Albumin thiolate anion is an intermediate in the formation of albumin-S-S-homocysteine

An elevated concentration of plasma total homocysteine is an independent risk factor for cardiovascular disease. Greater than 80% of circulating homocysteine is covalently bound to plasma protein by disulfide bonds. It is known that albumin combines with cysteine in circulation to form albumin-Cys(34)-S-S-Cys. Studies are now presented to show that the formation of albumin-bound homocysteine proceeds through the generation of an albumin thiolate anion. Incubation of human plasma with l-(35)S-homocysteine results in the association of >90% of the protein-bound (35)S-homocysteine with albumin as shown by nonreduced SDS-polyacrylamide gel electrophoresis. Treatment of the complex with beta-mercaptoethanol results in near quantitative release of the bound l-(35)S-homocysteine, demonstrating that the binding of homocysteine to albumin is through a disulfide bond. Furthermore, using an in vitro model system to study the mechanisms of this disulfide bond formation, we show that homocysteine binds to albumin in two steps. In the first step homocysteine rapidly displaces cysteine from albumin-Cys(34)-S-S-Cys, forming albumin-Cys(34) thiolate anion and homocysteine-cysteine mixed disulfide. In the second step, albumin thiolate anion attacks homocysteine-cysteine mixed disulfide to yield primarily albumin-Cys(34)-S-S-Hcy and to a much lesser extent albumin-Cys(34)-S-S-Cys. The results clearly suggest that when reduced homocysteine enters circulation, it attacks albumin-Cys(34)-S-S-Cys to form albumin-Cys(34) thiolate anion, which in turn, reacts with homocysteine-cysteine mixed disulfide or homocystine to form albumin-bound homocysteine.     

Cross-talk between Cys34 and lysine residues in human serum albumin revealed by N-homocysteinylation

Protein N-homocysteinylation involves a post-translational modification by homocysteine (Hcy)-thiolactone. In humans, about 70% of circulating Hcy is N-linked to blood proteins, mostly to hemoglobin and albumin. It was unclear what protein site(s) were prone to Hcy attachment and how N-linked Hcy affected protein function. Here we show that Lys(525) is a predominant site of N-homocysteinylation in human serum albumin in vitro and in vivo. We also show that the reactivity of albumin lysine residues, including Lys(525), is affected by the status of Cys(34). The disulfide forms of circulating albumin, albumin-Cys(34)-S-S-Cys and albumin-Cys(34)-S-S-Hcy, are N-homocysteinylated faster than albumin-Cys(34)-SH. Although N-homocysteinylations of albumin-Cys(34)-SH and albumin-Cys(34)-S-S-Cys yield different primary products, subsequent thiol-disulfide exchange reactions result in the formation of a single product, N-(Hcy-S-S-Cys)-albumin-Cys(34)-SH. We also show that N-homocysteinylation affects the susceptibility of albumin to oxidation and proteolysis. The data suggest that a disulfide at Cys(34) of albumin promotes conversion of N-(Hcy-SH)-albumin-Cys(34)-SH to a proteolytically sensitive form N-(Hcy-S-S-Cys)-albumin-Cys(34)-SH, which would facilitate clearance of the N-homocysteinylated form of mercaptoalbumin.     

The control of hyperhomocysteinemia through thiol exchange mechanisms by mesna

In hyperhomocysteinemic patients, after reaction with homocysteine-albumin mixed disulfides (HSS-ALB), mesna (MSH) forms the mixed disulfide with Hcy (HSSM) which can be removed by renal clearance, thus reducing the plasma concentration of total homocysteine (tHcy). In order to assess the HSS-ALB dethiolation via thiol exchange reactions, the distribution of redox species of cysteine, cysteinylglycine, homocysteine and glutathione was investigated in the plasma of healthy subjects: (i) in vitro, after addition of 35 μM reduced homocysteine (HSH) to plasma for 72 h, followed by MSH addition (at the concentration range 10–600 μM) for 25 min; (ii) in vivo, after oral treatment with methionine (methionine, 200 mg/kg body weight, observation time 2–6 h). In both experiments the distribution of redox species, but not the total amount of each thiol, was modified by thiol exchange reactions of albumin and cystine, with changes thermodynamically related to the pKa values of thiols in the corresponding mixed disulfides. MSH provoked a dose–response reversal of the redox state of aged plasma, and the thiol action was confirmed by in vivo experiments. Since it was observed that the dimesna production could be detrimental for the in vivo optimization of HSSM formation, we assume that the best plasma tHcy lowering can be obtained at MSH doses producing the minimum dimesna concentration in each individual.     

