Aminothiol Levels Research Articles

Assessment of the Concentrations of Selected Aminothiols in Patients after COVID-19.

Background: Data show that due to endothelial damage and thrombogenic effects, severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection may accelerate the development of atherosclerosis and increase the risk of cardiovascular diseases (CVDs). The impaired metabolism of aminothiols increases oxidative stress, as these molecules are involved in antioxidant defense as well as in thiol redox control. In this study, total levels of selected aminothiols (i.e., cysteine (Cys), homocysteine (HCy), and glutathione) in convalescents after coronavirus disease of 2019 (COVID-19) were evaluated. The analyses were made according to the sex of the patients, time from COVID-19 onset, and COVID-19 severity. Methods: The study group consisted of 212 patients after COVID-19. Levels of total aminothiols were assessed in the blood plasma using high-performance liquid chromatography (HPLC). Results: The mean Cys concentrations were higher in men than in women (229.92 µmol/L ± 51.54 vs. 210.35 µmol/L ± 41.90, respectively; p = 0.003). Differences in Cys levels were also noticed in the total study group between patients distinguished due to time from disease onset (226.82 µmol/L ± 40.57 in <12 weeks, 232.23 µmol/L ± 47.99 in patients 12-24 weeks, and 208.08 µmol/L ± 48.43 in patients >24 weeks; p = 0.005). In addition, over 11% of total patients 12-24 weeks from disease onset had Cys levels above 300 µmol/L compared to almost 4% of patients <12 weeks and 2% of patients >24 weeks (p = 0.046). In sex-adjusted subgroups, significant differences due to time from COVID-19 were found in Cys levels in women (p = 0.004) and in glutathione levels in men (p = 0.024). None of the aminothiol levels differed between the subgroups based on the severity of COVID-19. Conclusions: Men had overall higher Cys levels than women. Cys levels were lower >24 weeks after COVID-19 onset than in the earlier period after disease onset. A partial elevation in Cys levels 12-24 weeks after the disease onset may contribute to the increase in CVD risk in the post-COVID-19 period.

The Diagnostic and Prognostic Roles Played by Homocysteine and Other Aminothiols in Patients with Chronic Kidney Disease.

We examined standard clinical and laboratory biochemical parameters, as well as the levels of aminothiols in the blood and urine (homocysteine (Hcy), cysteine (Cys), S-adenosylmethionine (SAM), and S-adenosylhomocysteine (SAH)) via capillary electrophoresis in patients with CKD at stages II-V. Patient outcomes were assessed after five years. To complete forecasting, correlation and ROC analysis were performed. It was found that the levels of Cys and Hcy in blood plasma were earlier markers of CKD starting from stage II, while the levels of SAM and SAM/SAH in urine made it possible to differentiate between CKD at stages II and III. Blood plasma Hcy and urinary SAM and SAM/SAH correlated with mortality, but plasma Hcy concentrations were more significant. Thus, plasma Hcy, urine SAM, and SAM/SAH can be considered to be potential diagnostic and prognostic markers in patients with CKD.

Technical note: simultaneous determination of amino thiols in pig tissue by ultra-high performance liquid chromatography with fluorescence detection.

