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).

Utilizing methylglyoxal and D-lactate in urine to evaluate saikosaponin C treatment in mice with accelerated nephrotoxic serum nephritis.

The relationship between methylglyoxal (MGO) and D-lactate during saikosaponin C (SSC) treatment of mice with accelerated nephrotoxic serum (NTS) nephritis was investigated. NTS nephritis was induced by administration of anti-basement membrane antibodies to C57BL/6 mice and three dosages of SSC were administered for 14 days. Proteinuria, blood urea nitrogen, serum creatinine, renal histology, urinary MGO and d-lactate changes were examined. Compared to the NTS control group, the middle dosage (10 mg/kg/day) of SSC significantly alleviated the development of nephritis based on urine protein measurements (34.40 ± 6.85 vs. 17.33 ± 4.79 mg/day, p<0.05). Pathological observation of the glomerular basement membrane (GBM) revealed monocyte infiltration, hypertrophy, and crescents were alleviated, and injury scoring also showed improved efficacy for the middle dose of SSC during nephritis (7.92 ± 1.37 vs. 3.50 ± 1.14, p<0.05). Moreover, the significant decreases in urinary levels of MGO (24.71 ± 3.46 vs. 16.72 ± 2.36 µg/mg, p<0.05) and D-lactate (0.31 ± 0.04 vs. 0.23 ± 0.02 µmol/mg, p<0.05) were consistent with the biochemical and pathological examinations. This study demonstrates that MGO and D-lactate may reflect the extent of damage and the efficacy of SSC in NTS nephritis; further studies are required to enable clinical application.

MG-HCr, the Methylglyoxal-Derived Hydroimidazolone of Creatine, a Biomarker for the Dietary Intake of Animal Source Food.

In the course of the Maillard reaction in vivo or in food, creatine reacts with the 1,2-dicarbonyl compound methylglyoxal to N-(4-methyl-5-oxo-1-imidazolin-2-yl)sarcosine (MG-HCr). We studied whether the urinary excretion of MG-HCr is affected by its intake with meat or by the intake of creatine and subsequent in vivo formation of MG-HCr. Therefore, 24 h urine of 30 subjects with different dietary habits was analyzed with HPLC-MS/MS. The daily MG-HCr excretion via urine varied between omnivores (0.39-9.67 µmol/day, n = 24), vegetarians (0.18-0.97 µmol/day, n = 19), and vegans (0.10-0.27 µmol/day, n = 8). An intervention study with 18 subjects demonstrated the bioavailability of MG-HCr (ca. 54%) from 200 g of heated meat and its quick excretion with urine. A creatine intervention of 0.44 g did not increase MG-HCr excretion. Thus, the differences in MG-HCr excretion between different diets are mainly caused by the dietary uptake of MG-HCr. We additionally found MG-HCr in milk and egg products, where it is formed during heat treatment. This partly explains differences in MG-HCr excretion of vegetarians and vegans. Hence, MG-HCr in urine is a short-term marker for the intake of heat-processed animal source food.

Effect of prednisolone on glyoxalase 1 in an inbred mouse model of aristolochic acid nephropathy using a proteomics method with fluorogenic derivatization-liquid chromatography-tandem mass spectrometry.

