Capillary electrophoresis in the evaluation of aminothiols in body fluids

Thiols play a fundamental role in cell biology, biochemistry and pharmacology. Altered thiol levels in body fluids are linked to specific pathological conditions. Glutathione is the most abundant intracellular low-molecular-mass thiol, playing an essential role in protecting cells from toxic species; other relevant thiol-containing compounds are homocysteine (Hcy), cysteine (Cys), cysteinylglycine (CysGly). Plasma aminothiols can be bound to proteins but they also occur free in the disulfide (symmetrical and mixed) and in the reduced forms. The simultaneous determination of these aminothiols, their precursor and metabolites is a useful tool in studying oxidative stress, metabolic and redox regulation. Many capillary electrophoresis methods have been proposed for this purpose, the aim of the present review is to support researchers in the choice of suitable methods for the determination of thiols in body fluids evaluating the different approaches and technologies proposed from the literature.     

Survival curves of glutathione synthetase deficient human fibroblasts: correlation between radiosensitivity in hypoxia and glutathione synthetase activity

The role of intracellular non-protein bound sulphydryl compounds (NPSH), and in particular that of glutathione (GSH), in the response of cells to ionizing radiation under different O2 concentrations has been assessed using cell strains deficient in glutathione synthetase and exhibiting different NPSH levels. The cell strains used originated from patients with 5-oxoprolinuria and from their relatives (heterozygotes and proficient homozygotes). No correlation has been found between NPSH and GSH concentrations and radiosensitivity under oxic, aerobic and hypoxic conditions. However, a highly significant correlation has been observed between radiosensitivity under hypoxic conditions (and therefore the oxygen enhancement ratio) and the glutathione synthetase activity, suggesting that synthesis of GSH is required after irradiation. In order to explain our results we postulated, beside radical processes, the existence of a GSH-dependent enzymatic repair mechanism for N2 type damage. Hypoxic radio-sensitivity measured with survival curves would result from the interaction of both competition and biochemical repair processes.     

Changes in organ nonprotein sulfhydryl and glutathione concentrations during acute and chronic administration of inorganic lead to chicks

The effects of dietary and injected lead (Pb) on organ nonprotein sulfhydryl (NPSH) and glutathione (GSH) concentrations in the chick were studied. Lead acetate·3H₂O was administered either in the diet for 3 wk at 2000 ppm Pb or by intraperitoneal (ip) injection of 3-wkold chicks with 52 mg Pb/100 g body wt. In Exp. 1, NPSH concentrations in liver and kidney were increased by both dietary and injected Pb in comparison to chicks not receiving Pb. Thigh muscle NPSH was decreased by injected Pb, whereas dietary Pb had no effect. In Expt. 2, whole blood and plasma NPSH were measured at 0, 0.5, 1.0, 2.0, and 4.0 h following ip Pb injection. Both whole blood and plasma NPSH were increased by 30 min. Whole blood NPSH concentrations plateaued at 30 min, and plasma NPSH continued to rise for 2 h. In Expt. 3, injected Pb increased hepatic NPSH, but not GSH concentrations. The ratio of GSH/NPSH was therefore lowered. The incorporation of [1-¹⁴C]glycine into hepatic GSH was stimulated by injected Pb. Buthionine sulfoximine (BSO), an inhibitor of GSH synthesis, reduced hepatic NPSH and [¹⁴C]glycine incorporation in Pb-treated chicks to below control (non-Pb injected) values. In Expt. 4, dietary Pb fed for 3 wk increased the hepatic concentrations of both NPSH and GSH such that the ratio of GSH/NPSH was unchanged in comparison to chicks not fed Pb. The data suggest that the initial response to acute Pb intoxication may involve a mobilization of nonprotein thiols via the interorgan translocation system for GSH. Such a response would help to maintain adequate levels of GSH in organs crucial to detoxification.     

p-Aminophenol-induced liver toxicity: tentative evidence of a role for acetaminophen