Sulfur amino acid nutrition and metabolism are linked to animal disease. While validated methods for the determination of amino thiol levels in plasma or serum are available, there is a dearth of validated methods for their measurement in tissue. A robust and reproducible ultra-high performance liquid chromatography method has been validated for the simultaneous determination of concentrations of cysteine (Cys), cysteinylglycine (CysGly), homocysteine (Hcys), γ-glutamylcysteine (γ-GluCys), and glutathione (GSH) in pig tissue. Tissue was homogenized and deproteinized with trichloroacetic acid. Amino thiols in the acid-soluble fraction of the tissue homogenate were reduced with tris-(2-carboxyethyl)-phosphine hydrochloride and derivatized with 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (ABD-F). Amino thiols were resolved under reversed-phase gradient conditions on a Waters Acquity BEH C18 column (1.7 µm, 2.1 mm × 100 mm) within 4.5 min and detected with fluorescence. The peak area ratio of analyte to 2-mercaptopropionylglycine internal standard, added to external calibration standards and samples, was used to develop linear calibration curves. Linear calibrations were performed over the range of 15-1,500 nmol/g for Cys, CysGly, Hcys, and γ-GluCys and 150-15,000 nmol/g for GSH. Linearity, lower limit of detection, lower limit of quantitation, accuracy, precision, sample stability, and carryover were evaluated. We demonstrate excellent linearity for all analytes within their respective concentration range (r2 > 0.99) and excellent recovery of amino thiols from spiked samples (mean ± SD across tissues; Cys, 100.0 ± 2.2%; CysGly, 95.4 ± 5.1%; Hcys, 96.6 ± 2.0%; γ-GluCys, 102.2 ± 2.7%; and GSH, 100.6 ± 3.3%). The intra-day and inter-day precisions did not exceed 5% and 10%, respectively. Repeated freezing and thawing of tissue homogenate did not affect measured amino thiol concentrations, ABD-labeled amino thiols were stable for 1 wk after derivatization, and there was no sample carryover across consecutive injections. We confirm the identity of each ABD-labeled amino thiol with Orbitrap mass spectrometry. Finally, we apply the method to the determination of amino thiol concentrations in liver and jejunum tissues in newly weaned pigs and show that despite elevated Cys and maintained GSH concentrations in liver, both γ-GluCys and GSH decline in jejunum of weaned pigs.

Oxidative Stress Is Associated With Diastolic Dysfunction in Women With Ischemia With No Obstructive Coronary Artery Disease.

BackgroundWomen with signs and symptoms of ischemia and no obstructive coronary artery disease often have evidence of diastolic dysfunction. Oxidative stress (OS) is associated with cardiovascular risk factors and adverse outcomes. The relationship between systemic OS and diastolic dysfunction is unknown.Methods and ResultsA subgroup of women (n=75) with suspected ischemia and no obstructive coronary artery disease who had both cardiac magnetic resonance imaging and OS measurements were enrolled in the WISE‐CVD (Women Ischemia Syndrome Evaluation—Coronary Vascular Dysfunction) study. Left ventricular end‐diastolic pressure was measured invasively. Left ventricular end‐diastolic volume and peak filling rate were assessed using cardiac magnetic resonance imaging. Aminothiol levels of plasma cystine and glutathione were measured as markers of OS. Spearman correlation and linear regression analyses were conducted. The group mean age was 54±11 years, and 61% had a resting left ventricular end‐diastolic pressure >12 mm Hg. Cystine levels correlated negatively with the peak filling rate (r=−0.31, P=0.007) and positively with left ventricular end‐diastolic pressure (r=0.25; P=0.038), indicating that increased OS was associated with diastolic dysfunction. After multivariate adjustment including multiple known risk factors for diastolic dysfunction and cardiovascular medications, cystine levels continued to be associated with peak filling rate (β=−0.27, P=0.049) and left ventricular end‐diastolic pressure (β=0.25; P=0.035). Glutathione levels were not associated with indices of diastolic function.Conclusions OS, measured by elevated levels of cystine, is associated with diastolic dysfunction in women with evidence of ischemia and no obstructive coronary artery disease, indicating the role of OS in patients with ischemia and no obstructive coronary artery disease. Its role in the progression of heart failure with preserved ejection fraction should be explored further.

