Analysis of Total Thiols in the Urine of a Cystathionine ß-Synthase-Deficient Mouse Model of Homocystinuria Using Hydrophilic Interaction Chromatography.

Homocysteine and related thiols (cysteine, cysteinylglycine, and glutathione) in the urine of a cystathionine ß-synthase (CBS)-deficient mouse model were quantified using hydrophilic interaction chromatography with fluorescence detection. Urine samples were incubated with tris(2-carboxyethyl) phosphine to reduce disulfide bonds into thiols. After deproteinization, thiols were fluorescently derivatized with ammonium 7-fluoro-2,1,3-benzoxadiazole-4-sulfonate (SBD-F). Homocysteine, cysteine, cysteinylglycine, and glutathione in mouse urine were analyzed using an amide-type column with a mobile phase of acetonitrile/120 mM ammonium formate buffer (pH 3.0) (81:19). The developed method was well-validated. Thiol concentrations in the urine of CBS-wild type (-WT), -heterozygous (-Hetero), and -knockout (-KO) mice were quantified using the developed method. As expected, total homocysteine concentration in CBS-KO mice was significantly higher than that in CBS-WT and CBS-Hetero mice. The developed method shows promise for diagnoses in preclinical and clinical studies.

Increased Urinary 3-Mercaptolactate Excretion and Enhanced Passive Systemic Anaphylaxis in Mice Lacking Mercaptopyruvate Sulfurtransferase, a Model of Mercaptolactate-Cysteine Disulfiduria.

Mercaptopyruvate sulfurtransferase (Mpst) and its homolog thiosulfate sulfurtransferase (Tst = rhodanese) detoxify cyanide to thiocyanate. Mpst is attracting attention as one of the four endogenous hydrogen sulfide (H2S)/reactive sulfur species (RSS)-producing enzymes, along with cystathionine ß-synthase (Cbs), cystathionine γ-lyase (Cth), and cysteinyl-tRNA synthetase 2 (Cars2). MPST deficiency was found in 1960s among rare hereditary mercaptolactate-cysteine disulfiduria patients. Mpst-knockout (KO) mice with enhanced liver Tst expression were recently generated as its model; however, the physiological roles/significances of Mpst remain largely unknown. Here we generated three independent germ lines of Mpst-KO mice by CRISPR/Cas9 technology, all of which maintained normal hepatic Tst expression/activity. Mpst/Cth-double knockout (DKO) mice were generated via crossbreeding with our previously generated Cth-KO mice. Mpst-KO mice were born at the expected frequency and developed normally like Cth-KO mice, but displayed increased urinary 3-mercaptolactate excretion and enhanced passive systemic anaphylactic responses when compared to wild-type or Cth-KO mice. Mpst/Cth-DKO mice were also born at the expected frequency and developed normally, but excreted slightly more 3-mercaptolactate in urine compared to Mpst-KO or Cth-KO mice. Our Mpst-KO, Cth-KO, and Mpst/Cth-DKO mice, unlike semi-lethal Cbs-KO mice and lethal Cars2-KO mice, are useful tools for analyzing the unknown physiological roles of endogenous H2S/RSS production.

Label-free gold nanorods sensor array for colorimetric detection and discrimination of biothiols in human urine samples.

Biothiols play important roles in regulating redox balance in biological systems, but their discrimination is challengeable. In this work, a colorimetric nanosensing array for biothiols was established, which was composed of gold nanorods (AuNRs) and metal ions (Hg2+, Pb2+, Cu2+, Ag+). By employing label-free AuNRs as the colorimetric probe, and the color and spectral changes of AuNRs as the output signal, principal component analysis (PCA) was applied to processing the signal and generating a clustering map. Due to the different binding affinity between biothiols and metal ions, AuNRs exhibited a unique pattern to form a fingerprint-like colorimetric array, which was able to discriminate five biothiols by the naked eyes. This strategy combines PCA and sensor array to achieve rapid and accurate discrimination and detection of biothiols. In addition, the method shows the great potential in analysis of biothiols in human urine samples.

A robust and versatile mass spectrometry platform for comprehensive assessment of the thiol redox metabolome.

