A robust ultra-performance liquid chromatography-tandem mass spectrometry method for simultaneous determination of 10 components in glutathione cycle.

Glutathione (GSH) is an important antioxidant that is generated and degraded via the GSH cycle. Quantification of the main components in the GSH cycle is necessary to evaluate the process of GSH. In this study, a robust ultra-performance liquid chromatography-tandem mass spectrometry method for the simultaneous quantification of 10 components (GSH; γ-glutamylcysteine; cysteinyl-glycine; n-acetylcysteine; homocysteine; cysteine; cystine; methionine; glutamate; pyroglutamic acid) in GSH cycle was developed. The approach was optimized in terms of derivative, chromatographic, and spectrometric conditions as well as sample preparation. The unstable thiol groups of GSH, γ-glutamylcysteine, cysteinyl-glycine, n-acetylcysteine, cysteine, and homocysteine were derivatized by n-ethylmaleimide. The derivatized and underivatized analytes were separated on an amino column with gradient elution. The method was further validated in terms of selectivity (no interference), linearity (R2 > 0.99), precision (% relative standard deviation [RSD%] range from 0.57 to 10.33), accuracy (% relative error [RE%] range from -3.42 to 10.92), stability (RSD% < 5.68, RE% range from -2.54 to 4.40), recovery (RSD% range from 1.87 to 7.87) and matrix effect (RSD% < 5.42). The validated method was applied to compare the components in the GSH cycle between normal and oxidative stress cells, which would be helpful in clarifying the effect of oxidative stress on the GSH cycle.

Evaluation of N-acetylcysteine and glutathione as quenching agents for the analysis of halogenated disinfection by-products.

Disinfection by-products (DBPs), formed from the reactions of disinfectants with natural organic matter and halides in drinking water, were considered to be cytotoxic and genotoxic, and might trigger various cancers. The relatively low concentration of DBPs in finished water (low µg/L or even ng/L levels) and the interference from water matrix inhibited in situ determination of DBPs. Moreover, the further formation and degradation of DBPs by disinfectants during the holding time (several hours to several days) from sample collection to analysis could adversely affect the determination of DBPs. To obtain accurate, precise and reliable data of DBP occurrence and formation, robust and reliable sample preservation is indispensable. However, the commonly used quenching agents (e.g., sodium sulfite, sodium thiosulfate, and ascorbic acid) for sample preservation can decompose reactive DBPs by reductive dehalogenation. This study evaluated the performance of N-acetylcysteine (NAC) and glutathione (GSH) as quenching agents for the analysis of halogenated DBPs by investigating the stoichiometry of the disinfectant-quenching agent reaction, the formation of DBPs during chlor(am)ination of NAC or GSH, and the effects of NAC or GSH on the stability of 18 individual DBPs and total organic halogen (TOX). Based on the results of this study, NAC and GSH were considered to be ideal quenching agents for the analysis of most DBPs and TOX, except halonitromethanes.

Fabrication of a "Selenium Signature" Chemical Probe-Modified Paper Substrate for Simultaneous and Efficient Determination of Biothiols by Paper Spray Mass Spectrometry.

Significant efforts have been made to develop robust and reliable methods for simultaneous biothiols determination in different matrices, but there still exist the problems such as easy oxidation, tedious derivatization, and difficulty in discrimination, which brings unsatisfactory results in their accuracy and fast quantification in biological samples. To overcome these problems, a simultaneous biothiols detection method combining a "selenium signature" chemical probe and paper spray mass spectrometry (PS-MS) was proposed. In the strategy, the modified-paper substrate is used to enhance the analytical performance. Chemical probe Ebselen-NH2 that has a specific response to biothiols was designed and covalently fixed on the surface of an oxidized paper substrate. By the identification of derivatized product with distinctive selenium isotope distribution and employment of the optimized PS-MS method, qualitative and quantitative analysis of five biothiols including glutathione (GSH), cysteine (Cys), cysteinylglycine (CysGly), N-acetylcysteine (Nac), and homocysteine (Hcy) were realized. Biothiols in plasma and cell lysates were measured with satisfactory results. The established method not only provides a novel protocol for simultaneous determination of biothiols, but also is helpful for understanding the biological and clinical roles played by these bioactive small molecules.