Prednisolone is involved in glucose homeostasis and has been used for treatment for aristolochic acid (AA) nephropathy (AAN), but its effect on glycolysis in kidney has not yet been clarified. This study aims to investigate the effect in terms of altered proteins after prednisolone treatment in a mice model of AAN using a proteomics technique. The six-week C3H/He female mice were administrated AA (0.5 mg/kg/day) for 56 days. AA+P group mice were then given prednisolone (2 mg/kg/day) via oral gavage for the next 14 days, and AA group mice were fed water instead. The tubulointerstitial damage was improved after prednisolone treatment comparing to that of AA group. Kidney homogenates were harvested to perform the proteomics analysis with fluorogenic derivatization-liquid chromatography-tandem mass spectrometry method (FD-LC-MS/MS). On the other hand, urinary methylglyoxal and D-lactate levels were determined by high performance liquid chromatography with fluorescence detection. There were 47 altered peaks and 39 corresponding proteins on day 14 among the groups, and the glycolysis-related proteins, especially glyoxalase 1 (GLO1), fructose-bisphosphate aldolase B (aldolase B), and triosephosphate isomerase (TPI), decreased in the AA+P group. Meanwhile, prednisolone decreased the urinary amount of methylglyoxal (AA+P: 2.004 ± 0.301 µg vs. AA: 2.741 ± 0.630 µg, p < 0.05), which was accompanied with decrease in urinary amount of D-lactate (AA+P: 54.07 ± 5.45 µmol vs. AA: 86.09 ± 8.44 µmol, p < 0.05). Prednisolone thus alleviated inflammation and interstitial renal fibrosis. The renal protective mechanism might be associated with down-regulation of GLO1 via reducing the contents of methylglyoxal derived from glycolysis. With the aid of proteomics analysis and the determination of methylglyoxal and its metabolite-D-lactate, we have demonstrated for the first time the biochemical efficacy of prednisolone, and urinary methylglyoxal and its metabolite-D-lactate might be potential biomarkers for AAN.

Glyoxal and methylglyoxal as urinary markers of diabetes. Determination using a dispersive liquid-liquid microextraction procedure combined with gas chromatography-mass spectrometry.

Glyoxal (GO) and methylglyoxal (MGO) are α-oxoaldehydes that can be used as urinary diabetes markers. In this study, their levels were measured using a sample preparation procedure based on salting-out assisted liquid-liquid extraction (SALLE) and dispersive liquid-liquid microextraction (DLLME) combined with gas chromatography-mass spectrometry (GC-MS). The effect of the derivatization reaction with 2,3-diaminonaphthalene, the addition of acetonitrile and sodium chloride to urine, and the DLLME step using the acetonitrile extract as dispersant solvent and carbon tetrachloride as extractant solvent were carefully optimized. Quantification was performed by the internal standard method, using 5-bromo-2-chloroanisole. The intraday and interday precisions were lower than 6%. Limits of detection were 0.12 and 0.06ngmL-1, and enrichment factors 140 and 130 for GO and MGO, respectively. The concentrations of these α-oxoaldehydes in urine were between 0.9 and 35.8ngg-1 levels (creatinine adjusted). A statistical comparison of the analyte contents of urine samples from non-diabetic and diabetic patients pointed to significant differences (P=0.046, 24 subjects investigated), particularly regarding MGO, which was higher in diabetic patients. The novelty of this study compared with previous procedures lies in the treatment of the urine sample by SALLE based on the addition of acetonitrile and sodium chloride to the urine. The DLLME procedure is performed with a sedimented drop of the extractant solvent, without a surfactant reagent, and using acetonitrile as dispersant solvent. Separation of the analytes was performed using GC-MS detection, being the analytes unequivocal identified. The proposed procedure is the first microextraction method applied to the analysis of urine samples from diabetic and non-diabetic patients that allows a clear differentiation between both groups using a simple analysis.

Glyoxal and methylglyoxal determination in urine by surfactant-assisted dispersive liquid-liquid microextraction and LC.

AIM: Two important markers of oxidative stress, glyoxal and methylglyoxal, are preconcentrated from human urine by surfactant-assisted dispersive liquid-liquid microextraction and separated by LC-fluorescence. METHODS/RESULTS: Derivatization was carried out overnight with 0.8 mM 2,3-diaminonaphthalene at 4°C. For surfactant-assisted dispersive liquid-liquid microextraction, 500 µl buffer solution (pH 10.5) and 25 µl 0.03 M Triton X-114 were added to 2.5 ml of the sample and the mixture was made up to 10 ml before the rapid injection of 75 µl 1-undecanol (extractant solvent) and 0.5 ml ethanol (dispersant solvent). CONCLUSION: The method can be applied to analyze glyoxal and methylglyoxal in urine with LOD of 13 and 16 ng/l, respectively, and recoveries in the 88-103% range.