Several diseases are associated with perturbations in redox signaling and aberrant hydrogen sulfide metabolism, and numerous analytical methods exist for the measurement of the sulfur-containing species affected. However, uncertainty remains about their concentrations and speciation in cells/biofluids, perhaps in part due to differences in sample processing and detection principles. Using ultrahigh-performance liquid chromatography in combination with electrospray-ionization tandem mass spectrometry we here outline a specific and sensitive platform for the simultaneous measurement of 12 analytes, including total and free thiols, their disulfides and sulfide in complex biological matrices such as blood, saliva and urine. Total assay run time is < 10 min, enabling high-throughput analysis. Enhanced sensitivity and avoidance of artifactual thiol oxidation is achieved by taking advantage of the rapid reaction of sulfhydryl groups with N-ethylmaleimide. We optimized the analytical procedure for detection and separation conditions, linearity and precision including three stable isotope labelled standards. Its versatility for future more comprehensive coverage of the thiol redox metabolome was demonstrated by implementing additional analytes such as methanethiol, N-acetylcysteine, and coenzyme A. Apparent plasma sulfide concentrations were found to vary substantially with sample pretreatment and nature of the alkylating agent. In addition to protein binding in the form of mixed disulfides (S-thiolation) a significant fraction of aminothiols and sulfide appears to be also non-covalently associated with proteins. Methodological accuracy was tested by comparing the plasma redox status of 10 healthy human volunteers to a well-established protocol optimized for reduced/oxidized glutathione. In a proof-of-principle study a deeper analysis of the thiol redox metabolome including free reduced/oxidized as well as bound thiols and sulfide was performed. Additional determination of acid-labile sulfide/thiols was demonstrated in human blood cells, urine and saliva. Using this simplified mass spectrometry-based workflow the thiol redox metabolome can be determined in samples from clinical and translational studies, providing a novel prognostic/diagnostic platform for patient stratification, drug monitoring, and identification of new therapeutic approaches in redox diseases.

A ratiometric nanoprobe based on silver nanoclusters and carbon dots for the fluorescent detection of biothiols.

Ratiometric fluorescent probes could eliminate the influence from experimental factors and improve the detection accuracy. In this article, a ratiometric nanoprobe was constructed based on silver nanoclusters (AgNCs) with nitrogen-doped carbon dots (NCDs) and used for the detection of biothiols. The fluorescence peak of AgNCs was observed at 650nm with excitation wavelength at 370nm. In order to construct the ratiometric fluorescent probe, NCDs with the excitation and emission wavelengths at 370nm and 450nm were selected. After adding AgNCs, the fluorescence of NCDs was quenched. The mechanism of the fluorescence quenching was studied by fluorescence, UV-Vis absorption and the fluorescence lifetime spectra. The results indicated that the quenching could be ascribed to the inner filter effect (IFE). With the addition of biothiols, the fluorescence of AgNCs at 650nm decreased due to the breakdown of AgNCs, and the fluorescence of NCDs at 450nm recovered accordingly. Thus, the relationship between the ratio of the fluorescence intensities (I450/I650) and biothiol concentration was used to establish the determination method for biothiols. Cysteine (Cys) was taken as the model of biothiols, and the working curve for Cys was I450/I650=0.60CCys-1.86 (CCys: µmol/L) with the detection limit of 0.14µmol/L (S/N=3). Then, the method was used for the detection of Cys in human urine and serum samples with satisfactory accuracy and recovery ratios. Furthermore, the probe could be applied for the visual semi-quantitative determination of Cys by naked eyes.

Tandem derivatization combined with salting-out assisted liquid-liquid microextraction for determination of biothiols in urine by gas chromatography-mass spectrometry.

Detection of polar organic compounds (POCs) using gas chromatography (GC) is not straightforward due to high polarity, hydrophilicity, and low volatility of POCs. In this study, we report a tandem microwave-assisted derivatization method combined with salting-out assisted liquid-liquid microextraction (SALLME) to modify successively the polar groups of POCs in protic and aprotic solvents. Biothiols (cysteine and homocysteine) served as a proof of concept for this method because they possess three polar groups (thiol, amine, and carboxyl); the derivatizing reagent was 3,4,5-trifluorobenzyl bromide (Br-TFB) for alkylation. The solubility of the POCs in the protic or aprotic reaction medium affected the number of TFB molecules attached. Using the tandem derivatization with Br-TFB, the thiol and amine groups of biothiols were alkylated in the protic system, and the carboxylic groups of biothiols were alkylated in the aprotic system. The developed method was then successfully applied to measure biothiols in human urine. Because of the complex urine matrix and the lack of urine samples without endogenous biothiols, the standard addition method was utilized to avoid the matrix effect, check the recovery, and calculate the initial biothiol content in the urine. Regarding the linearity of the standard addition curves, the coefficient of determination was >0.996, and the linear regression showed satisfactory reproducibility with a relative standard deviation <3.9% for the slope and <8.8% for the intercept. The levels of cysteine and homocysteine in healthy human urine ranged from 28.8 to 111µmolL-1 and from 1.28 to 3.73µmolL-1, respectively. The proposed method effectively increased the sensitivity of GC-MS assays of water-soluble compounds in human urine.

