tandem mass spectrometry (UHPLC-DAD-MS(n)), was proposed for rapid and high-throughput screening of natural MGO scavengers directly from the crude extract of Polygonum cuspidatum Sieb. et Zucc, a well-known traditional Chinese medicine which was used for treatment of diabetic complications. The hypothesis is that upon reaction with MGO, the peak areas of components with MGO scavenging potency in the chromatogram will be significantly reduced or disappear, and the structural characterization could be achieved by UHPLC-DAD-MS(n) hyphenated technique. First of all, 12 compounds in P. cuspidatum were well separated within shorter time (~12 min) than previous methods and identified, and two of them, i.e. 3,5,4'-trihydroxystilbene-3-O-(6″-galloyl)-glucoside (3) and emodin-8-O-(6'-malonyl)-glucoside (8) were firstly reported ingredients. After incubation with MGO, four stilbene derivatives were demonstrated to possess potential MGO trapping activities. Furthermore, it was proved that both polydatin (piceid) and resveratrol exhibited effective MGO-trapping capacity by UHPLC analysis, and they could significantly inhibit the formation of advanced glycation end products (AGEs) in the human serum albumin (HSA)-MGO assay, indicating that they were potential candidate agents for delaying and preventing diabetic complications. Additionally, MGO trapping mechanism exploration by UHPLC-MS(n) showed that the positions 2 and 4 of the A ring of stilbene were major active sites for trapping MGO to form both mono- and di-MGO adducts, however, the glucosylation of the hydroxyl group would significantly decrease the trapping efficiency. Collectively, the current work provides a very promising method for rapid discovery of natural MGO scavengers directly from complex matrices such as herbal medicines with huge resources.

Methylglyoxal content in drinking coffee as a cytotoxic factor.

A causal relationship between metabolic syndrome and methylglyoxal (MG) has been suggested. Consumption of coffee and other types of beverages has been known to produce MG, thus resulting in both nutritional and health concerns. The purpose of this study was to determine the ideal combination of coffee, cream, and sugar in order to minimize MG consumption. Four types of black coffee were tested: espresso, bold, mild, and a decaffeinated mild roast. Sugar and/or cream were added to the coffee samples to test whether MG levels were altered. Using high-performance liquid chromatography, the concentration of MG in various coffee samples was determined. The espresso coffee sample was found to contain the highest level of MG at 230.9 microM. The bold coffee roast had the 2nd highest amount of MG, followed by the mild and decaffeinated varieties. Adding cream to bold coffee significantly reduced its MG level in comparison to the coffee sample without cream. On the other hand, the addition of sugar to the bold coffee did not further increase the MG level in the samples. The cellular damaging effect of MG was shown as there were decreased numbers of cultured HEK-293 cells after 24 h of MG treatment (100 and 300 microM), which is consistent with an increased cell apoptosis induced by MG treatment (100 and 300 microM). Due to the overconsumption of exogenous MG, drinking an excess of any type of coffee poses health risks.

Identification of 4-oxo-2-hexenal and other direct mutagens formed in model lipid peroxidation reactions as dGuo adducts.

We searched for mutagens that react with 2'-deoxyguanosine (dGuo) in model systems of lipid peroxidation. To autoxidation systems of methyl linoleate (model of omega-6 fat), methyl alpha-linolenate (MLN) (model of omega-3 fat), and commercial salad oil, dGuo was added. The reaction mixtures were analyzed by HPLC. Six adducts were detected, and their structures were determined by 1H and 13C NMR, UV, and mass spectra and by comparison with synthetic authentic samples. The mutagens that reacted with dGuo to form these adducts were proposed as glyoxal, glyoxylic acid, ethylglyoxal, and 4-oxo-2-hexenal (4-OHE). The formation of 8-hydroxy-dGuo, an oxidized product of dGuo, was also detected in the model reaction mixtures. Among them, glyoxal and glyoxylic acid are known mutagens, while ethylglyoxal and 4-OHE, produced from MLN, have not been reported as mutagens thus far. We confirmed the mutagenic activity of 4-OHE with Salmonella strains, TA100 and TA104, without S9 mix. These compounds may be involved in lipid peroxide-related cancers.

Contribution of coffee aroma constituents to the mutagenicity of coffee.

About 40 coffee aroma constituents belonging to the classes of dicarbonyls, sulphur-containing compounds, furfuryls, N-heterocyclics and others were systematically evaluated in three Ames tester strains. Only aliphatic dicarbonyl compounds showed notable direct mutagenic activity, which mainly affected 'base-pair substitution' in Ames tester strains TA100 and TA102. Very weak effects were also seen with some N-heterocyclics, mainly affecting frameshift tester strain TA98 upon metabolic activation. However, it was shown that these N-heterocyclics do not contribute substantially to the mutagenicity in coffee. The hydrogen peroxide and methylglyoxal contents of coffee were determined up to 26 hr after preparation. Their concentrations tended to decrease whereas mutagenic activity decreased significantly with time in tester strains TA100 and TA102. It is concluded that several highly labile coffee constituents contribute to the bacterial mutagenicity and also that the synergism between hydrogen peroxide and methylglyoxal is not the main factor. The absence of coffee mutagenicity/carcinogenicity in rodents with these highly reactive coffee aroma compounds can be explained in part by detoxification of microsomal enzyme systems.

Simple and sensitive determination of methylglyoxal in biological samples by gas chromatography with electron-capture detection.

Methylglyoxal was allowed to react with 4,5-dichloro-1,2-phenylenediamine, and the 6,7-dichloro-2-methylquinoxaline formed was determined by gas chromatography with electron-capture detection. The standard curve of the quinoxaline was linear up to 160 pmol/ml. The recoveries of methylglyoxal from coffee and rat liver homogenate were 84.1 and 77.6%, respectively. This procedure was very selective and so sensitive that as little as 9 fmol of the quinoxaline could be measured in biological and food samples.

Methylglyoxal in beverages and foods: its mutagenicity and carcinogenicity.

Methylglyoxal was found to induce mutations in Salmonella typhimurium strains TA100, TA102 and TA104, gene conversion in Saccharomyces cerevisiae strain D7, and diphtheria toxin resistance mutation in Chinese hamster lung cells. Methylglyoxal was detected in coffee, whiskey, a soft drink, toasted bread and soy sauce. The mutagenicity of methylglyoxal was suppressed by sulfite, cysteine, glutathione and dithiothreitol, and enhanced by hydrogen peroxide. Methylglyoxal induced sarcomas in rats at the site of its subcutaneous injection.