p-Aminophenol (PAP) is a widely used industrial chemical and a metabolite of analgesics, such as acetaminophen (APAP). It was found recently that PAP, a known nephrotoxicant, could cause acute hepatotoxicity in mice but not in rats. The mechanism of hepatotoxicity is not known. The aim of this study was to investigate the role of N-acetylation of PAP to APAP in PAP-induced toxicity. Male C57BL/6 mice injected intraperitoneally (i.p.) with various doses of PAP were sacrificed at 12 hours for measurement of serum glutamic-pyruvic transaminase (GPT) and sorbitol dehydrogenase (SDH) levels and determination of the extent of hepatic nonprotein sulfhydryl (NPSH) and glutathione (GSH) depletion. Plasma levels of APAP and its metabolites were measured by HPLC after PAP administration. p-Aminophenol depleted NPSH in a dose- and time-dependent manner. Depletion of NPSH in mouse liver occurred at PAP doses above 400 mg/kg. Buthionine sulfoximine (BSO), an inhibitor of GSH synthesis, potentiated the PAP-induced hepatotoxicity. Ascorbate, a reducing agent, did not affect PAP-induced hepatotoxicity and NPSH depletion. After PAP treatment, APAP and its sulfate and glucuronide conjugates as well as GSH conjugates (APAP-cysteine and APAP-mercapturate) were detected in the plasma. The results suggest the roles of GSH and N-acetylation of PAP to APAP in PAP-induced hepatotoxicity.     

巯基参与胃粘膜防御机制

本工作研究了胃粘膜非蛋白质巯基物质(NPSH)在粘膜防御功能中的作用。结果表明,酸性乙醇灌胃或冷冻加束缚应激引起大鼠胃粘膜 NPSH 含量显著下降;补充含-SH 的化合物半胱胺或还原型谷胱甘肽可防止酸性乙醇引起的胃粘膜损伤;在酸性乙醇灌胃或应激后,胃粘膜谷胱甘肽还原酶活性明显降低,并与 NPSH 含量的下降在时间上一致;丙二醛含量在酸性乙醇灌胃后显著升高,自由基清除剂二甲亚砜可减轻胃粘膜损伤。上述结果提示,胃粘膜NPSH 可能通过对自由基的清除作用参与粘膜的局部防御机制;谷胱甘肽还原酶活性下降和自由基生成增加所导致的粘膜 NPSH 含量下降可能是损伤发生过程的重要环节。     

A new HPLC method for the simultaneous determination of oxidized and reduced plasma aminothiols using coulometric electrochemical detection

A new method has been developed that is capable of providing a complete profile of the most common monothiols and disulfides present in plasma or tissue extracts. The method utilizes reversed phase ion-pairing high performance liquid chromatography coupled with coulometric electrochemical detection to simultaneously quantify free oxidized and reduced aminothiols or total aminothiols after chemical reduction. The method is extremely sensitive, with limits of detection in the 5 fmol/mL range for monothiols and 50 fmol/mL for dithiols. The interassay and intraassay coefficients of variation for total and free aminothiols ranged between 1.2 and 5.8%. The mean recoveries for total and plasma aminothiols ranged between 97.1 and 102.8%. The aminothiols are quantified directly, without derivatization, and include methionine, homocysteine, homocystine, cystathionine, cysteine, cystine, cysteinylglycine, and oxidized and reduced glutathione. Because a complete aminothiol profile of metabolites in both the remethylation (anabolic) and transulfuration (catabolic) pathways of homocysteine metabolism can be determined simultaneously, this new method should be useful in determining the metabolic etiology of homocysteinemia and in designing appropriate nutritional intervention strategies. Basic research applications of this method should lead to an increased understanding of the metabolic pathology of aminothiol imbalance.     

The effects of mercuric chloride and sodium selenite on glutathione and total nonprotein sulfhydryls in the kidney of the black bullhead (Ictalurus melas)

1. Mercuric chloride injections (10 μmol/kg) caused renal nonprotein sulfhydryls (NPSH) to decrease within 3 hr and then increase by 72 hr after injection.2. Selenium injections (10 μmol/kg) caused renal NPSH increases.3. The Se-promoted increases in renal NPSH more than compensated for the Hg-induced decreases when the two elements were simultaneously injected.4. This Se-promoted increase in renal NPSH may explain why Se promotes renal uptake of Hg when the two elements are simultaneously injected.5. Glutathione accounted for most (77–95%) of the NPSH.     

Evidences for adduct formation between intracellular non-protein thiols and nitroazoles possessing an alpha,beta-unsaturated carbonyl side chain and the effects on radiosensitization of hypoxic cells