Surface plasmon resonance based spectrophotometric determination of medicinally important thiol compounds using unmodified silver nanoparticles.

The determination of thiol based biological molecules and drugs, such as cysteine (Cys) (I), α-lipoic acid (II), and sodium 2-sulfanylethane sulphonate (Mesna (III)) in human plasma are becoming progressively more important due to the growing body of knowledge about their essential role in numerous biological pathways. Herein we demonstrate a sensitive colorimetric sensor for the determination of medicinally important thiol drugs based on aggregation of the citrate capped silver nanoparticles (Ag NPs). This approach exploited the high affinity of thiols towards the Ag NPs surface which could tempt replacement of the citrate shell by the thiolate shell of target molecules, resulting in aggregation of the NPs through intermolecular electrostatic interaction or hydrogen-bonding. Because of aggregation, the plasmon band at around 400nm decreases gradually, along with the appearance of a new band connoting a red shift. The calibration curves are derived from the intensity ratios of A530/A400, which display a linear relation in the range of 1µM-150µM, 5µM-200µM and 10µM-130µM, respectively. The obtained detection limits (3σ) were found to be 1.5µM, 5.6µM and 10.2µM for compound I-III, respectively. The proposed method has been successfully applied for the detection of thiol compounds in real samples.

A versatile method for analysis of saliva, plasma and urine for total thiols using HPLC with UV detection.

A simple and rapid HPLC method using 2-chloro-1-methyllepidinium tetrafluoroborate (CMLT) as a derivatization reagent was developed for simultaneous determination of homocysteine (Hcy), glutathione (GSH), γ-glutamylcysteine (γ-GluCys), cysteinylglycine (CysGly), N-acetylcysteine (NACys) and cysteine (Cys) in human saliva, plasma and urine. Separation of the analytes was achieved in just 7min using an HPLC, followed by UV detection at 355nm. Chromatographic separation was accomplished on Aeris PEPTIDE XB-C18 (150mm×4.6mm, 3.6µm) column from Phenomenex with a gradient elution: 0-4.0min, 7-30% B; 4.0-5.5min, 30-7% B; 5.5-7.5min, 7% B; (A: B, v/v); (A) 0.5% CH3COOH and (B) EtOH. Mobile phase was delivered at a flow rate 1.0mLmin(-1). Linearity in detector response for total thiols was observed over the range of 0.1-20µmolL(-1) for Hcy, GSH and γ-GluCys, 0.25-50µmolL(-1) for NACys and CysGly and 5-300 for Cys. The LOQ values for Hcy, GSH, γ-GluCys, NACys, CysGly and Cys were 0.05, 0.05, 0.10, 0.06, 0.12 and 0.08µmolL(-1), respectively. The method was successfully implemented to analysis of the samples donated by 15 apparently healthy volunteers and 10 patients.

A CE-LIF method based on long wavelength fluorescence labeling for the analysis of thiols in human urine.

A rapid and robust CE method using a long wavelength fluorescent reagent 1,7-dimethyl-3,5-distyryl-8-phenyl-(2-maleimide)difluoroboradiaza-s-indacene as the labeling reagent has been developed for the simultaneous determination of thiols, including glutathione, cysteine, homocysteine, N-acetylcysteine, cysteinylglycine, and penicillamine. The derivatization reaction was carried out in 14 mmol/L pH 8.5 borate buffer at 30°C for 6 min and the labeled thiols derivatives were separated with the running buffer containing 30 mmol/L pH 7.4 phosphate, 30% v/v acetonitrile and 8 mmol/L SDS within 12 min. Detection limits ranged from 0.4 to 2.4 nmol/L. To demonstrate the capability of this method, it was applied to the analysis of thiols in human urine with recoveries of 92.4-105.6%. The derivatization reaction was much faster at milder conditions, and the analysis was rapider. Moreover, with excitation wavelength at long wavelength region, background interference from samples was reduced effectively. The present method seems to be a potential choice for quantifying thiols in human urine.