Global Profiling of Toxicologically Relevant Metabolites in Urine: Case Study of Reactive Aldehydes.

Among the numerous unknown metabolites representative of our exposure, focusing on toxic compounds should provide more relevant data to link exposure and health. For that purpose, we developed and applied a global method using data independent acquisition (DIA) in mass spectrometry to profile specifically electrophilic compounds originating metabolites. These compounds are most of the time toxic, due to their chemical reactivity toward nucleophilic sites present in biomacromolecules. The main line of cellular defense against these electrophilic molecules is conjugation to glutathione, then metabolization into mercapturic acid conjugates (MACs). Interestingly, MACs display a characteristic neutral loss in MS/MS experiments that makes it possible to detect all the metabolites displaying this characteristic loss, thanks to the DIA mode, and therefore to highlight the corresponding reactive metabolites. As a proof of concept, our workflow was applied to the toxicological issue of the oxidation of dietary polyunsaturated fatty acids, leading in particular to the formation of toxic alkenals, which lead to MACs upon glutathione conjugation and metabolization. By this way, dozens of MACs were detected and identified. Interestingly, multivariate statistical analyses carried out only on extracted HRMS signals of MACs yield a better characterization of the studied groups compared to results obtained from a classic untargeted metabolomics approach.

Prospective randomized multicentre comparison on sibling oocytes comparing G-Series media system with antioxidants versus standard G-Series media system.

RESEARCH QUESTION: Does the inclusion of three antioxidants (A3), acetyl-l-carnitine (ALC), N-acetyl-l-cysteine (NAC) and alpha-lipoic acid (ALA) improve human embryo development and pregnancy potential? DESIGN: Prospective randomized multicentre comparison of sibling oocytes. A total of 1563 metaphase II oocytes from 133 patients in two IVF centres. Day 3 embryo and day 5/6 blastocyst quality were assessed. Good embryo quality on day 3 was defined as 8 to 10 cells with even cells and low fragmentation; good quality blastocysts as 3BB or greater. Clinical outcome was assessed on transfers of fresh or vitrified-warmed blastocyst on day 5. RESULTS: Of the two-pronuclei, 40.7% (G-Series) and 50.2% (G-Series with A3 group) resulted in good quality embryos on day 3 (P < 0.05). The implantation rate by fetal sac was 39.2% and 50.6%, and by fetal heartbeat was 37.8% and 47.1% for the G-Series and G-Series with A3 group, respectively. When stratified by female patient age, patients 35-40 years had an implantation rate by fetal sac and heart of 23.5% in the G-Series compared with 57.5% (P < 0.05) and 50.0% (P < 0.05) in the A3 group. The ongoing pregnancies in patients 35-40 years were significantly higher in the A3 group (50%) compared with the control (25.8%) (P < 0.05). CONCLUSIONS: The presence of antioxidants during IVF and embryo culture for patients 35-40 years resulted in a significant increase in implantation and pregnancy rate. Supplementation of antioxidants to IVF and culture media may therefore improve the viability of human embryos in assisted reproductive technologies, plausibly through the reduction of oxidative stress.

An ultra-high-performance liquid chromatography tandem mass spectrometry method for oxidative stress biomarker analysis in wastewater.