Determination of homocysteine, penicillamine, and their symmetrical and mixed disulfides by liquid chromatography with electrochemical detection

A method for the determination of D-penicillamine, homocysteine, homocystine, penicillamine-homocysteine mixed disulfide, and penicillamine disulfide in human plasma and urine is described. The method involves separation of the various thiols and disulfides by high-performance liquid chromatography with detection by a dual Hg/Au amalgam electrochemical detector. D-Penicillamine and homocysteine are detected at the downstream electrode; the disulfides are first reduced to thiols at the upstream electrode and then the thiols are detected at the downstream electrode. Hydrodynamic voltammograms were measured for the various thiols and disulfides to determine optimum settings for the electrochemical detector, and the effect of mobile phase parameters on retention times was studied to optimize the separation. A convenient method for the preparation of calibration solutions of penicillamine-homocysteine mixed disulfide by thiol/disulfide exchange with standardization of the solution by H NMR spectroscopy is described. Detection limits are below the concentrations of homocystine and penicillamine-homocysteine mixed disulfide reported to be present in the plasma and urine, respectively, of homocystinuric patients under treatment with D-penicillamine.     

Results of oral methionine loads in normal subjects and patients with liver diseases using an analyzer short program

Control subjects and patients with liver diseases (cirrhosis, fatty liver) were given an oral methionine load with 100 mg L-Met/kg body weight. Amino acid chromatography was made by a short-program particularly suitable for the diagnosis of hereditary disorders of methionine metabolism. Met-tolerance in blood plasma as well as cystathionine, homocystine and the mixed disulfide homocysteine-cysteine in plasma and urine were investigated. Methylmalonic acid excretion in the urine was determined by gas chromatography. Patients with liver diseases showed some pathological changes of methionine tolerance after the load. However, cystathionine and homocysteine could not be demonstrated. No methylmalonic acid excretion occurred in normal subjects and patients with liver diseases after the methionine load.     

Thiol-disulfide interchange between cystine and N-2-mercaptoethyl-1, 3-diaminopropane as a potential treatment for cystinuria

The radioprotective compound WR2721 is a thiophosphate, which, when administered orally, is activated at the acid pH of the stomach to its free thiol (MDP). The free thiol is a mucolytic compound which acts via the reduction of disulfide bonds of mucin molecules. An equimolar mixture of MDP and cysteine, in urine at pH 6.0 and 37 degrees C, when oxidized by molecular oxygen, preferentially forms the soluble mixed disulfide between MDP and cysteine. The disulfide cystine will undergo thiol-disulfide interchange with MDP; as a result, cystine crystals are effectively dissolved. Moreover, in the presence of catalytic amounts of free thiol, the disulfide of MDP will undergo thiol-disulfide interchange with cystine to dissolve cystine crystals. The mixed disulfide of MDP with cysteine is soluble in urine at pH 6.0 and 37 degrees C to at least 100 mg/ml. Chromatographic procedures which permit the analysis of MDP and its mixed disulfide derivatives as MDP-sulfonic acid are described. By these procedures, it was demonstrated that 20% of a single oral dose of WR2721 was excreted as MDP derivatives in the urine of normal volunteers. These procedures will permit the evaluation of WR2721 in the treatment of cystinuria.     