Determination of thiol metabolites in human urine by stable isotope labeling in combination with pseudo-targeted mass spectrometry analysis.

Precursor ion scan and multiple reaction monitoring scan (MRM) are two typical scan modes in mass spectrometry analysis. Here, we developed a strategy by combining stable isotope labeling (IL) with liquid chromatography-mass spectrometry (LC-MS) under double precursor ion scan (DPI) and MRM for analysis of thiols in 5 types of human cancer urine. Firstly, the IL-LC-DPI-MS method was applied for non-targeted profiling of thiols from cancer samples. Compared to traditional full scan mode, the DPI method significantly improved identification selectivity and accuracy. 103 thiol candidates were discovered in all cancers and 6 thiols were identified by their standards. It is worth noting that pantetheine, for the first time, was identified in human urine. Secondly, the IL-LC-MRM-MS method was developed for relative quantification of thiols in cancers compared to healthy controls. All the MRM transitions of light and heavy labeled thiols were acquired from urines by using DPI method. Compared to DPI method, the sensitivity of MRM improved by 2.1-11.3 folds. In addition, the concentration of homocysteine, γ-glutamylcysteine and pantetheine enhanced more than two folds in cancer patients compared to healthy controls. Taken together, the method demonstrated to be a promising strategy for identification and comprehensive quantification of thiols in human urines.

Monitoring urinary metabolites resulting from sulfur mustard exposure in rabbits, using highly sensitive isotope-dilution gas chromatography-mass spectrometry.

A highly sensitive method for the determination of sulfur mustard (SM) metabolites thiodiglycol (TDG) and thiodiglycol sulfoxide (TDGO) in urine was established and validated using isotope-dilution negative-ion chemical ionization (NICI) gas chromatography-mass spectrometry (GC-MS). TDGO in the samples was reduced with TiCl3, and then determined together with TDG as a single analyte. The sample preparation procedures, including two solid-phase-extraction (SPE) clean-up steps, were optimized to improve the sensitivity of the method. The limits of detection (LOD) for both TDG and TDG plus TDGO (TDG + TDGO) were 0.1 ng mL(-1), and the limits of quantitation (LOQ) for both were 0.3 ng mL(-1). The method was used in a rabbit cutaneous SM exposure model. Domestic rabbits were exposed to neat liquid SM at three dosage levels (0.02, 0.05, and 0.15 LD50), and the urinary excretion of four species of hydrolysis metabolites, namely free TDG, free plus conjugated TDG (total TDG), free TDG + TDGO, and free plus conjugated TDG + TDGO (total TDG + TDGO), was evaluated to investigate the metabolic processes. The total urinary excretion profiles of the metabolites, including the peak time, time window, and dose-response and time-response relationships, were clarified. The results revealed that the concentrations of TDG and TDG + TDGO in the urine increased quickly and then decreased rapidly in the first two days after SM exposure. The cumulative amount of total TDG + TDGO excreted in urine during the first five days accounted for 0.5-1% of the applied dose of SM. It is also concluded that TDG and TDGO in urine existed mainly in free form, the levels of glucuronide and of sulfate conjugates of TDG or TDGO were very low, and most hydrolysis metabolites were present in the oxidized form (TDGO). The study indicates that the abnormal increase of TDG and TDGO excretion levels can be used as a diagnostic indicator and establishes a reference time-window for retrospective analysis and sampling after SM exposure.

Analytical methods involving separation techniques for determination of low-molecular-weight biothiols in human plasma and blood.

Low-molecular-weight biothiols such as homocysteine, cysteine, and glutathione are metabolites of the sulfur cycle and play important roles in biological processes such as the antioxidant defense network, methionine cycle, and protein synthesis. Thiol concentrations in human plasma and blood are related to diseases such as cardiovascular disease, neurodegenerative disease, and cancer. The concentrations of homocysteine, cysteine, and glutathione in plasma samples from healthy human subjects are approximately in the range of 5-15, 200-300, and 1-5 µM, respectively. Glutathione concentration in the whole blood is in the millimolar range. Measurement of biothiol levels in plasma and blood is thought to be important for understanding the physiological roles and biomarkers for certain diseases. This review summarizes the relationship of biothiols with certain disease as well as pre-analytical treatment and analytical methods for determination of biothiols in human plasma and blood by using high-performance liquid chromatography and capillary electrophoresis coupled with ultraviolet, fluorescence, or chemiluminescence detection; or mass spectrometry.