Reported herein is the development of an analytical method for the detection of four oxidative stress biomarkers in wastewater using ultra-high-performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS) and solid phase extraction (SPE). The following four biomarkers of oxidative stress and lipid peroxidation have been investigated: hydroxynonenal-mercapturic acid (HNE-MA), 8-iso-prostglandin F2beta (8-iso-PGF2ß), 8-nitroguanine (8-NO2Gua) and 8-hydroxy-2-deoxyguanosine (8-OHdG). The method showed very good performance: accuracy (> 87%), precision (> 90%), method quantification limits (1.3-3.0 ng L-1) and biomarker stability in wastewater in the case of HNE-MA, 8-OHdG and 8-iso-PGF2ß. In contrast, 8-NO2Gua was found to be less stable in wastewater, which affected its method performance: accuracy (> 63%), precision (> 91%) and method quantification limits (85.3 ng L-1). Application of the developed method resulted in, for the first time, HNE-MA being successfully observed and quantified within wastewater over a study period of a week (displayed average daily loads per capita of 48.9 ± 4.1 mg/1000/people/day). 8-iso-PGF2ß was detected with good intensity but could not be quantified due to co-elution with other isomers. 8-OHdG was detected, albeit at < MQL. This study demonstrates the potential for expanding on the possible endogenous biomarkers of health used in urban water fingerprinting to aid in measuring health in near-real time on a community-wide scale.

Simultaneous determination of total homocysteine, cysteine, glutathione, and N-acetylcysteine in brain homogenates by HPLC.

We have developed a simple, fast, accurate, and cheap method for the simultaneous determination of total cysteine, homocysteine, glutathione, and N-acetylcysteine in brain homogenates based on the reduction of disulfide bonds by tris(2-carboxyethyl) phosphine, pre-column derivatization of free thiol groups with 2-chloro-1-methylquinolinium tetrafluoroborate followed by ion-pair reversed-phase high-performance liquid chromatography separation with ultraviolet detection. The separation of thiol derivatives was achieved in 10 min. Linearity was observed in the range of 10-300, 0.7-10, 2-30, and 3-20 µmol/L homogenate with a limit of detection of 3.7, 0.2, 0.8, and 1.2 µmol/L homogenate for cysteine, homocysteine, glutathione, and N-acetylcysteine, respectively. The precision, calculated as relative standard deviation, was in the range of 1.21-4.77, 1.53-14.35, 0.47-1.92, and 1.61-8.95% for cysteine, homocysteine, glutathione, and N-acetylcysteine, respectively. The presented method was successfully applied to the selective determination of total amino thiols in pig brain tissue samples.

Determination of Diclofenac and N-Acetylcysteine by an Optimized Luminescence Method Using CdS Quantum Dots as Sensitizers.

A simple, sensitive and selective chemiluminescence method is proposed for the determination of Acetylcysteine and Diclofenac in pharmaceutical samples. The method is based on the measuring light intensity of NaHCO3-H2O2 @ CdS chemiluminescence system in the absence and presence of Acetylcysteine and Diclofenac. It was found that the intensity of chemiluminescence is increased by addition of acetylcysteine while the light intensity is decreased with increasing diclofenac concentration. The CdS quantum dots nanoparticles were characterized by X-ray diffraction (XRD), Scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), UV-Vis spectrophotometry and Fluorescence spectroscopy. The optimum conditions for maximizing the CL emission intensity were studied by means of a Box-Behnken experimental design combined with response surface modeling (RSM) and quadratic programming. Under the optimal conditions, results for measurement of diclofenac was found as follows: linear range 1 × 10-5 - 1 × 10-9 Molar with R2 = 0.98, limit of detection 5 × 10-10 Molar and relative standard deviation of 2.4% for diclofenac; and for N-acetylcysteine was : linear range 1 × 10-6 - 1 × 10-9 Molar, the limit of detection 5 × 10-10 Molar, and the relative standard deviation of 3.4%.

[Biomarkers of internal exposure to toxicologically relevant contaminants in food]. / Biomarker der internen Exposition gegenüber toxikologisch relevanten Kontaminanten in Lebensmitteln.

