Rafał Głowacki

Determination of endogenous thiols and thiol drugs in urine by HPLC with ultraviolet detection

Analysis of urine for endogenous thiols and thiol drugs content by HPLC with ultraviolet detection is addressed. Other methodologies for detection and determination of thiols in urine are only mentioned. Outline of metabolism, role of main biological thiols in physiological and pathological processes and their reference concentrations in urine are presented. In particular, urine sample preparation procedures, including reduction of thiol disulfides, chemical derivatization and reversed-phase HPLC separation steps are discussed. Some experimental details of analytical procedures for determination of endogenous thiols cysteine, cysteinylglycine, homocysteine, N-acetylcysteine, thioglycolic acid; and thiol drugs cysteamine, tiopronin, D-penicillamine, captopril, mesna, methimazole, propylthiouracil and thioguanine are reviewed.

Urinary excretion measurement of cysteine and homocysteine in the form of their S-pyridinium derivatives by high-performance liquid chromatography with ultraviolet detection

Several human diseases, in particular metabolic disorders, often lead to the accumulation of characteristic metabolites in plasma, urine and cells. The selected diseases of this type include cystinuria and homocystinuria. In the typical laboratory diagnosis of these two diseases, a positive nitroprusside test is followed by quantitative analysis of urine cysteine and homocysteine in order to differentiate between cystinuria and homocystinuria. A sensitive and reproducible assay for total urine cysteine and homocysteine has been developed. The essential steps in the assay include conversion of disulphides to free thiols with tributylphosphine, conjugation of the thiols with 2-chloro-1-methyl pyridinium iodide, separation of S-pyridinium derivatives of cysteine and homocysteine from other endogenous urine thiol derivatives by reversed-phase high-performance liquid chromatography, and detection and quantitation by spectrophotometry. The method has a sensitivity of 4 pmol and is reproducible, intra- and inter-day coefficients of variation are from 1.37 to 4.14% and from 2.38 to 5.01%, respectively. The mean concentration of total urine cysteine and homocysteine in healthy donors (7 men and 7 women) were for women. 92.0 +/- 45.8 and 16.4 +/- 4.8 respectively, and for men 120.9 +/- 46.6 and 21.5 +/- 7.4 nmol/ml, respectively. Total urine homocysteine represents approximately 17.7% of cysteine in the urine of normal individuals.

Ultraviolet derivatization of low-molecular-mass thiols for high performance liquid chromatography and capillary electrophoresis analysis.

Thiols play an important role in metabolic processes of all living creatures and their analytical control is very important in order to understand their physiological and pathological function. Among a variety of methods available to measure thiol concentrations, chemical derivatization utilizing a suitable labeling reagent followed by liquid chromatographic or electrophoretic separation is the most reliable means for sensitive and specific determination of thiol compounds in real world samples. Ultraviolet detection is, for its simplicity, commonly used technique in liquid chromatography and capillary electrophoresis, and consequently many ultraviolet derivatization reagents are in used. This review summarizes HPLC and CE ultraviolet derivatization based methods, including pre-analytical considerations, procedures for sample reduction, derivatization, and separation of the primary biological aminothiols--cysteine, homocysteine, cysteinylglycine and glutathione, and most important thiol-drugs in pharmaceutical formulations and biological samples. Cognizance of the biochemistry involved in the formation of the analytes is taken.

Oxidative/Nitrative Modifications of Plasma Proteins and Thiols from Patients with Schizophrenia