Reactivity of a number of nitroazole derivatives bearing an alpha,beta-unsaturated carbonyl group on the side chain toward non-protein thiols (NPSH) was examined both in the phosphate buffer solution and in the biological system. These alpha,beta-unsaturated compounds reacted with NPSH, such as glutathione (GSH) and L-cysteine (Cys), in the buffer solution to afford the 1,4-addition products. The reaction gave a second-order rate constant. The adducts of methyl 4-(2'-nitroimidazol-1'-yl)crotonate (1) with GSH and Cys were isolated and characterized as two diastereomers (7a,b and 8a,b) in ca. 1:1 ratio, respectively. Similarly, exposure of EMT6/KU cells to 1 at 1.0 mM for 1 h resulted in depletion of the intracellular NPSH by more than 80%. Over 50% of the depleted NPSH was attributed to the formation of the conjugated diastereomeric adducts. On the other hand, incubation of EMT6/KU cells with 1 at 1.0 mM under hypoxic conditions before X-ray irradiation caused concurrently a sharp reduction of the shoulder of the dose-survival curves (reduced the extrapolation number (n) from 8.0 to ca. 1.0) and an increase in the slope (decreased the mean lethal dose (Do) to ca. 50% of the control level). The observed effects of 1 on the dose-survival curves were due to the NPSH depletion through the Michael addition occurred in the cellular system. A fairly linear relationship was obtained between the n value and the reduced intracellular NPSH level. It indicated that the shoulder effect of the dose-survival curves of hypoxic cells should be the result of the NPSH depletion by the alpha,beta-unsaturated carbonyl group attached to the nitroazoles.     

Effect of different amino acidic pretreatments that protect the kidney against papillary necrosis induced by bromoethylamine on differential distribution of renal nonprotein sulfhydryls

Content of nonprotein sulfhydryls (NPSH) was found to be higher in rat renal cortex than in external medulla and papilla. Administration of bromoethylamine (BEA), at a dose that produces extensive papillary necrosis and minor effects in the other renal segments, induced a significant reduction in NPSH levels of renal cortex and external medulla, with no changes in the papilla. Treatment with N-acetyl-L-cysteine (NAC) elicited an increase in papillary NPSH and a decrease in the cortex, with opposite changes being observed with an amino acid mixture of glutamine, glycine, and cystine (AM). Similar results were found in animals pretreated with NAC or AM prior to BEA intoxication. These pretreatments protect the cortex, external medulla, and papilla from the necrosis induced by BEA. It is suggested that protection of BEA-induced renal necrosis by NAC or AM pretreatments might be due to different mechanisms, with NPSH playing direct or indirect roles, respectively.     

AtATM3 is involved in heavy metal resistance in Arabidopsis

AtATM3, an ATP-binding cassette transporter of Arabidopsis (Arabidopsis thaliana), is a mitochondrial protein involved in the biogenesis of iron-sulfur clusters and iron homeostasis in plants. Our gene expression analysis showed that AtATM3 is up-regulated in roots of plants treated with cadmium [Cd(II)] or lead (II); hence, we investigated whether this gene is involved in heavy metal tolerance. We found that AtATM3-overexpressing plants were enhanced in resistance to Cd, whereas atatm3 mutant plants were more sensitive to Cd than their wild-type controls. Moreover, atatm3 mutant plants expressing 35S promoter-driven AtATM3 were more resistant to Cd than wild-type plants. Since previous reports often showed that the cytosolic glutathione level is positively correlated with heavy metal resistance, we measured nonprotein thiols (NPSH) in these mutant plants. Surprisingly, we found that atatm3 contained more NPSH than the wild type under normal conditions. AtATM3-overexpressing plants did not differ under normal conditions, but contained less NPSH than wild-type plants when exposed to Cd(II). These results suggest a role for AtATM3 in regulating cellular NPSH level, a hypothesis that was further supported by our gene expression study. Genetic or pharmacological inhibition of glutathione biosynthesis led to the elevated expression of AtATM3, whereas expression of the glutathione synthase gene GSH1 was increased under Cd(II) stress and in the atatm3 mutant. Because the closest homolog of AtATM3 in fission yeast (Schizosaccharomyces pombe), HMT1, is a vacuolar membrane-localized phytochelatin-Cd transporter, it is tempting to speculate that glutathione-Cd(II) complexes formed in the mitochondria are exported by AtATM3. In conclusion, our data show that AtATM3 contributes to Cd resistance and suggest that it may mediate transport of glutamine synthetase-conjugated Cd(II) across the mitochondrial membrane.     

Determination of total plasma homocysteine and related aminothiols by gas chromatography with flame photometric detection

A selective and sensitive method for the determination of total homocysteine (Hcy) and related aminothiols, such as cysteine (Cys) and cysteinylglycine (CysGly), in plasma samples by gas chromatography (GC) has been developed. After reduction of the sample with sodium borohydride, the liberated Hcy and other aminothiols were converted to their N,S-diisopropoxycarbonyl methyl ester derivatives and measured by GC with flame photometric detection using a DB-17 capillary column. The calibration curves were linear over the range 0.5–10 nmol for Hcy and CysGly, and over the range 5–100 nmol for Cys, and the correlation coefficients were above 0.996. Using this method, total plasma Hcy, Cys and CysGly could be directly analysed without prior clean-up of the sample and without any interference from coexisting substances. Overall recoveries of Hcy and other aminothiols added to plasma samples were 95–106%. Analytical results for the determination of total plasma Hcy, Cys and CysGly from normal subjects are presented.     