The assessment of health risks resulting from the intake of genotoxic carcinogens in food depends essentially on a valid exposure assessment. The reliability of the external exposure estimation is restricted by various factors, e. g. inaccurate data from dietary protocols and variations of food contaminant contents. As an alternative, the individual internal exposure to genotoxic substances may be described by specific biomarkers in different matrices. For example, mercapturic acids formed after glutathione conjugation of electrophilic metabolites can be detected in the urine. This typically reflects the exposure to the parent compound over a period of one to two days. The determination of adducts in the blood proteins serum albumin (SA) and hemoglobin (Hb) allows for conclusions to be drawn about the external exposure within the last three weeks (SA) or within the last four months (Hb). Protein adducts are used routinely in occupational medicine as biomarkers of internal exposure to substances in the ambient air of the workplace. The availability of increasingly sensitive analytical techniques also makes it possible to detect numerous adducts in proteins from human blood samples that are formed after the continuous intake of very small doses of toxic substances from foods. Here, we present the current state of science exemplified by protein adducts of the food contaminants acrylamide, aflatoxin B1 and glycidol. The biomarker can be used in the future to investigate previously unknown relationships between internal exposure and disease incidences.

Mercapturic acids: recent advances in their determination by liquid chromatography/mass spectrometry and their use in toxicant metabolism studies and in occupational and environmental exposure studies.

This review describes recent selected HPLC/MS methods for the determination of urinary mercapturates that are useful as noninvasive biomarkers in characterizing human exposure to electrophilic industrial chemicals in occupational and environmental studies. High-performance liquid chromatography/mass spectrometry is a sensitive and specific method for analysis of small molecules found in biological fluids. In this review, recent selected mercapturate quantification methods are summarized and specific cases are presented. The biological formation of mercapturates is introduced and their use as indicators of metabolic processing of reactive toxicants is discussed, as well as future trends and limitations in this area of research.

Effect of N-acetyl cysteine and glycine supplementation on growth performance, glutathione synthesis, anti-oxidative and immune ability of Nile tilapia, Oreochromis niloticus.

An 8-week feeding trial was conducted to evaluate the effect of N-acetyl cysteine (NAC) and glycine supplementation on growth performance, glutathione (GSH) synthesis, anti-oxidative and immune ability of Nile tilapia, Oreochromis niloticus. Four practical diets were formulated, control, control +0.2% NAC, control +0.5% glycine, control +0.2% NAC +0.5% glycine. Each diet was randomly assigned to quadruplicate groups of 30 fish (approximately 9.5 g). The weight gain and specific growth rate were significantly increased with the supplementation of NAC and glycine. While they had no effect on feed efficiency feed intake and survival. Glutathion peroxidase (GPx) was increased by NAC and γ-glutamine cysteine synthase (γ-GCS) in plasma were increased by glycine. After the feeding trail, fish were challenged by Streptococcus iniae, fish fed the diet supplemented with NAC obtained significantly higher survival rate after 72 h challenge test. NAC also decreased malonaldehyde (MDA) in liver, increased glutathione S-transferase (GST) activity in plasma, up-regulated mRNA expression of Superoxide dismutase (SOD) and GPx in liver and headkidney. Dietary supplementation of glycine increased the anti-oxidative ability of tilapia through increase anti-oxidative enzyme activity (SOD, glutathione reductase, myeloperoxidase) and up-regulate anti-oxidative gene expression (SOD). Immune ability only enhanced by the supplementation of NAC through increased interleukin-1ß (IL-1ß) mRNA expression. These results clearly indicated that the supplementation of NAC and glycine can significantly improve the growth performance of tilapia, and NAC also enhance the anti-oxidative and immune capacity of tilapia, glycine could only enhance the anti-oxidative ability.

Precise determination of N-acetylcysteine in pharmaceuticals by microchip electrophoresis.