Diet low in advanced glycation end products increases insulin sensitivity in healthy overweight individuals: a double-blind, randomized, crossover trial.

BACKGROUND: The consumption of advanced glycation end products (AGEs) has increased because of modern food processing and has been linked to the development of type 2 diabetes in rodents. OBJECTIVE: We determined whether changing dietary AGE intake could modulate insulin sensitivity and secretion in healthy, overweight individuals. DESIGN: We performed a double-blind, randomized, crossover trial of diets in 20 participants [6 women and 14 men; mean ± SD body mass index (in kg/m(2)): 29.8 ± 3.7]. Isoenergetic- and macronutrient-matched diets that were high or low in AGE content were alternately consumed for 2 wk and separated by a 4-wk washout period. At the beginning and end of each dietary period, a hyperinsulinemic-euglycemic clamp and an intravenous glucose tolerance test were performed. Dietary, plasma and urinary AGEs N(€)-(carboxymethyl)lysine (CML), N(€)-(carboxyethyl)lysin (CEL), and methylglyoxal-derived hydroimadazolidine (MG-H1) were measured with the use of mass spectrometry. RESULTS: Participants consumed less CML, CEL, and MG-H1 during the low-AGE dietary period than during the high-AGE period (all P < 0.05), which was confirmed by changes in urinary AGE excretion. There was an overall difference in insulin sensitivity of -2.1 mg · kg(-1) · min(-1) between diets (P = 0.001). Insulin sensitivity increased by 1.3 mg · kg(-1) · min(-1) after the low-AGE diet (P = 0.004), whereas it showed a tendency to decrease by 0.8 mg · kg(-1) · min(-1) after the high-AGE diet (P = 0.086). There was no difference in body weight or insulin secretion between diets (P = NS). CONCLUSIONS: A diet that is low in AGEs may reduce the risk of type 2 diabetes by increasing insulin sensitivity. Hence, a restriction in dietary AGE content may be an effective strategy to decrease diabetes and cardiovascular disease risks in overweight individuals. This trial was registered at clinicaltrials.gov as NCT00422253.

Trapping Methylglyoxal by Genistein and Its Metabolites in Mice.

Increasing evidence supports dicarbonyl stress such as methylglyoxal (MGO) as one of the major pathogenic links between hyperglycemia and diabetic complications. In vitro studies have shown that dietary flavonoids can inhibit the formation of advanced glycation end products (AGEs) by trapping MGO. However, whether flavonoids can trap MGO in vivo and whether biotransformation limits the trapping capacity of flavonoids remain virtually unknown. In this study, we investigated whether genistein (GEN), the major soy isoflavone, could trap MGO in mice by promoting the formation of MGO adducts of GEN and its metabolites. Two different mouse studies were conducted. In the acute study, a single dose of MGO and GEN were administered to mice via oral gavage. In the chronic study, MGO was given to mice in drinking water for 1 month and then GEN was given to mice for 4 consecutive days via oral gavage. Two mono-MGO adducts of GEN and six mono-MGO adducts of GEN phase I and microbial metabolites were identified in mouse urine samples from these studies using liquid chromatography/electrospray ionization tandem mass spectrometry. The structures of these MGO adducts were confirmed by analyzing their MS(n) (n = 1-4) spectra as well as by comparing them with the tandem mass spectra of authentic standards. All of the MGO adducts presented in their phase II conjugated forms in mouse urine samples in the acute and chronic studies. To our knowledge, this is the first in vivo evidence to demonstrate the trapping efficacy of GEN in mice and to show that the metabolites of GEN remain bioactive.

Elucidation of the etiology and characteristics of pink urine in young healthy subjects.