Spectrophotometric method for the determination of total thiols in human urine.

Thiols have been of enduring interest for many years because of their role in biological and pharmacological processes. Monitoring of total thiols content is very important in order to understand their function in living organisms. This paper describes the spectrophotometric method for the determination of total thiols concentration in urine. The method is based on derivatization with 1-benzyl-2-chloropyridinium bromide and ultraviolet detection of S-pyridinium derivatives at 316 nm. The analytical recovery and RSD values for precision within the calibration range were from 95.7 to 102.9% and from 2.1 to 8.4%, respectively. The concentration of total thiols normalized against creatinine for 38 apparently healthy subjects (19 women and 19 men) occurred in the range 17.2-73.7 and 25.7-83.6 mmol/mol creatinine, respectively. There was no difference in the urinary excretion of thiols in men and women, but there was a significant statistical correlation between urine total thiols and age in the studied group.

Characterization of thiol-conjugated metabolites of ginger components shogaols in mouse and human urine and modulation of the glutathione levels in cancer cells by [6]-shogaol.

SCOPE: Shogaols, a series of major constituents in dried ginger with the most abundant being [6]-, [8]-, and [10]-shogaols, show much higher anticancer potencies than gingerols. Previously, we reported the mercapturic acid pathway as a major metabolic route for [6]-shogaol in mice. However, it is still unclear how the side chain length affects the metabolism of shogaols and how shogaols are metabolized in humans. METHODS AND RESULTS: We first investigate the metabolism of [10]-shogaol in mouse urine, and then investigate the biotransformation of shogaols in human urine. Our results show that eight major thiol-conjugated metabolites of [10]-shogaol were detected in mouse urine, while six major thiol-conjugated metabolites of [6]-shogaol, two thiol-conjugated metabolites of [8]-shogaol, and two thiol-conjugated metabolites of [10]-shogaol were detected in urine collected from human after drinking ginger tea, using LC/ESI-MS/MS. Our results clearly indicate the mercapturic acid pathway is a major metabolic route for [10]-shogaol in mice and for shogaols in human. Furthermore, we also investigated the regulation of glutathione (GSH) by [6]-shogaol in human colon cancer cells HCT-116. Our results show [6]-shogaol, after initially depleting glutathione levels, can subsequently restore and increase GSH levels over time. CONCLUSION: Shogaols are metabolized extensively in mouse and human to form thiol-conjugated metabolites and GSH might play an important role in the cancer-preventive activity of ginger.

Identification of phase II metabolites of thiol-conjugated [6]-shogaol in mouse urine using high-performance liquid chromatography tandem mass spectrometry.

Ginger is frequently consumed as a spice and has numerous medicinal properties. Extensive research has characterized the anti-inflammatory, antioxidant, and antitumor activities of ginger. Previously, we reported the mercapturic acid pathway as a major metabolic route of [6]-shogaol in mice and the thiol conjugates of [6]-shogaol existed in the glucuronidated and sulfated forms in mouse urine. However, their structures are still unknown. In the present study, we further investigated the phase II metabolism of thiol-conjugated [6]-shogaol in mouse urine, in which we identified sixteen phase II metabolites of thiol-conjugated [6]-shogaol: 5-cysteinyl-[6]-shogaol glucuronide (9), 5-N-acetylcysteinyl-[6]-shogaol glucuronide (10), 5-cysteinylglycinyl-[6]-shogaol glucuronide (11), 5-methylthio-[6]-shogaol glucuronide (12), 5-cysteinyl-M6 glucuronide (13 and 14), 5-cysteinyl-M6 sulfate (15 and 16), 5-N-acetylcysteinyl-M6 glucuronide (17 and 18), 5-cysteinylglycinyl-M6 glucuronide (19 and 20), 5-cysteinylglycinyl-M6 sulfate (21 and 22), and 5-methylthio-M6 glucuronide (23 and 24) using liquid chromatography/electrospray ionization tandem mass spectrometry. The structures of these metabolites were confirmed by analyzing their MS(n) (n=1-4) spectra as well as comparing with the tandem mass spectra of authentic standards. To the best of our knowledge, this is the first report involving identification of phase II urinary metabolites of [6]-shogaol in mice.

Hg2+-mediated aggregation of gold nanoparticles for colorimetric screening of biothiols.