Plasma homocysteine and aminothiol levels in idiopathic epilepsy patients receiving antiepileptic drugs

Abstract Objective Homocysteine is a sulfur containing amino acid that is formed during methionine metabolism. Patients under long-term antiepileptic drug treatment often have hyperhomocysteinemia. These patients have low levels of serum folate, vitamin B12 and vitamin B6, all of which are associated with homocysteine metabolism. We have investigated the effects of valproic acid and new generation antiepileptic drugs (lamotrigine and levetiracetam) on plasma levels of homocysteine and aminothiols as well as serum vitamin B12 and folic acid. Materials and methods Forty-seven idiopathic epileptic patients on antiepileptic drugs were compared with 38 age-matched healthy controls. Commercial immunoassay methods were used for vitamin B12 and folic acid analyses. Homocysteine, cysteine, cysteinylglycine and glutathione levels were determined by high performance liquid chromatography. Results There was no significant difference in patient and control values in terms of vitamin B12, folic acid and homocysteine. Valproic acid and lamotrigine seemed to effect aminothiol redox status. Glutathione levels of epileptic patients receiving valproic acid and lamotrigine were higher than controls. Conclusion Our results suggest that redox homeostasis may be impaired and glutathione synthesis increased in response to the oxidative stress caused by antiepileptic drug use.

A colorimetric and ratiometric dual-site fluorescent probe with 2,4-dinitrobenzenesulfonyl and aldehyde groups for imaging of aminothiols in living cells and zebrafish

Aminothiol levels are indicative of cellular and physiological statuses. A colorimetric and ratiometric dual-site fluorescent probe, NL-S, has been designed and synthesized. NL-S contains both a 2,4-dinitrobenzenesulfonyl group and an aldehyde group as recognition units for aminothiols. NL-S exhibited highly selective and sensitive detection of aminothiols, with a 27–44-fold enhancement of the fluorescent signal observed within 30 min of the addition of up to 20 equiv. of cysteine (Cys), homocysteine (Hcy) or glutathione (GSH) in aqueous media. The probe undergoes a turn-on excited-state intramolecular proton transfer process derived from cleavage and condensation reactions. The limits of detection for Cys, Hcy and GSH were 0.114, 0.146 and 0.202 μM, respectively. NL-S with negligible cytotoxicity was successfully employed to fluorescent imaging of aminothiols in living cells and zebrafish, suggesting good potential of NL-S in biological applications.

Association between oxidative stress and atrial fibrillation

Oxidative stress (OS) may be a key mechanism underlying the development of atrial fibrillation (AF) in experimental studies, but data in humans remain limited. Systemic OS can be estimated by measurements of circulating levels of the aminothiols including glutathione, cysteine, and their oxidized products. We tested the hypothesis that the redox potentials of glutathione (EhGSH) and cysteine will be associated with prevalent and incident AF. Plasma levels of aminothiols were measured in 1439 patients undergoing coronary angiography, of whom 148 (10.3%) had a diagnosis of AF. After a median follow-up of 6.3 years, 104 of 917 patients (11.5%) developed incident AF. Multivariate logistic regression and Cox regression models were used to determine whether OS markers were independent predictors of prevalent and incident AF after adjustment for traditional risk factors, heart failure, coronary artery disease, and high-sensitivity C-reactive protein level. For each 10% increase in EhGSH, the odds of prevalent AF was 30% higher (odds ratio [OR] 1.3; 95% confidence interval [CI] 1.1-1.7; P = .02) and 90% higher (OR 1.9; 95% CI 1.3-2.7; P = .004) when the median was used as a cutoff. The EhGSH level above the median was more predictive of chronic AF (OR 4.0; 95% CI 1.3-12.9; P = .01) than of paroxysmal AF (OR 1.7; 95% CI 1.1-2.7; P = .03). Each 10% increase in EhGSH level was associated with a 40% increase in the risk of incident AF (hazard ratio 1.4; 95% CI 1.1-1.7; P = .01). Increased OS measured by the redox potentials of glutathione is associated with prevalent and incident AF. Therapies that modulate OS need to be investigated to treat and prevent AF.