Protein-thiol substitution or protein dethiolation by thiol/disulfide exchange reactions: the albumin model

Dethiolation experiments of thiolated albumin with thionitrobenzoic acid and thiols (glutathione, cysteine, homocysteine) were carried out to understand the role of albumin in plasma distribution of thiols and disulfide species by thiol/disulfide (SH/SS) exchange reactions. During these experiments we observed that thiolated albumin underwent thiol substitution (Alb-SS-X+RSH<-->Alb-SS-R+XSH) or dethiolation (Alb-SS-X+XSH<-->Alb-SH+XSSX), depending on the different pK(a) values of thiols involved in protein-thiol mixed disulfides (Alb-SS-X). It appeared in these reactions that the compound with lower pK(a) in mixed disulfide was a good leaving group and that the pK(a) differences dictated the kind of reaction (substitution or dethiolation). Thionitrobenzoic acid, bound to albumin by mixed disulfide (Alb-TNB), underwent rapid substitution after thiol addition, forming the corresponding Alb-SS-X (peaks at 0.25-1 min). In turn, Alb-SS-X were dethiolated by the excess nonprotein SH groups because of the lower pK(a) value in mixed disulfide with respect to that of other thiols. Dethiolation of Alb-SS-X was accompanied by formation of XSSX and Alb-SH up to equilibrium levels at 35 min, which were different for each thiol. Structures by molecular simulation of thiolated albumin, carried out for understanding the role of sulfur exposure in mixed disulfides in dethiolation process, evidenced that the sulfur exposure is important for the rate but not for determining the kind of reaction (substitution or dethiolation). Our data underline the contribution of SH/SS exchanges to determine levels of various thiols as reduced and oxidized species in human plasma.     

Oxidation of cysteine and homocysteine by bovine albumin

The autooxidation of cysteine and homocysteine to their disulfide forms was determined by measuring the time course of thiol groups disappearance. We found the oxidative chemistry of cysteine and homocysteine to be quite different. In the absence of added Cu(II), cysteine autooxidized at a slower rate than homocysteine, though in its presence cysteine oxidation was much faster, homocysteine being found to be a poor responder to copper catalysis. Albumin speeded up the spontaneous oxidation of both aminothiols, the reaction being faster with cysteine than with homocysteine. The copper content of different albumins was found to be highly variable, ranging from 12.75 to 0.64 microg Cu(II)/g albumin. We propose that copper bound to albumin possesses redox cycling activity to perform cysteine oxidation since: (i) copper elimination by copper chelators markedly reduces oxidation; and (ii) a positive correlation exists between the albumin copper content and the oxidation reaction rate.     

Direct observation of covalent adducts with Cys34 of human serum albumin using mass spectrometry

The interactions of the unpaired thiol residue (Cys34) of human serum albumin (HSA) with low-molecular-weight thiols and an Au(I)-based antiarthritic drug have been examined using electrospray ionization mass spectrometry. Early measurements of the amount of HSA containing Cys34 as the free thiol suggested that up to 30% of circulating HSA bound cysteine as a mixed disulfide. It has also been suggested that reaction of HSA with cysteine, occurs only on handling and storage of plasma. In our experiments, there were three components of HSA in freshly collected plasma from normal volunteers, HSA, HSA+cysteine, and HSA+glucose in the ratio approximately 50:25:25. We addressed this controversy by using iodoacetamide to block the free thiol of HSA in fresh plasma, preventing its reaction with plasma cysteine. When iodoacetamide was injected into a vacutaner tube as blood was collected, the HSA was modified by iodoacetamide, with 20-30% present as the mixed disulfide with cysteine (HSA+cys). These data provide strong evidence that 20-30% of HSA in normal plasma contains one bound cysteine. Reaction of HSA with [Au(S(2)O(3))(2)](3-) resulted in formation of the adducts HSA+Au(S(2)O(3)) and HSA+Au. Reaction of HSA with iodoacetamide prior to treatment with [Au(S(2)O(3))(2)](3-) blocked the formation of gold adducts.     

Hydrolysis of homocysteine thiolactone results in the formation of Protein-Cys-S-S-homocysteinylation