BACKGROUND: Pink urine syndrome (PUS) is attributed to the precipitation of uric acid caused by low urinary pH (U-pH). However, the reasons for the lower U-pH are unclear. OBJECTIVES: To investigate the occurrence of PUS and verified the cause of U-pH reduction. METHODS: Participants comprised 4,940 students who had undergone a physical examination. Data on the presence [PUS (+)] or absence [PUS (-)] of PUS, as well as age, gender, body mass index (BMI), blood pressure (BP), heart rate (HR), and U-pH were collected. Of these participants, 300 randomly selected individuals were evaluated for their waist circumference, as well as their levels of urinary C-peptide, angiotensinogen, methylglyoxal, thiobarbituric acid-reactive substances (TBARS), and Na(+) excretion. Independent risk factors of lower U-pH were decided by a multiple-regression analysis. RESULTS: PUS was observed in 216 students (4.4 %). A greater number of men comprised the PUS (+) group compared with the PUS (-) group, and subjects in this group had high BMI, BP, and HR values, as well as low U-pH. A logistic regression analysis revealed that the BMI and U-pH were independent risk factors for PUS (+). The decrease of U-pH was closely related to the progress of chronic kidney disease (CKD). BMI value was related to PUS (+) in the CKD (-) subjects. On the other hand, low U-pH was related to PUS (+) in the CKD (+) subjects. All factors other than HR showed a significant negative correlation with U-pH. However, multiple-regression analysis revealed that TBARS and angiotensinogen were independent risk factors. CONCLUSION: Obesity and lower U-pH were each independently related to PUS, whereas increased intrarenal oxidative stress and exacerbation of the renin-angiotensin system activation were associated with the lowering of U-pH. U-pH low value is related to potential CKD.

Methylglyoxal (MG) and cerebro-renal interaction: does long-term orally administered MG cause cognitive impairment in normal Sprague-Dawley rats?

Methylglyoxal (MG), one of the uremic toxins, is a highly reactive alpha-dicarbonyl compound. Recent clinical studies have demonstrated the close associations of cognitive impairment (CI) with plasma MG levels and presence of kidney dysfunction. Therefore, the present study aims to examine whether MG is a direct causative substance for CI development. Eight-week-old male Sprague-Dawley (SD) rats were divided into two groups: control (n = 9) and MG group (n = 10; 0.5% MG in drinking water), and fed a normal diet for 12 months. Cognitive function was evaluated by two behavioral tests (object exploration test and radial-arm maze test) in early (4-6 months of age) and late phase (7-12 months of age). Serum MG was significantly elevated in the MG group (495.8 ± 38.1 vs. 244.8 ± 28.2 nM; p < 0.001) at the end of study. The groups did not differ in cognitive function during the course of study. No time-course differences were found in oxidative stress markers between the two groups, while, antioxidants such as glutathione peroxidase and superoxide dismutase activities were significantly increased in the MG group compared to the control. Long-term MG administration to rats with normal kidney function did not cause CI. A counter-balanced activation of the systemic anti-oxidant system may offset the toxicity of MG in this model. Pathogenetic significance of MG for CI requires further investigation.

High-performance liquid chromatography determination of glyoxal, methylglyoxal, and diacetyl in urine using 4-methoxy-o-phenylenediamine as derivatizing reagent.

Bioanalytical relevance of glyoxal (Go) and methylglyoxal (MGo) arises from their role as biomarkers of glycation processes and oxidative stress. The third compound of interest in this work is diacetyl (DMGo), a component of different food products and alcoholic beverages and one of the small α-ketoaldehydes previously reported in urine. The original idea for the determination of the above compounds by reversed phase high-performance liquid chromatography (HPLC) with fluorimetric detection was to use 4-methoxy-o-phenylenediamine (4MPD) as a derivatizing reagent and diethylglyoxal (DEGo) as internal standard. Acetonitrile was added to urine for matrix precipitation, and derivatization reaction was carried out in the diluted supernatant at neutral pH (40 °C, 4 h); after acidification, salt-induced phase separation enabled recovery of the obtained quinoxalines in the acetonitrile layer. The separation was achieved within 12 min using a C18 Kinetex column and gradient elution. The calibration detection limits for Go, MGo, and DMGo were 0.46, 0.39, and 0.28 µg/L, respectively. Within-day precision for real-world samples did not exceed 6%. Several urine samples from healthy volunteers, diabetic subjects, and juvenile swimmers were analyzed. The sensitivity of the procedure proposed here enabled detection of differences between analyte concentrations in urine from patients at different clinical or exposure-related conditions.