Effect of Angiotensin II Type I Receptor Blockade with Valsartan on Carotid Artery Atherosclerosis: A Double Blind Randomized Clinical Trial Comparing Valsartan and Placebo (EFFERVESCENT)

Progression of atherosclerosis is associated with a greater risk for adverse outcomes. Angiotensin II plays a key role in the pathogenesis and progression of atherosclerosis. We aimed to investigate the effects of angiotensin II type-1 receptor blockade with Valsartan on carotid wall atherosclerosis, with the hypothesis that Valsartan will reduce progression of atherosclerosis. Subjects (n = 120) with carotid intima-media thickness >0.65 mm by ultrasound were randomized (2:1) in a double-blind manner to receive either Valsartan or placebo for 2 years. Bilateral T2-weighted black-blood carotid magnetic resonance imaging was performed at baseline, 12 and 24 months. Changes in the carotid bulb vessel wall area and wall thickness were primary endpoints. Secondary endpoints included changes in carotid plaque thickness, plasma levels of aminothiols, C-reactive protein, fibrinogen, and endothelium-dependent and -independent vascular function. Over 2 years, the carotid bulb vessel wall area decreased with Valsartan (-6.7, 95% CI [-11.6, -1.9] mm(2)) but not with placebo (3.4, 95% CI [-2.8, 9.6] mm(2)), P = .01 between groups. Similarly, mean wall thickness decreased with Valsartan (-0.18, 95% CI [-0.30, -0.06] mm), but not with placebo (0.08, 95% CI [-0.07, 0.23] mm), P = .009 between groups. Furthermore, plaque thickness decreased with Valsartan (-0.35, 95% CI [-0.63, -0.08] mm) but was unchanged with placebo (+0.28, 95% CI [-0.11, 0.69] mm), P = .01 between groups. These findings were unaffected by statin therapy or changes in blood pressure. Notably, there were significant improvements in the aminothiol cysteineglutathione disulfide, and trends to improvements in fibrinogen levels and endothelium-independent vascular function. In subjects with carotid wall thickening, angiotensin II type-1 receptor blockade was associated with regression in carotid atherosclerosis. Whether these effects translate into improved outcomes in subjects with subclinical atherosclerosis warrants investigation.

Close relationship between redox state of human serum albumin and serum cysteine levels in non-diabetic CKD patients with various degrees of renal function.

Human mercaptalbumin (HMA) acts as a covalent-carrier protein for sulfur-containing amino acids, cysteine, and glutathione. In addition, its sulfhydryl residue reacts with peroxyl radicals. In this study, we evaluated the redox state of human serum albumin and its relationship with serum amino thiol levels in non-diabetic chronic kidney disease (CKD) patients with various degrees of renal function. High performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC-MS) with selected ion monitoring were used to analyze serum samples collected from 37 non-dialysis patients with primary glomerulonephritis without diabetes mellitus (DM) or other systemic diseases (non-diabetic CKD patients). Total cysteine and homocysteine plasma levels increased with decreasing renal function and showed a significant negative correlation with glomerular filtration rate. The protein-bound ratio of serum cysteine also changed with the degree of renal dysfunction. The HPLC fraction of human mercaptalbumin (HMA) (%) was significantly lower in nondiabetic CKD patients than in healthy subjects. The redox state of human serum albumin (i.e., HMA %) correlated significantly with the total serum cysteine level. The HPLC fraction of HMA (%) closely correlated with the serum cysteine level in non-diabetic CKD patients. An increase in oxidized cysteine with impaired renal function and a reduced plasma redox capacity associated with a decrease in the reduced form of serum albumin (HMA %) may be important risk factors for promoting long-term complications in patients with renal dysfunction.