A novel microchip electrophoresis method for the rapid and high-precision determination of N-acetylcysteine, a pharmaceutically active ingredient, in mucolytics has been developed. Isotachophoresis separations were carried out at pH 6.0 on a microchip with conductivity detection. The methods of external calibration and internal standard were used to evaluate the results. The internal standard method effectively eliminated variations in various working parameters, mainly run-to-run fluctuations of an injected volume. The repeatability and accuracy of N-acetylcysteine determination in all mucolytic preparations tested (Solmucol 90 and 200, and ACC Long 600) were more than satisfactory with the relative standard deviation and relative error values <0.7 and <1.9%, respectively. A recovery range of 99-101% of N-acetylcysteine in the analyzed pharmaceuticals predetermines the proposed method for accurate analysis as well. This work, in general, indicates analytical possibilities of microchip isotachophoresis for the quantitative analysis of simplified samples such as pharmaceuticals that contain the analyte(s) at relatively high concentrations.

Distribution, enrichment, and source identification of selected heavy metals in surface sediments of the Siran River, Mansehra, Pakistan.

To assess the trace metal pollution in the Siran River, sediments were collected from 12 sites, from the left and right banks of the river in 2013. The concentrations, accumulation, distribution pattern, and pollution status of heavy metals in sediments were investigated using geoaccumulation index (I geo) and enrichment factor (EF). The toxic risk of heavy metals was assessed using interim sediment quality guidelines (ISQGs), portable effect level (PEL), threshold effect level (TEL), and toxic effect threshold (TET). I geo and EF values showed that sediments were loaded with Ni, Cd, Pb, and Co and no obvious variations were found among the left and right banks of the river. The EF and I geo values were found in order of Co > Pb > Ni > As > Cd > Cu > Zn > Fe and Cd > Co > Pb > Ni > As > Fe > Zn > Cu > Mn, respectively. Furthermore, multivariate statistical analysis like inter-metal correlation, cluster analysis (CA), and principal component analysis (PCA) results revealed that geogenic and anthropogenic activities were major sources of sediment contamination in the study area. These results indicated that more attention should be paid to the inner loads of sediment in order to achieve improvements in reservoir water quality after the control of external pollution.

Quinones as novel chemiluminescent probes for the sensitive and selective determination of biothiols in biological fluids.

Altered plasma aminothiol concentrations are thought to be a valuable risk indicator and are interestingly utilized for routine clinical diagnosis and for the monitoring of various metabolic disorders and human diseases, and accordingly there is a need for an accurate and reliable assay capable of simultaneously determining aminothiols including glutathione (GSH), N-acetylcysteine (NAC), homocysteine (Hcys), and cysteine (Cys) in human plasma. Herein, a highly sensitive, selective, and very fast HPLC-chemiluminescence (HPLC-CL) coupled method is reported, exploiting for the first time the strong nucleophilicity and high reactivity of aminothiols toward quinones for a CL assay. The unique redox-cycling capability of quinone and/or Michael addition adducts, thioether-quinone conjugates, was utilized to establish a novel analytical method based on the reaction of adducts with dithiothreitol (DTT) to liberate reactive oxygen species (ROS), which are detected by using a luminol-CL assay. Specimen preparation involved the derivatization of aminothiols with menadione (MQ) for 5 minutes at room temperature. A unique green chemistry synthesis of thioether-quinones in HEPES buffer (pH 8.5) was introduced by using our reaction methodology without needing any hazardous organic solvent or catalyst. The aminothiol-MQ adducts were separated using solid-phase extraction followed by isocratic elution on an ODS column. Linearity was observed in the range of 2.5-500, 5-500, 10-1500, and 20-2000 nM with detection limits (S/N of 3) of 3.8, 4.2, 8, and 16 (fmol per injection) for GSH, NAC, Hcys, and Cys, respectively. The method was successfully applied for the selective determination of aminothiols in human plasma from healthy people and patients with rheumatic arthritis and diabetes mellitus. The obtained results postulated the usefulness of our method for investigating the relationship between aminothiol metabolism and related human disorders.

Quantification of 3-MCPD and its mercapturic metabolite in human urine: validation of an LC-MS-MS method and its application in the general population.