An increased level of homocysteine, a reactive thiol amino acid, is associated with several complex disorders and is an independent risk factor for cardiovascular disease. A majority (>80%) of circulating homocysteine is protein bound. Homocysteine exclusively binds to protein cysteine residues via thiol disulfide exchange reaction, the mechanism of which has been reported. In contrast, homocysteine thiolactone, the cyclic thioester of homocysteine, is believed to exclusively bind to the primary amine group of lysine residue leading to N-homocysteinylation of proteins and hence studies on binding of homocysteine thiolactone to proteins thus far have only focused on N-homocysteinylation. Although it is known that homocysteine thiolactone can hydrolyze to homocysteine at physiological pH, surprisingly the extent of S-homocysteinylation during the exposure of homocysteine thiolactone with proteins has never been looked into. In this study, we clearly show that the hydrolysis of homocysteine thiolactone is pH dependent, and at physiological pH, 1 mM homocysteine thiolactone is hydrolysed to ~0.71 mM homocysteine within 24 h. Using albumin, we also show that incubation of HTL with albumin leads to a greater proportion of S-homocysteinylation (0.41 mol/mol of albumin) than N-homocysteinylation (0.14 mol/mol of albumin). S-homocysteinylation at Cys³⁴ of HSA on treatment with homocysteine thiolactone was confirmed using LC-MS. Further, contrary to earlier reports, our results indicate that there is no cross talk between the cysteine attached to Cys³⁴ of albumin and homocysteine attached to lysine residues.     

The Redox State of Glutathione,Cysteine and Homocysteine in the Extracellular Fluid in the Skin

Glutathione, the most abundant low-molecular weight thiol in the skin, has been shown to protect the skin from both photobiological and chemical injury. The thiols, glutathione in particular, have also been shown to be crucially involved in defence against contact allergens. Since the levels of extracellular thiol concentrations are important determinants of intracellular thiol status, we have compared the normal concentrations and the redox status of the main low-molecular weight thiol components in the extracellular fluid at the dermo-epidermal junction with the corresponding plasma levels. In their sulfhydryl form, all three thiols, i.e. glutathione, cysteine and homocysteine, were more abundant in experimental skin blister fluid than in plasma, as were the free disulfides of glutathione and homocysteine, whereas the free disulfides of cysteine were about the same in blister fluid and in plasma. Protein mixed disulfide levels were higher in plasma than in blister fluid. The present results provide information concerning the extracellular defence in the skin.     

The redox state of glutathione, cysteine and homocysteine in the extracellular fluid in the skin

Glutathione, the most abundant low-molecular weight thiol in the skin, has been shown to protect the skin from both photobiological and chemical injury. The thiols, glutathione in particular, have also been shown to be crucially involved in defence against contact allergens. Since the levels of extracellular thiol concentrations are important determinants of intracellular thiol status, we have compared the normal concentrations and the redox status of the main low-molecular weight thiol components in the extracellular fluid at the dermo-epidermal junction with the corresponding plasma levels. In their sulfhydryl form, all three thiols, i.e. glutathione, cysteine and homocysteine, were more abundant in experimental skin blister fluid than in plasma, as were the free disulfides of glutathione and homocysteine, whereas the free disulfides of cysteine were about the same in blister fluid and in plasma. Protein mixed disulfide levels were higher in plasma than in blister fluid. The present results provide information concerning the extracellular defence in the skin.     

Regulation of redox forms of plasma thiols by albumin in multiple sclerosis after fasting and methionine loading test

Increases in plasma concentrations of total homocysteine (tHcy) have recently been reported in multiple sclerosis (MS) as the alteration of the methionine cycle for the onset of autoimmune diseases. Homocysteine (Hcy) and cysteine (Cys) are generated by the methionine cycle and transsulfuration reactions. Their plasma levels are subjected to complex redox changes by oxidation and thiol/disulfide (SH/SS) exchange reactions regulated by albumin. The methionine loading test (MLT) is a useful in vivo test to assay the functionality of the methionine cycle and transsulfuration reactions. Time courses of redox species of Cys, cysteinylglycine (CGly), Hcy, and glutathione have been investigated in plasma of MS patients versus healthy subjects after an overnight fasting, and 2, 4, and 6 h after an oral MLT (100 mg/kg body weight), to detect possible dysfunctions of the methionine cycle, transsulfuration reactions and alterations in plasma distribution of redox species. After fasting, the MS group showed a significant increase in cysteine-protein mixed disulfides (bCys) and total Cys (tCys). While plasma bCys and tCys in MS group remained elevated after methionine administration when compared to control, cystine (oxCys) increased significantly with respect to control. Although increased plasma concentrations of bCys and tCys at fasting might reflect an enhance of transsulfuration reactions in MS patients, this was not confirmed by the analysis of redox changes of thiols and total thiols after MLT. This study has also demonstrated that albumin-dependent SH/SS exchange reactions are a potent regulation system of thiol redox species in plasma.     