In this work, we report a colorimetric assay for the screening of biothiols including glutathione (GSH), cysteine (Cys), and homocysteine (Hcys) based on Hg(2+)-mediated aggregation of gold nanoparticles (AuNPs). Hg(2+) can induce aggregation of thiol-containing naphthalimide (1) capped AuNPs due to the cross-linking interactions from the resulting "thymine-Hg(2+)-thymine" (T-Hg(2+)-T) analogous structure. When Hg(2+) is firstly treated with biothiols, followed by mixing with 1-capped AuNPs suspension, AuNPs undergo a transformation from an aggregation to a dispersion state depending on the concentration of biothiols. This anti-aggregation or re-dispersion of AuNPs is due to the higher affinity of Hg(2+) for biothiols relative to compound 1. The corresponding color variation in the process of anti-aggregation of AuNPs can be used for the quantitative screening of biothiols through UV-vis spectroscopy or by the naked eye. Under optimized conditions, a good linear relationship in the range of 0.025-2.28 µM is obtained for GSH, 0.035-1.53 µM for Cys, and 0.040-2.20 µM for Hcys. The detection limits of this assay for GSH, Cys, and Hcys are 17, 9, and 18 nM, respectively. This colorimetric assay exhibits a high selectivity and sensitivity with tunable dynamic range. The proposed method has been successfully used in the determination of total biothiol content in human urine samples.

Use of isotope differential derivatization for simultaneous determination of thiols and oxidized thiols by liquid chromatography tandem mass spectrometry.

Here we report a new isotopic pair of derivatization reagents, ω-bromoacetonylquinolinium bromide (BQB) and d(7)-ω-bromoacetonylquinolinium bromide (d(7)-BQB). BQB and d(7)-BQB both rapidly and selectively reacted with thiols in acidic medium within 3min with the aid of a microwave. Reduced thiols and total thiols in urine were labeled with BQB and d(7)-BQB, respectively. The BQB- and d(7)-BQB-labeled urine samples were then mixed and separated on a HILIC (hydrophilic interaction chromatography) column followed by electrospray ionization tandem mass spectrometry (ESI-MS/MS) detection. The new strategy, which we have named isotope differential derivatization, allows us to simultaneously determine thiols and oxidized thiols in a single run. Compared with positive mode ESI detection of unlabeled thiols, the positive mode ESI-MS signal intensities of BQB-labeled thiols were found to increase by 10-, 20-, and 40-fold for cysteine (Cys), homocysteine (HCys), and glutathione (GSH), respectively (unlabeled N-acetylcysteine (Nac) is difficult to detect by ESI-MS in positive mode due to its low ionization efficiency). The detection limits calculated at a signal-to-noise ratio of 3 were found to be 8.02, 1.56, 0.833, and 3.27nmol/L for Cys, HCys, Nac, and GSH, respectively. Recoveries of thiols and disulfides from spiked urine samples were between 80% and 105%. The method was successfully used to determine thiols and oxidized thiols in urine samples of 25 healthy volunteers.

Fenofibrate, homocysteine and renal function.

Fibrates or PPAR alpha agonists, in particular fenofibrate, are known to increase homocysteine levels (Hcy). A 3 to 5 micromol/L increase in Hcy is commonly observed within the first few weeks of fenofibrate treatment; it then persists in plateau when treatment is continued and is reversible upon its cessation. Since its description in 1999, this pharmacological effect attracted a great deal of attention as epidemiological studies in most populations have shown that elevated Hcy levels i.e.Hcy> or =15 micromol/L are associated with an increased risk of cardiovascular events (CVD), venous thromboembolic events (VTE) and possibly cognitive disorders and bone fractures. Chronic kidney disease is also associated with elevated Hcy levels and since fenofibrate increases creatinine levels by about 10-12 micromol/L, a relationship between Hcy and creatinine was postulated. Animal studies have shown that the Hcy increase is PPARalpha dependent but to date animal or human studies have not provided a clear mechanism. In particular, fenofibrate treatment does not change vitamin B levels; however, vitamin B supplements reduce fenofibrate-induced Hcy elevation but not the concomitant cysteine elevation. Similarly, the increase in creatinine with fenofibrate only partially accounted for by a reduction in glomerular filtration rate (GFR) since creatinine production is also increased by 5-10%. In the FIELD study, a placebo-controlled study in 9795 patients with type 2 diabetes, fenofibrate over 5 years reduced non-fatal cardiovascular events and microvascular events such as albuminuria, the need for laser treatment for proliferative retinopathy or maculopathy and amputations but did not reduce fatal events. The increase in Hcy was indeed much larger that what would be explained by creatinine elevation and independent from baseline kidney function. Although baseline Hcy and creatinine levels were associated with subsequent risk of CVD, as suggested by epidemiology, their respective elevation was not. Of interest, after withdrawal of fenofibrate, a potential renoprotective effect was unmasked, as estimated GFR was 5 ml/min/1.73 m2 higher in previous fenofibrate-allocated patients than in previous placebo-allocated patients. There was no suggestion that Hcy elevation was associated with VTE (which were increased by an unknown mechanism) or bone disorders. In conclusion, the discrepancy between the role of baseline Hcy levels in epidemiology and the absence of effect when altering its levels by either decreasing them with vitamin B supplements or increasing them with fenofibrate, suggests that the risk factor(s) behind homocysteine should be found. Nevertheless, other studies are also needed to understand the mechanism and the implications of the moderate homocysteine and creatinine elevations with fenofibrate and other PPARalpha agonists.