Objective: The level of various specific biomarkers of oxidative stress in plasma from schizophrenic patients, as well as biomarkers (the level of isoprostanes) in urine in schizophrenic patients was described. The aim of our present study was to evaluate biomarkers of oxidative stress by oxidative/nitrative modifications of plasma proteins (by measuring the level of carbonyl groups and 3-nitrotyrosine in proteins) from patients with schizophrenic disorders and from control group. We also investigated the level of low-molecular-weight thiols [glutathione (GSH), cysteine (CSH), cysteinylglycine (CGSH) and homocysteine] in plasma obtained from schizophrenic patients and from healthy volunteers. Patients hospitalized in the 1st and 2nd Psychiatric Department of the Medical University in Lodz, Poland were interviewed with a special questionnaire (treatment, course of diseases, dyskinesis and other extrapyramidal syndromes). According to DSM-IV criteria, all patients had a diagnosis of paranoid type. They were treated with antipsychotic drugs (clozapine, risperidone, olanzapine). The mean duration of schizophrenia was about 5 years. Methods: Levels of carbonyl groups and 3-nitrotyrosine residues in plasma proteins were measured by ELISA and a competition ELISA, respectively. High-performance liquid chromatography was used to analyze free thiols in plasma. Results: We observed a statistically increased level of biomarkers of oxidative/nitrative stress such as carbonyl groups or 3-nitrotyrosine in plasma proteins from schizophrenic patients. In schizophrenic patients the amount of homocysteine in plasma was higher compared with the control group; the level of GSH, CSH and CGSH was decreased. This indicates that reactive oxygen species and reactive nitrogen species may stimulate oxidative/nitrative modifications of plasma proteins in schizophrenic patients. Conclusion: Considering the data presented in this study, we suggest that the amount of carbonyl groups and 3-nitrotyrosine in plasma proteins may be important indicators of protein damage in vivo in schizophrenia.

Fully automated method for simultaneous determination of total cysteine, cysteinylglycine, glutathione and homocysteine in plasma by HPLC with UV absorbance detection

A fully automated HPLC method for the simultaneous determination of total thiols in plasma samples has been developed. The method involves reductive conversion of disulfides to their reduced counterparts with the use of tris(2-carboxyethyl)phosphine. After reduction the newly formed sulfhydryl groups are reacted with 2-chloro-1-methylquinolinium tetrafluoroborate to form 2-S-quinolinium derivatives followed by deproteinization by dialysis. The reaction products are separated by reversed-phase HPLC, detected and quantified by UV absorbance detection at 355nm. The recommended HPLC procedure enables measurement of four main plasma aminothiols cysteine, cysteinylglycine, glutathione, and homocysteine with low imprecision (mean relative standard deviations within calibration range, 3.47%, 5.34%, 4.25% and 3.26%, respectively) and good sensitivity. Accuracy, expressed as the mean measured amount as percentage of added amount, was within 97.5-103.0%, 98.3-102.5%, 96.3-99.5% and 97.1-99.1%, respectively. The lower limit of quantification for all thiols was 0.5microM. The whole unattended instrument acquisition time amounts 13min.

2-CHLORO-1-METHYLQUINOLINIUM TETRAFLUOROBORATE AS AN EFFECTIVE AND THIOL SPECIFIC UV-TAGGING REAGENT FOR LIQUID CHROMATOGRAPHY

A new, highly reactive, and thiol specific derivatization reagent for liquid chromatographic analysis has been developed. The reagent, 2-chloro-1-methylquinolinium tetrafluoroborate, reacts instantaneously with hydrophilic thiols in water under mild conditions. The 2-S-quinolinium derivatives, resulting from this reaction, are stable thioethers exhibiting well defined absorption maximum at 348 nm and molar absorptivity coefficient about 2 × 10[4] L mol −1cm−1. It is shown that under reversed-phase high performance liquid chromatography conditions with gradient elution, six thiols possessing different functional groups, can be baseline separated and quantified within 4 min in one analytical run. The lower limits of detection and quantitation of the analytes (20 μL injection volume) are within 0.3–1.5 pmol and 0.5–4.0 pmol, respectively. The assays are linear in the range of 0.025–8 nmol/mL with correlation coefficients values close to 0.9999. Synthesis of 2-chloro-1-methylquinolinium tetrafluoroborate, as well as isolation of the 2-S-quinolinium derivative of one model thiol, are described.

Facile and sensitive method for the determination of mesna in plasma by high-performance liquid chromatography with ultraviolet detection

The use of 2-chloro-1-methylquinolinium tetrafluoroborate, an ultraviolet tagging reagent, for the ion-pair, reversed-phase high-performance liquid chromatography of mesna in human plasma is reported. In order to achieve this objective optimization of the two-step procedure, derivatization and separation of mesna S-quinolinium derivative from that of other thiols present in plasma and internal standard, was investigated. The derivatization was optimized in terms of pH, reagent excess and time of the reaction, and the mobile phase in terms of ion-pairing reagent concentration, pH, organic modifier content and temperature. Baseline separation was achieved on an analytical Waters Nova-Pak C18 (150x3.9 mm, 5 microm) column with a mobile phase consisting of pH 2.3 0.05 M trichloroacetic acid-acetonitrile (89:11, v/v) pumped at 1.2 ml/min. The peak height ratios of the mesna derivative to that of the internal standard (thiomalic acid) varied linearly with the concentration of the analyte added to normal plasma with a correlation coefficient of 0.9997. The lower limits of detection and quantitation were 40 pmol/ml (0.8 pmol on-column) and 160 pmol/ml (3.2 pmol on-column), respectively. The intra-run imprecision and inaccuracy were from 1.3 to 2.4 and from 1.3 to 2.0%, respectively.