Plasma Levels of Aminothiols, Nitrite, Nitrate, and Malondialdehyde in Myelodysplastic Syndromes in the Context of Clinical Outcomes and as a Consequence of Iron Overload

The role of oxidative stress in the initiation and progression of myelodysplastic syndromes (MDS) as a consequence of iron overload remains unclear. In this study we have simultaneously quantified plasma low-molecular-weight aminothiols, malondialdehyde, nitrite, and nitrate and have studied their correlation with serum iron/ferritin levels, patient treatment (chelation therapy), and clinical outcomes. We found significantly elevated plasma levels of total, oxidized, and reduced forms of cysteine (P < 0.001) , homocysteine (P < 0.001), and cysteinylglycine (P < 0.006) and significantly depressed levels of total and oxidized forms of glutathione (P < 0.03) and nitrite (P < 0.001) in MDS patients compared to healthy donors. Moreover, total (P < 0.032) and oxidized cysteinylglycine (P = 0.029) and nitrite (P = 0.021) differed significantly between the analyzed MDS subgroups with different clinical classifications. Malondialdehyde levels in plasma correlated moderately with both serum ferritin levels (r = 0.78, P = 0.001) and serum free iron levels (r = 0.60, P = 0.001) and were significantly higher in patients with iron overload. The other analyzed compounds lacked correlation with iron overload (represented by serum iron/ferritin levels). For the first time our results have revealed significant differences in the concentrations of plasma aminothiols in MDS patients, when compared to healthy donors. We found no correlation of these parameters with iron overload and suggest the role of oxidative stress in the development of MDS disease.

Assessment of plasma aminothiol levels and the association with recurrent atherothrombotic events in patients hospitalized for an acute coronary syndrome: a prospective study

The aminothiols homocysteine and, to a lesser extent, cysteine have been associated with adverse cardiovascular outcome, whereas glutathione, as an antioxidant, may protect against atherosclerosis and thrombosis. Potentially, the combined assessment of these aminothiols may provide a more accurate association with future cardiovascular outcome. We evaluated the association between recurrent atherothrombotic events and the concentration of total plasma cysteine, homocysteine, and glutathione and their combination. Respective aminothiols were measured by high-performance liquid chromatography in blood plasma of consecutive first-day survivors admitted for an acute coronary syndrome between April 2002 and January 2004. The combined score was calculated using the combination of the individual aminothiols. The end point was the composite of cardiovascular death, myocardial infarction, and/or stroke. A cohort of 375 consecutive patients (median age 66 years, 66% male) were followed for a median duration of 2.7 years. The end point occurred in 82 patients (22%). In univariate analyses, all aminothiols were significantly associated with the composite end point. After correction for possible confounders, only cysteine and glutathione remained significantly associated. The strongest association with the end point was observed for the combined score (adjusted hazard ratio, 1.40 per standard deviation increase; p=0.005). Although homocysteine is generally considered the aminothiol of interest with respect to cardiovascular disease, in our prospective study, only cysteine and glutathione appeared independently associated with recurrent atherothrombotic events. Moreover, we showed that an imbalance in the combination of aminothiols could be of more importance than investigating the individual metabolites.

Effects of vitamin C and E supplementation on oxidative stress and liver toxicity in rats fed a low-fat ethanol diet

We compared the preventive capacity of high intakes of vitamin C (VC) and vitamin E (VE) on oxidative stress and liver toxicity in rats fed a low-fat ethanol diet. Thirty-two Wistar rats received the low fat (10% of total calories) Lieber-DeCarli liquid diet as follows: either ethanol alone (Alc group, 36% of total calories) or ethanol in combination with VC (Alc + VC group, 40 mg VC/100 g body weight) or VE (Alc + VE group, 0.8 mg VE/100 g body weight). Control rats were pair-fed a liquid diet with the Alc group. Ethanol administration induced a modest increase in alanine aminotransferase (ALT), aspartate aminotransferase (AST), conjugated dienes (CD), and triglycerides but decreased total radical-trapping antioxidant potential (TRAP) in plasma. VE supplementation to alcohol-fed rats restored the plasma levels of AST, CD, and TRAP to control levels. However, VC supplementation did not significantly influence plasma ALT, AST, or CD. In addition, a significant increase in plasma aminothiols such as homocysteine and cysteine was observed in the Alc group, but cysteinylglycine and glutathione (GSH) did not change by ethanol feeding. Supplementing alcohol-fed rats with VC increased plasma GSH and hepatic S-adenosylmethionine, but plasma levels of aminothiols, except GSH, were not influenced by either VC or VE supplementation in ethanol-fed rats. These results indicate that a low-fat ethanol diet induces oxidative stress and consequent liver toxicity similar to a high-fat ethanol diet and that VE supplementation has a protective effect on ethanol-induced oxidative stress and liver toxicity.