Metabolism and disposition of [14C]dibromoacetonitrile in rats and mice following oral and intravenous administration.

1. Tissue distribution, metabolism, and disposition of oral (0.2-20 mg/kg) and intravenous (0.2 mg/kg) doses of [2-(14)C]dibromoacetonitrile (DBAN) were investigated in male rats and mice. 2. [(14)C]DBAN reacts rapidly with rat blood in vitro and binds covalently. Prior depletion of glutathione (GSH) markedly diminished loss of DBAN. Chemical reaction with GSH readily yielded glutathionylacetonitrile. 3. About 90% of the radioactivity from orally administered doses of [(14)C]DBAN was absorbed. After intravenous administration, 10% and 20% of the radioactivity was recovered in mouse and rat tissues, respectively, at 72 h. After oral dosing, three to four times less radioactivity was recovered, but radioactivity in stomach was mostly covalently bound. 4. Excretion of radioactivity into urine exceeded that in feces; 9-15% was exhaled as labeled carbon dioxide and 1-3% as volatiles in 72 h. 5. The major urinary metabolites were identified by liquid chromatography-mass spectrometry, and included acetonitrile mercaptoacetate (mouse), acetonitrile mercapturate, and cysteinylacetonitrile. 6.The primary mode of DBAN metabolism is via reaction with GSH, and covalent binding may be due to reaction with tissue sulphydryls.

Dietary administration of sodium arsenite to rats: relations between dose and urinary concentrations of methylated and thio-metabolites and effects on the rat urinary bladder epithelium.

Based on epidemiological data, chronic exposure to high levels of inorganic arsenic in drinking water is carcinogenic to humans, inducing skin, urinary bladder and lung tumors. In vivo, inorganic arsenic is metabolized to organic methylated arsenicals including the highly toxic dimethylarsinous acid (DMA(III)) and monomethylarsonous acid (MMA(III)). Short-term treatment of rats with 100 microg/g trivalent arsenic (As(III)) as sodium arsenite in the diet or in drinking water induced cytotoxicity and necrosis of the urothelial superficial layer, with increased cell proliferation and hyperplasia. The objectives of this study were to determine if these arsenic-induced urothelial effects are dose responsive, the dose of arsenic at which urothelial effects are not detected, and the urinary concentrations of the arsenical metabolites. We treated female F344 rats for 5 weeks with sodium arsenite at dietary doses of 0, 1, 10, 25, 50, and 100 ppm. Cytotoxicity, cell proliferation and hyperplasia of urothelial superficial cells were increased in a dose-responsive manner, with maximum effects found at 50 ppm As(III). There were no effects at 1 ppm As(III). The main urinary arsenical in As(III)-treated rats was the organic arsenical dimethylarsinic acid (DMA(V)). The thio-metabolites dimethylmonothioarsinic acid (DMMTA(V)) and monomethylmonothioarsinic acid (MMMTA(V)) were also found in the urine of As(III)-treated rats. The LC(50) concentrations of DMMTA(V) for rat and human urothelial cells in vitro were similar to trivalent oxygen-containing arsenicals. These data suggest that dietary As(III)-induced urothelial cytotoxicity and proliferation are dose responsive, and the urothelial effects have a threshold corresponding to the urinary excretion of measurable reactive metabolites.