Metabolic transit of dietary methylglyoxal.

Methylglyoxal (MGO) is responsible for the pronounced antibacterial activity of manuka honey, in which it may reach concentrations up to 800 mg/kg. As MGO formed in vivo is discussed to play a role in diabetic complications, the metabolic transit of dietary MGO was studied within a 3 day dietary recall with four healthy volunteers. Determination of MGO in 24 h urine was performed with GC-MS after derivatization with O-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine, and D-lactate was quantified enzymatically. Following a diet virtually free from MGO and other glycation compounds, a defined amount of MGO (500 µmol in manuka honey) was administered in the morning of day 2. Renal excretion was between 0.1 and 0.4 µmol/day for MGO and between 50 and 220 µmol/day for D-lactate. No influence on excretion of both compounds was observed following administration of MGO. To investigate the stability of MGO under physiological conditions, a simulated in vitro gastrointestinal digestion was performed with MGO-containing honey. After 8 h of in vitro digestion, only 5-20% of the initial methylglyoxal was recovered. This indicates that dietary MGO is rapidly degraded during the digestion process in the intestine and, therefore, exerts no influence on the MGO level in vivo.

Relationship of methylglyoxal-adduct biogenesis to LDL and triglyceride levels in diabetics.

AIMS: Protein glycation leading to advanced glycation-endproducts (AGE) is enhanced in diabetes by increased blood glucose and collateral endogenous production of reactive α-dicarbonyls. Among AGE precursors, methylglyoxal (MG) is considered as one of the key intermediates. We hypothesized it to be a common product of both carbonyl and oxidative stress, and investigated its biogenesis in relation to glycemic and lipid status in diabetic patients. METHODS: Serum and urine MG-adducts were measured by competitive immunofluorometric assay in 83 diabetic and 20 healthy subjects. KEY FINDINGS: A significant association of MG-adducts serum level with LDL (r=0.31;p=0.003) was observed. A correlation between LDL-c, HDL-C and PPG as independent variables and serum MG-adducts as a dependent variable was found (p<0.014) using multiple stepwise regression, whereas urine albumin/creatinine ratio was independently associated with urine MG-adducts. LDL cut-off >3.0mmol/l discriminated patients with higher serum MG-adducts (p=0.0052), although there was no between-subgroup difference in glycemic control. Patients on statin therapy had a lower MG-adduct level. The positive relationship between LDL-c and MG-adducts (r=0.38;p=0.042) was noted in patients free of statin treatment, whereas an inverse tendency was found in the statin-treated subgroup. SIGNIFICANCE: Significant relationship between LDL and MG-adduct production, as well as tight correlation between triglycerides and urinary MG-adduct excretion suggest that the lipoxidation and glyceraldehyde-3-phosphate route, along with the glycolytic pathway, might be an important source of MG generation. The glycotoxin methylglyoxal seems to be a common factor linking hyperglycemia and intensive lipolysis, two dominant metabolic changes in diabetes.

A novel capillary electrophoretic method for determining methylglyoxal and glyoxal in urine and water samples.

Two non-electroactive biomarkers methylglyoxal (MGo) and glyoxal (Go) in urine and environmental water samples were determined for the first time by capillary electrophoresis with amperometric detection (CE-AD) after derivatizing with an electroactive compound 2-thiobarbituric acid. Experimental conditions of derivatization and CE-AD detection were optimized. Highly linear response was obtained for these two biomarkers over three orders of magnitude with good correlation (r(2)>0.999). The limits of detection (LODs) and limits of quantitation (LOQs) of MGo and Go were 0.2microgL(-1) and 1.0microgL(-1), 0.5microgL(-1) and 2.0microgL(-1), respectively. The average recovery and relative standard deviation (RSD) were within the range of 90.9-101.3% and 0.7-2.2%, respectively. The proposed CE-AD method provides a reliable and sensitive quantitative evaluation of MGo and Go in real sample matrices by employing relatively simple and inexpensive instrument.