A Native‐Chemical‐Ligation‐Mechanism‐Based Ratiometric Fluorescent Probe for Aminothiols

Thiol-containing amino acids (aminothiols) such as cysteine (Cys) and homocysteine (Hcy) play a key role in various biological processes including maintaining the homeostasis of biological thiols. However, abnormal levels of aminothiols are associated with a variety of diseases. The native chemical ligation (NCL) reaction has attracted great attention in the fields of chemistry and biology. NCL of peptide segments involves cascade reactions between a peptide-α-thioester and an N-terminal cysteine peptide. In this work, we employed the NCL reaction mechanism to formulate a Förster resonance energy transfer (FRET) strategy for the design of ratiometric fluorescent probes that were selective toward aminothiols. On the basis of this new strategy, the ratiometric fluorescent probe 1 for aminothiols was judiciously designed. The new probe is highly selective toward aminothiols over other thiols and exhibits a very large variation (up to 160-fold) in its fluorescence ratio (I(458)/I(603)). The new fluorescent probe is capable of ratiometric detection of aminothiols in newborn calf and human serum samples and is also suitable for ratiometric fluorescent imaging of aminothiols in living cells.

The differential effect of statins on oxidative stress and endothelial function: Atorvastatin versus pravastatin

Atherogenic risk in subjects with metabolic syndrome is partly mediated by increased oxidative stress and subsequent endothelial dysfunction. Clinical trials have demonstrated differences in outcomes between subjects receiving lipophilic statins (atorvastatin) compared with hydrophilic statins (pravastatin). However, whether these findings are attributable to differences in the doses administered or to nonlipid-lowering pleiotropic effects of statins on oxidative stress and vascular function remains unknown. We hypothesized that equipotent doses of these two statins will have divergent effects on markers of oxidative stress and endothelial function. Thirty-six subjects with hyperlipidemia and metabolic syndrome and/or diabetes were randomized in a double-blind manner to either pravastatin 80 mg or atorvastatin 10 mg daily. Oxidative stress (dROMs assay that measures lipid hydroperoxides, plasma thiobarbituric acid reactive substances [TBARS], and aminothiol levels) and brachial artery flow-mediated dilation (FMD) were measured at baseline and after 12 weeks of statin therapy. Statin therapy reduced serum low-density lipoprotein cholesterol levels equally in both groups. Atorvastatin therapy was associated with a significant reduction in TBARS (P= .006) and dROMs levels (P= .02), which was not observed in subjects treated with pravastatin. Endothelial function improved with statin therapy (P= .02), but there was no difference between the statin groups. In hyperlipidemic subjects with metabolic syndrome, atorvastatin is associated with a greater reduction in lipid markers of oxidation compared with pravastatin. Whether these effects are responsible for the outcome differences in trials comparing these agents needs further investigation.