A new method for the simultaneous quantitative determination in human urine of 3-monochloropropane-1,2-diol (3-MCPD), a toxic food contaminant, and its metabolite, 2,3-dihydroxypropyl mercapturic acid (DHPMA), was developed and validated. After urine dilution, the analytes were separated on an Atlantis®dC18 column and quantified by liquid chromatography-tandem mass spectrometry using isotopically labelled internal standards. The limits of quantification (S/N = 10) were 1.90 and 2.21 µg/L for 3-MCPD and DHPMA, respectively. Intra- and inter-day precision were lower than 6 % for each compound. Matrix effects were evaluated. Due to the high sensitivity and good accuracy of the method, 3-MCPD and DHPMA were found in 67 and 100 % of urine samples of healthy subjects, respectively.

Analysis of 18 urinary mercapturic acids by two high-throughput multiplex-LC-MS/MS methods.

Mercapturic acids (MAs) are metabolic end products formed from conjugates between glutathione and electrophilic compounds. MAs are, therefore, suitable biomarkers of exposure to toxicants, which are either electrophiles by themselves or metabolized to electrophilic intermediates. We developed and validated two LC-MS/MS methods which allow the complementary, rapid, and sensitive determination of MAs derived from acrolein, acrylamide, acrylonitrile, benzene, 1,3-butadiene, crotonaldehyde, N,N-dimethylformamide, ethylene, ethylene oxide, vinyl chloride, propylene oxide, styrene, toluene as well as methylating and ethylating agents. Since separate determinations of single or small groups of MAs are time-consuming and expensive, we multiplexed several individual methods into two LC-MS/MS methods covering 18 individual mercapturic acids. Method validation according to FDA guidelines showed excellent results in terms of robustness, accuracy, and sensitivity of the methods. Moreover, the use of a minimal, simple, and straightforward sample cleanup procedure further accelerated the analytical workflow, which allows a time- and cost-efficient analysis of up to 18 MAs derived from various toxicants in environmental levels. The methods were applied to urine samples derived from a strictly diet-controlled clinical study, including 25 smoking and 25 non-smoking subjects. Significant increase in the urine concentrations in smokers as compared to non-smokers (p < 0.01; Student t test) was observed for 13 individual MAs. Moreover, a dose dependence was obtained for the majority of the analytes. In conclusion, the newly developed assays represent a powerful tool for the fast and reliable quantification of 18 MAs in clinical studies. A first method application suggests several suitable biomarkers for nine relevant toxicants in tobacco smoke.

A Voltammetric Sensor Based on Modified Multi-Walled Carbon Nanotubes for N-Acetyl-L-Cysteine Determination in the Presence of Tryptophan Using 4-Chlorocatechol as a Homogenous Electrochemical Catalyst.

Simultaneous determination of N-acetyl-L-cysteine (NAC) and Tryptophan (Trp) has been studied at the surface of glassy carbon electrode (GCE) modified with multi-walled carbon nanotubes (MWCNTs) in the presence of 4-chlorocatechol as homogenous electrochemical catalyst in aqueous media. The electrocatalytic properties of GCE modified with MWCNTs in the presence of 4-chlorocatechol toward the electrocatalytic oxidation of NAC and Trp was studied using cyclic voltammetry (CV), double-step potential chronoamperometry and differential pulse voltammetry (DPV). The results has shown that NAC participates in Michael type addition reaction with electrogenerated quinone from electrooxidation of 4-chlorocatechol at MWCNT/GCE to form the corresponding thioquinone derivative. The reoxidation of the adduct leads to increase in the oxidative current which is proportional to the concentration of NAC. Differential pulse voltammogram peak currents of NAC and Trp increased linearly with their concentration at the ranges of 5-60 µM and 5-50 µM, respectively and the detection limits for NAC and Trp were sequentially 3.427 µM and 2.494 µM. The proposed method was successfully used for the determination of NAC in pharmaceutical samples and the obtained results were found to be reasonable.