Liquid chromatographic determination of total homocysteine in blood plasma with photometric detection

A rapid and sensitive method for quantification of homocysteine total forms and glutathione levels in blood plasma via HPLC was developed. Dithiotreitol as a water soluble agent has been used as a reductant for both protein and nonprotein disulphides. Dithiotreitol reacts with the mixed disulphides under 60 degrees C treatment within 10 min. Reduced aminothiols and homocystein were easily derivated with 5,5'-dithiobis-(2-nitrobenzoic acid) and the resultant ultraviolet absorbance within 330 nm was detected by the HPLC method. The concentration of total plasma homocysteine was significantly higher in groups of patients: with the end stage of renal disease: 45.5+/-40.9 micromol/l (n=79), with cerebral vascular disorders 12.3+/-7.0 micromol/l (n=65), and with coronary atherosclerosis 15.4+/-10.9 micromol/l (n=15) than that in healthy subjects (6.2+/-1.74 micromol/l, n=20). Some major advantages of the method include: simultaneous measurement of both total homocysteine and total glutathione, no loss of oxidized form during processing of blood plasma for aminothiols measurement, use of protein-bound aminothiols solution as a calibrator.     

The importance of NPSH on the radiosensitizing effect of oxygen in Chinese hamster V-79 cells

The radiosensitivity of Chinese hamster V-79-171B fibroblasts increased more rapidly with increasing partial pressure of oxygen when the cell cultures had low endogenous levels of non-protein sulphydryl (NPSH), about 5 mumol per cell compared with about 15 mumol per cell. There was a good correlation between initial NPSH content and sensitization by oxygen concentrations between 0.06 and 0.7 per cent.     

Reactions of pyrzdoxal 5'-phosphate, 6-aminocaproic acid, cysteine, and penicilamine. Models for reactions of Schiff base linkages in pyridoxal 5'-phosphate-requiring enymes.

Schiff base formation, transaldimination, and reaction with aminothiols are important reactions which occur on the surface of pyridoxal-P-requiring enzymes. As a first step in assessing the role of the protein in these reactions, models for these reactions were studied in the absence of enzyme at 25 degrees, gamma/2 0.28. The reaction of 6-aminocaproic acid with pyridoxal-P to form the Schiff base N6-(P-pyridoxylidene)-aminocaproic acid was studied as a model for formation of Schiff base on the holoenzyme...     

Sulfhydryl groups during the S phase: comparison of cells from G 1 , plateau-phase G 1 , and G 0

The non-protein sulfhydryl (NPSH) content of cells moving into S from G₁, plateau phase G₁, and G₀ was measured. Chinese hamster ovary (CHO) cells accumulated in G₁ by growth into plateau phase contain only one-fourth the NPSH concentration of cycling C₁ cells or G₁ cells accumulated by brief growth in isoleucine-deficient medium. Upon dilution of plateau cultures with fresh medium, cellular NPSH content increases rapidly, reaching the same level as that in cycling cells within four hours. This increase is prevented by cycloheximide but not by actinomycin D or hydroxyurea. Neither CHO cells cycling in vitro nor salivary gland G₀ cells stimulated with isoproterenol in vivo show significant changes in intracellular NPSH concentrations during S phase. This suggests that the concentration of intracellular NPSH (glutathione) remains constant during the cell cycle except when cells are grown to plateau phase in exhausted or deficient medium, in which case normal degradation exceeds synthesis and the gross level falls until fresh medium is provided and synthesis, apparently on preexisting RNA templates, accelerates.     

General characteristics and comparative evaluation of the radioprotective properties of aryl alkyl amine adrenomimetics in experiments on mice]

The radioprotective effect (RPE) of some arylalkylamines (AAAs) was studied in experiments on mice. Mesaton and its close analogues were injected subcutaneously 15 minutes prior to irradiation at a dose of 800 rad. The protective effect is exerted by AAAs in low doses (25--50 mumole/kg), the compounds show stable and high RPE (80--80% survival, dose reduction factor being 1.3--1.4) and low toxicity (LD50 = 4--8 mumole/kg). AAAs studied are not less effective than aminothiols. Their pharmacological spectrum--K = LD50/ED50 (200--500) is superior to that of known aminothiols and indolylalkylamines.     