Oxidative stress is associated with impaired arterial elasticity

AimsArterial stiffening may lead to hypertension, greater left ventricular after-load and adverse clinical outcomes. The underlying mechanisms influencing arterial elasticity may involve oxidative injury to the vessel wall. We sought to examine the relationship between novel markers of oxidative stress and arterial elastic properties in healthy humans. Methods and resultsWe studied 169 subjects (mean age 42.6±14 years, 51.6% male) free of traditional cardiovascular risk factors. Indices of arterial stiffness and wave reflections measured included carotid-femoral Pulse Wave Velocity (PWV), Augmentation Index (Aix) and Pulse Pressure Amplification (PPA). Non-free radical oxidative stress was assessed as plasma oxidized and reduced amino-thiol levels (cysteine/cystine, glutathione/GSSG) and their ratios (redox potentials), and free radical oxidative stress as derivatives of reactive oxygen metabolites (dROMs). Inflammation was assessed as hsCRP and interleukin-6 levels.The non-free radical marker of oxidative stress, cystine was significantly correlated with all arterial indices; PWV (r=0.38, p<0.001), Aix (r=0.35, p<0.001) and PPA (r=−0.30, p<0.001). Its redox potential, was also associated with PWV (r=0.22, p=0.01), while the free radical marker of oxidative stress dROMS was associated with Aix (r=0.25, p<0.01). After multivariate adjustment for age, gender, arterial pressure, height, weight, heart rate and CRP, of these oxidative stress markers, only cystine remained independently associated with PWV (p=0.03), Aix (p=0.01) and PPA (p=0.05). ConclusionsIn healthy subjects without confounding risk factors or significant systemic inflammation, a high cystine level, reflecting extracellular oxidant burden, is associated with increased arterial stiffness and wave reflections. This has implications for understanding the role of oxidant burden in pre-clinical vascular dysfunction.

An improved method for simultaneous analysis of aminothiols in human plasma by high-performance liquid chromatography with fluorescence detection

Altered levels of aminothiols in biological fluids are thought to be an important risk indicator for several diseases, and reliable methods for the accurate determination of aminothiols concentrations in plasma are thus required. In this paper ammonium 5-bromo-7-fluorobenzo-2-oxa-1,3-diazole-4-sulphonate (SBD-BF) is proposed as a convenient fluorogenic derivatizating reagent for the determination of aminothiols (cysteine, cysteinylglycine, homocysteine and glutathione) by HPLC with fluorescence detection. The reactions of SBD-BF with aminothiols at room temperature are about three-times faster than those of ammonium 7-fluorobenzo-2-oxa-1,3-diazole-4-sulphonate (the most frequently employed reagent) at 60 °C. The derivatives of SBD-BF with cysteine, cysteinylglycine, homocysteine and glutathione are easily separated by HPLC and their calibration curves show excellent linearity over the range 0.05–20 μmol/L with excellent r 2 values for all analytes. SBD-BF reacts with thiols under mild conditions, i.e. at 25 °C over about 30 min, and is proposed as a suitable fluorogenic reagent for thiol derivatization to be introduced in analytical clinical chemistry. The detection limits of Cys, Cys-Gly, Hcy and GSH at a signal-to-noise ratio of 5 were 0.1 μM for Cys, 0.01 μM for Cys-Gly and Hcy, and 0.02 μM for GSH. Furthermore, validation parameters of the proposed method are quite satisfactory. As an application of this method the determination of thiol derivatives in human plasma was carried out on a number of samples.

Comparison of carbamazepine and oxcarbazepine effects on aminothiol levels

The aim of our study was to investigate the effects of carbamazepine (CBZ) and oxcarbazepine (OXCBZ) on aminothiol levels, including homocysteine (Hcy), cysteine, and cysteinylglycine, in chronically treated patients. Epileptic patients receiving CBZ or OXCBZ were recruited as part of routine clinical practice. Demographic data and concomitant medications were recorded from the patient medical file. Sixty patients were included in the study; 30 patients were treated with CBZ and 30 with OXCBZ. Median Hcy level was significantly higher in CBZ-treated patients (20.6 micromol/l) than in OXCBZ-treated patients (14.0 micromol/l, p < 0.0001). No correlation was evidenced between antiepileptic drugs or metabolite levels and Hcy levels for each group. Less change observed with OXCBZ compared with CBZ on aminothiol levels could constitute an advantage for OXCBZ treatment in patients with other factors influencing Hcy levels and/or at high risk for cardiovascular diseases.