Chemical Biology of Homocysteine Thiolactone and Related Metabolites

Protein-related homocysteine (Hcy) metabolism produces Hcy-thiolactone, N-Hcy-protein, and N epsilon-homocysteinyl-lysine (N epsilon-Hcy-Lys). Hcy-thiolactone is generated in an error-editing reaction in protein biosynthesis when Hcy is erroneously selected in place of methionine by methionyl-tRNA synthetase. Hcy-thiolactone, an intramolecular thioester, is chemically reactive and forms isopeptide bonds with protein lysine residues in a process called N-homocysteinylation, which impairs or alters the proteins biological function. The resulting protein damage is exacerbated by a thiyl radical-mediated oxidation. N-Hcy-proteins undergo structural changes leading to aggregation and amyloid formation. These structural changes generate proteins, which are toxic and which induce an autoimmune response. Proteolytic degradation of N-Hcy-proteins generates N epsilon-Hcy-Lys. Levels of Hcy-thiolactone, N-Hcy-protein, and N epsilon-Hcy-Lys increase under pathological conditions in humans and mice and have been linked to cardiovascular and brain disorders. This chapter reviews fundamental biological chemistry of Hcy-thiolactone, N-Hcy-protein, and N epsilon-Hcy-Lys and discusses their clinical significance.

Determination by liquid chromatography of free and total cysteine in human urine in the form of its S-quinolinium derivative

A reversed-phase high-performance liquid chromatographic method for the determination of free and total cysteine in urine is described. The method involves reductive conversion of cysteine dimer and cysteine mixed disulphides to their reduced counterpart with the use of tri-n-butylphosphine, ultraviolet-labeling with 2-chloro-1-methylquinolinium tetrafluoroborate, and liquid chromatographic separation with isocratic conditions. In developing this method the following parameters were investigated and optimized: the time, pH and reagent excess in the derivatization step, and mobile phase buffer concentration, pH, organic modifier and column temperature in the separation step. The method provides quantitative information on free and total cysteine based on assays with derivatization before and after reduction with tri-n-butylphosphine. The calibration graph, obtained with the use of normal urine spiked with growing amounts of cystine, was linear over the concentration range covering most experimental and clinical cases. The assay has a low pmol detection and quantitation limits, low imprecision and high recovery. The method was validated for urine samples received from several donors. Cystine was chosen as a primary calibrator for these assays.

Liquid chromatographic assessment of total and protein-bound homocysteine in human plasma.

Homocysteine present in human blood plasma is derivatized with thiol selective ultraviolet labelling reagent, 2-chloro-1-methylpyridinium iodide, and separated from other plasma thiol derivatives by high-performance liquid chromatography (HPLC) with detection at 312 nm. The separation is carried out isocratically on LiChrospher RP-18 column using mobile phase consisting of pH 2.5 0.04 M trichloroacetic acid buffer and methanol in the ratio 9:1 (v/v) pumped at 0.5 ml min(-1) at 40 degrees C. The homocysteine S-pyridinium derivative elutes at 6.5 min. To determine total and protein-bound homocysteine it is necessary to cleave disulphide bounds by the use of tri-n-butylphosphine in order to form free sulfhydryl group. The method provides quantitative information on total and protein-bound homocysteine based on assays with derivatization after reduction of whole plasma, and derivatization after reduction of acid precipitated proteins. The calibration graph is linear over the concentration range covering most experimental and clinical cases. The assay has a low pmol sensitivity and is reproducible; intra- and inter-day, relative standard deviation range from 1.79 to 5.09% and from 2.80 to 5.60%, respectively. The method is applied to the determination of total and protein-bound homocysteine in the plasma of healthy individuals.