Validation of Armadillo officinalis Dumèril, 1816 (Crustacea, Isopoda, Oniscidea) as a bioindicator: in vivo study of air benzene exposure.

This study tests the potential for using Armadillo officinalis as a bioindicator of exposure to and activation of benzene metabolic pathways using an in vivo model. A. officinalis specimens collected in a natural reserve were divided into a control and three test groups exposed to 2.00, 5.32 or 9.09 µg/m(3) benzene for 24h. Three independent tests were performed to assess model reproducibility. Animals were dissected to obtain three pooled tissue samples per group: hepatopancreas (HEP), other organs and tissues (OOT), and exoskeleton (EXO). Muconic acid (MA), S-phenylmercapturic acid (S-PMA), two human metabolites of benzene, and changes in mtDNA copy number, a human biomarker of benzene exposure, were determined in each sample; benzene was determined only in EXO. MA was measured by high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection, S-PMA by triple quadrupole mass spectrometer liquid chromatography with electro spray ionization (LC-MS-ESI-TQD), mtDNA by real-time quantitative PCR and end-point PCR, and benzene by quadrupole mass spectrometer head-space gas chromatography (HSGC-MS). MA and S-PMA levels rose both in HEP and OOT; EXO exhibited increasing benzene concentrations; and mtDNA copy number rose in HEP but not in OOT samples. Overall, our findings demonstrate that A. officinalis is a sensitive bioindicator of air benzene exposure and show for the first time its ability to reproduce human metabolic dynamics.

Trace analysis of N-acetyl-L-cysteine using luminol-H2O2 chemiluminescence system catalyzed by silver nanoparticles.

N-Acetyl-L-cysteine (NAC) can inhibit the luminol-H2 O2 , reaction, which is catalyzed by silver nanoparticles. Based on this phenomenon a new method was developed for NAC determination. Under optimum conditions, a linear relationship between chemiluminescence intensity and NAC concentration was found in the range 0.034-0.98 µg/mL. The detection limit was 0.010 µg/mL (S/N =3), and the relative standard deviation (RSD) was <5% for 0.480 µg/mL NAC (n =5). This simple, sensitive and inexpensive method has been applied to measure the concentration of NAC in pharmaceutical tablets.

Role of aldose reductase in the metabolism and detoxification of carnosine-acrolein conjugates.

Oxidation of unsaturated lipids generates reactive aldehydes that accumulate in tissues during inflammation, ischemia, or aging. These aldehydes form covalent adducts with histidine-containing dipeptides such as carnosine and anserine, which are present in high concentration in skeletal muscle, heart, and brain. The metabolic pathways involved in the detoxification and elimination of these conjugates are, however, poorly defined, and their significance in regulating oxidative stress is unclear. Here we report that conjugates of carnosine with aldehydes such as acrolein are produced during normal metabolism and excreted in the urine of mice and adult human non-smokers as carnosine-propanols. Our studies show that the reduction of carnosine-propanals is catalyzed by the enzyme aldose reductase (AR). Carnosine-propanals were converted to carnosine-propanols in the lysates of heart, skeletal muscle, and brain tissue from wild-type (WT) but not AR-null mice. In comparison with WT mice, the urinary excretion of carnosine-propanols was decreased in AR-null mice. Carnosine-propanals formed covalent adducts with nucleophilic amino acids leading to the generation of carnosinylated proteins. Deletion of AR increased the abundance of proteins bound to carnosine in skeletal muscle, brain, and heart of aged mice and promoted the accumulation of carnosinylated proteins in hearts subjected to global ischemia ex vivo. Perfusion with carnosine promoted post-ischemic functional recovery in WT but not in AR-null mouse hearts. Collectively, these findings reveal a previously unknown metabolic pathway for the removal of carnosine-propanal conjugates and suggest a new role of AR as a critical regulator of protein carnosinylation and carnosine-mediated tissue protection.