Plasma total homocysteine and cysteine levels as cardiovascular risk factors in coronary heart disease

Objectives: As an important risk factor for coronary atherosclerosis, elevated plasma total homocysteine (t-hcy) concentration has recently received greater attention than have conventional risk factors. Though less reactive than homocysteine, cysteine (cys) is the most abundant plasma thiol and may function as an extracellular regulating factor of thiol/disulfide exchange in order to maintain an adequate redox status. An increase in the total amount of this compound may be noxious depending on environmental conditions. In the present study, the aim was to investigate changes of plasma total cysteine, homocysteine and other determinants in different types of coronary heart disease. Design and methods: Plasma total homocysteine (t-hcy), cysteine (t-cys), cysteinylglycine (t-cysgly), folic acid, vitamin B 12, lipid parameters, total protein, albumin and creatinine levels were studied in plasma from 68 patients with coronary heart disease and 42 healthy controls. After reduction of disulfide bonds with tri- n-buthylphosphine, plasma total thiols were assayed using high performance liquid chromatography (HPLC) and fluorescence detection following derivatization of sulfhydryl groups with 7-fluoro-benzo-2-oxa-1,3-diazole-4-sulfonate (SBD-F). Other parameters were determined by using commercial kits. Results: Plasma t-hcy and t-cys levels were higher in patients ( P<0.0001) than in controls, but t-cysgly was unchanged. Hcy and cys levels were correlated with age in the whole study population ( r=0.49, r=0.46, P<0.01). Plasma t-hcy positively correlated with plasma t-cys ( r=0.53, P<0.01) and t-cysgly ( r=0.49, P<0.01) in patients, and with plasma t-cys ( r=0.57, P<0.01) in controls. Postmenopausal women had higher t-cys and t-hcy levels than premenopausal women among the controls ( P<0.01). Folate and vitamin B 12 levels were similar in both patients and controls. Patients with vitamin B 12 levels below normal had higher plasma t-cys and t-cysgly levels ( P<0.05). Interestingly, control subjects with lower vitamin B 12 levels had lower plasma t-hcy levels ( P<0.05). Plasma total cholesterol, HDL-cholesterol, LDL-cholesterol, total protein, albumin and creatinine levels in patients and controls were within the normal range, but only HDL-cholesterol levels in patients were lower than in controls ( P<0.0001). Triglyceride and VLDL levels of patients were also higher than those of controls ( P<0.0001). Conclusions: Higher plasma total cysteine levels are as important as higher plasma total homocysteine levels. Both parameters are intercorrelated and may act synergistically. To discern their respective roles in atherosclerotic disease, these aminothiol levels have to be considered together.

Cystinosis. Intracellular cystine depletion by aminothiols in vitro and in vivo.

Certain aminothiols rapidly deplete cultured cystinotic skin fibroblasts of their abnormally high free (nonprotein) cystine pool. The free cystine content of these cells if reduced by over 90% in 1 h with 0.1 mM cysteamine. This is more rapid than previously known methods of removing free cystine from cystinotic fibroblasts. The disulfide, cystamine, is also able to deplete cystinotic cells of free cystine. A patient with nephropathic cystinosis and end-stage renal disease was treated with cysteamine, both intravenously and orally. Both methods of administration rapidly lowered the free cystine content of the patient's peripheral leukocytes. Study of the patient's urinary sulfur excretion did not conclusively determine the effect of this therapy on the total body cystine pool. Her renal status remained at end stage after 1 mo of oral cysteamine, when an episode of grand mal seizures prompted cessation of the study. Determination of the proper place of aminothiol therapy in this disease will depend upon further clinical trial with patients whose kidney function has not deteriorated to the point of irreversible change, accompanied by careful monitoring of plasma aminothiol levels.