High-performance liquid chromatographic determination of glyoxal and methylglyoxal in urine by prederivatization to lumazinic rings using in serial fast scan fluorimetric and diode array detectors.

Glyoxal and methylglyoxal are two important markers of oxidative stress and both are involved in the evaluation of several diseases. A new HPLC method for determining glyoxal and methylglyoxal in urine was developed. The method is based on the reaction of alpha-dialdehydes, glyoxal and methylglyoxal, with 5,6-diamino-2,4-hydroxypyrimidine sulfate in basic medium to form highly fluorescent lumazine derivatives. Creatinine was also included in the method even though it does not react with the reagent. The derivatives and creatinine are separated on a C(18) reversed-phase column with a mobile phase consisting of acetonitrile:citrate buffer, pH 6.0 (3:97 v/v). The flow rate was 1.0mLmin(-1) and the effluent was monitored photometrically at 250 nm for determination of creatinine and fluorimetrically at 500 nm (exciting at 330 nm) for determination of glyoxal and methylglyoxal derivatives. Recording time of the separation is less than 10 min. Determination of the analytes is performed in urine after incubation of the sample, with the reagent in alkaline medium, for 30 min at 60 degrees C. Urinary levels of glyoxal and methylglyoxal, expressed as glyoxal/creatinine and methylglyoxal/creatinine ratios, in healthy young women and men were determined. For women, values of 0.80+/-0.37 and 0.60+/-0.22 microg/mg of creatinine were found for glyoxal and methylglyoxal, respectively. For men, values of 0.63+/-0.15 and 0.49+/-0.05 microg/mg of creatinine were found for glyoxal and methylglyoxal, respectively. These results were also related to the body mass index of each individual.

Determination of urinary glyoxal and methylglyoxal by high-performance liquid chromatography.

Carbonyl stress compounds such as glyoxal and methylglyoxal have been recently attracting much attention because of their possible clinical significance in chronic and age-related diseases. A high-performance liquid chromatographic procedure has been developed for the simultaneous quantitation of glyoxal and methylglyoxal in human urine. The assay is based on the reaction of these compounds with 1,2-diamino-4,5-dimethoxybenzene to form fluorescent adducts, which are separated by reversed-phase high-performance liquid chromatography in a total run time of 45 minutes and quantitated fluorometrically using 2,3-pentanedione as an internal standard. Derivatization is performed for diluted urine (100-120 mOsm/kg H2O) under acidic conditions (pH 4.5) at 60 degrees C over a prolonged time (15 h) to maximize the yields. The assay is specific and sensitive enough to analyze urinary levels of glyoxal and methylglyoxal with the within- and between-day relative standard deviations of less than 5%. Urinary levels (mean +/- standard deviation, n = 16) of glyoxal and methylglyoxal in healthy subjects were 4.7 +/- 1.35 microg/mg creatinine, 2.2 +/- 0.65 microg/mg creatinine, respectively, the former being 2 to 3 times more than the latter in every subject. The glyoxal and methylglyoxal levels positively correlated with each other, which may suggest that the levels reflect the individual activity of glyoxalase by which both compounds are detoxified.

Simultaneous determination of formaldehyde and methylglyoxal in urine: involvement of semicarbazide-sensitive amine oxidase-mediated deamination in diabetic complications.