Simultaneous determination of total homocysteine, cysteine, cysteinylglycine, and glutathione in human plasma by high-performance liquid chromatography: application to studies of oxidative stress

A sensitive, reproducible, and robust high-performance liquid chromatography (HPLC) method has been validated for simultaneously determining total concentrations of the aminothiols homocysteine, cysteine, cysteinylglycine, and glutathione in human plasma. Plasma aminothiols are reduced via incubation with tris-(2-carboxyethyl)-phosphine hydrochloride, followed by protein precipitation with trichloroacetic acid and derivatization with ammonium-7-fluorobenzo-2-oxa-1,3-diazole-4-sulfonic acid. Separation of aminothiols and the internal standard mercaptopropionylglycine is achieved using reversed-phase HPLC conditions and fluorescence detection. Excellent linearity is observed for all analytes over their respective concentration ranges with correlation coefficients (r) > 0.99. The intra- and inter-day precision and accuracy were within +/-10%. This method utilizes an internal standard, employs phosphate buffered saline-based standards and quality controls, and demonstrates excellent plasma recovery and improved sensitivity. This assay is well suited for high-throughput quantitative determination of aminothiols in clinical studies, and is currently being used to support investigations of oxidative stress in patients with chronic kidney disease.     

Lead effects in the chick during selenium deficiency

1. Growing chicks (Gallus domesticus) were fed a selenium-deficient diet supplemented with 0 or 2000 ppm lead (Pb) and 0 or 0.1 ppm selenium (Se). 2. Selenium addition stimulated growth at 0 but not at 2000 ppm Pb, while Pb depressed growth at both levels of Se. 3. Selenium addition stimulated Se-dependent glutathione peroxidase (GSH-Px) activity in liver, but Pb was without effect on GSH-Px activity. 4. Lead addition increased non-protein sulfhydryl (NPSH) concentrations in liver, kidney and thigh muscle. NPSH levels were not altered by Se. 5. The reported antagonism between Pb and Se does not appear to be mediated through effects on GSH-Px or NPSH metabolism.     

The influence of thiols on the pre-irradiation incubation effect of nitroimidazoles in E. coli cells

The increase in the degree of radiosensitization of Escherichia coli cells following prolonged pre-irradiation incubation with nitroimidazoles is not correlated with the loss of intracellular non-protein thiols (NPSH) alone. The rates of reduction of the nitro compounds and the NPSH removal do not show strong dependencies on the lipophilicities of the nitroimidazoles whereas the highly lipophilic compound RGW-609 effects an increase in radiosensitization in a much shorter incubation time than the other nitroimidazoles. Exogenous dithiothreitol (DTT) increased the rate of reduction of misonidazole in the cells but did not alter the fraction converted to the amine. Added DTT (0.15 mmol dm-3) completely protected against the pre-irradiation incubation effect of misonidazole (2.5 mmol dm-3) when added at the start of the incubation but only partially protected when added before irradiation. It is suggested that NPSH can intercept metabolite(s) (or their precursors) of nitroimidazoles which can potentiate cell killing by radiation.     

Endogenous thiol levels in heterogeneous murine tumor cells as a function of the physiological state and the response to X-irradiation

The endogenous thiols (PSH, protein sulfhydryls; NPSH, nonprotein sulfhydryls; and GSH, glutathione) were measured in the 66 and 67 murine carcinoma cells growing under different physiological conditions in vitro (e.g., proliferation, P; nutrient-deprived quiescence QI; and QI cells stimulated by refeeding the monolayer in situ and assayed 4 (St4) and 14 (St14) h later). The aerobic radiation response was also studied as a function of the physiological state and thiol concentration. The changes in PSH levels suggest that the proportion of thiol-containing proteins changed whenever the cells were in transition between different physiological states (e.g., when QI cells were stimulated by refeeding, the proportion of PSH was elevated dramatically over either QI or P cells). The NPSH and GSH levels were both down significantly in the QI vs. P cells as was the total thiol level (PSH plus NPSH). Fourteen h but not 4 h after stimulation, the NPSH and GSH levels had returned to or exceeded the P-cell levels. Also, the proportion of GSH in the NPSH fraction varied as a function of the physiological state. The 66 and 67 QI cells were both more radiosensitive than the respective P cells. Also, the 66 cell radiation-induced cytotoxicity had returned to the P response by about 4 h after refeeding but the stimulated 67 cells had not. However, no overall correlation was apparent between the various aerobic radiation responses and the pool sizes of either the total thiols or of the various subsets of thiols. The depressed total thiol level and the increased radiosensitivity of the QI cells could represent a cause-and-effect relationship or these parameters could be independent phenomena only related indirectly through the reduced metabolic activity of the quiescent cells.