The deamination of methylamine and aminoacetone by semicarbazide-sensitive amine oxidase (SSAO) produces formaldehyde and methylglyoxal, respectively, which have been presumed to be involved in diabetic complications. A high-performance liquid chromatography procedure using 2,4-dinitrophenylhydrazine (DNPH) as a derivatizing agent is developed to determine endogenous formaldehyde, methylglyoxal, malondialdehyde, and acetaldehyde. The devised DNPH method is sensitive enough to analyze aldehyde levels in urine. An increase in the excretion of formaldehyde, methylglyoxal, and malondialdehyde is confirmed in streptozotocin-induced diabetic rats. Following the chronic administration of methylamine, the urinary levels of both formaldehyde and malondialdehyde (a product from lipid peroxidation) are found to be substantially increased. A potent selective SSAO inhibitor, (E)-2-(4-fluorophenethyl)-3-fluoroallylamine hydrochloride (MDL-72974A), reduced the formation of formaldehyde, methylglyoxal, and malondialdehyde. The increase of the cytotoxic aldehyde levels as a result of increased SSAO-mediated deamination may occur in some pathological conditions.

Deamination of methylamine and aminoacetone increases aldehydes and oxidative stress in rats.

Semicarbazide-sensitive amine oxidase (SSAO)-mediated deamination of methylamine and aminoacetone in vitro produces carbonyl compounds, such as formaldehyde and methylglyoxal, which have been proposed to be cytotoxic and may be responsible for some pathological conditions. An HPLC procedure was developed to assess different aldehydes, which were derivatized with 2,4-dinitrophenylhydrazine (DNPH). We have demonstrated in vivo deamination of methylamine and aminoacetone by examining the excretion of formaldehyde and methylglyoxal, respectively, in rats. Following chronic administration of methylamine, the urinary level of malondialdehyde (MDA), an end product of lipid peroxidation, was also found to be substantially increased. A selective SSAO inhibitor blocked the increase of MDA. The results support the idea that increased SSAO-mediated deamination of methylamine and aminoacetone can be a potential cytotoxic risk factor.

High-performance liquid chromatographic-fluorometric determination of glyoxal, methylglyoxal, and diacetyl in urine by prederivatization to pteridinic rings.

A sensitive and simple liquid chromatographic method to determine glyoxal, methylglyoxal, and diacetyl is reported. The method is based on the conversion to the corresponding pteridin derivatives (pterin, 6-methylpterin, and 6,7-dimethylpterin). The proposed method using fluorometric detection has been applied to the determination of the three alpha-dicarbonyl compounds in human urine. Linearity (peak area vs concentration of alpha-dicarbonyl) was observed at least up to 43 microM. Detection limits of 32 pmol for glyoxal, 11 pmol for methylglyoxal, and 99 pmol for diacetyl were calculated (20 microliters was injected). Levels of 132 microM for glyoxal and 15 microM for methylglyoxal were determined in normal urine samples, while diacetyl was not detected.

Relative hepatotoxicity of 2-(substituted phenyl)thiazoles and substituted thiobenzamides in mice: evidence for the involvement of thiobenzamides as ring cleavage metabolites in the hepatotoxicity of 2-phenylthiazoles.

The hepatotoxicity of the 3 isomers of para-substituted thiobenzamides and the 3 isomers of 2-(para-substituted phenyl)-4-methylthiazoles was evaluated in mice depleted of glutathione (GSH) by pretreatment with buthionine sulfoximine (BSO). In accordance with previous studies with the rat, p-methoxythiobenzamide was more toxic than thiobenzamide, and conversely p-chlorothiobenzamide was markedly less toxic as assessed by serum alanine aminotransferase (ALT) activity. The hepatotoxicity of 2-phenyl-4-methylthiazole was also altered by the addition of para-substituents to the phenyl ring in the same way as observed for thiobenzamide derivatives: the rank order of toxicity was 4-methylthiazoles having p-methoxyphenyl > phenyl >> p-chlorophenyl at the 2-position. This good correlation of the rank order of hepatotoxicity between series of 2-(para-substituted phenyl)-4-methylthiazoles and para-substituted thiobenzamides supports the concept that thiobenzamides as ring cleavage metabolites play a role in the hepatotoxicity of 2-phenylthiazole derivatives.