Homocysteine and cysteine - albumin binding in homocystinuria: assessment of cysteine status and implications for glutathione synthesis?

Summary. Measurement of plasma total cysteine rather than free dimeric cystine gives a better indication of cysteine status in homocystinuric patients. This is the result of displacement of cysteine from albumin by homocysteine and is related to the plasma homocysteine concentration. In control subjects the free/bound cyst(e)ine ratio was independent of albumin and total cysteine concentrations. In homocystinuric (HCU) patients both free and total cyst(e)ine values differed significantly from control values (P < 0.001) but whilst free cystine considerably overlapped control values the total cysteine concentrations were almost invariably lower. The possible consequences of this on glutathione synthesis was explored by assay of plasma total glutathione but no evidence for glutathione deficiency was found. Measurement of total cysteine, rather than free cystine, provides a better indication of cysteine status in HCU. Received February 1, 2001 Accepted November 13, 2001     

Distribution of oxidized and reduced forms of glutathione and cysteine in rat plasma

The distribution of the glutathionyl moiety between reduced and oxidized forms in rat plasma was markedly different than that for the cysteinyl moiety. Most of the glutathionyl moiety was present as mixed disulfides with cysteine and protein whereas most of the cysteinyl moiety was present as cystine. Seventy percent of total glutathione equivalents was bound to proteins in disulfide linkage. The distribution of glutathione equivalents in the acid-soluble fraction was 28.0% as glutathione, 9.5% as glutathione disulfide, and 62.6% as the mixed disulfide with the cysteinyl moiety. In contrast, 23% of total cysteine equivalents was protein-bound. The distribution of cysteine equivalents in the acid-soluble fraction was 5.9% as cysteine, 83.1% as cystine, and 10.8% as the mixed disulfide with the glutathionyl moiety. A first-order decline in glutathione occurred upon in vitro incubation of plasma and was due to increased formation of mixed disulfides of glutathione with cysteine and protein. This indicates that plasma thiols and disulfides are not at equilibrium, but are in a steady-state maintained in part by transport of these compounds between tissues during the inter-organ phase of their metabolism. The large amounts of protein-bound glutathione and cysteine provide substantial buffering which must be considered in analysis of transient changes in glutathione and cysteine. In addition, this buffering may protect against transient thiol-disulfide redox changes which could affect the structure and activity of plasma and plasma membrane proteins.     

Peroxynitrite oxidation of sulfhydryls. The cytotoxic potential of superoxide and nitric oxide.

Peroxynitrite anion (ONOO-) is a potent oxidant that mediates oxidation of both nonprotein and protein sulfhydryls. Endothelial cells, macrophages, and neutrophils can generate superoxide as well as nitric oxide, leading to the production of peroxynitrite anion in vivo. Apparent second order rate constants were 5,900 M-1.s-1 and 2,600-2,800 M-1.s-1 for the reaction of peroxynitrite anion with free cysteine and the single thiol of albumin, respectively, at pH 7.4 and 37 degrees C. These rate constants are 3 orders of magnitude greater than the corresponding rate constants for the reaction of hydrogen peroxide with sulfhydryls at pH 7.4. Unlike hydrogen peroxide, which oxidizes thiolate anion, peroxynitrite anion reacts preferentially with the undissociated form of the thiol group. Peroxynitrite oxidizes cysteine to cystine and the bovine serum albumin thiol group to an arsenite nonreducible product, suggesting oxidation beyond sulfenic acid. Peroxynitrous acid was a less effective thiol-oxidizing agent than its anion, with oxidation presumably mediated by the decomposition products, hydroxyl radical and nitrogen dioxide. The reactive peroxynitrite anion may exert cytotoxic effects in part by oxidizing tissue sulfhydryls.     

Disulfide stress: a novel type of oxidative stress in acute pancreatitis

Glutathione oxidation and protein glutathionylation are considered hallmarks of oxidative stress in cells because they reflect thiol redox status in proteins. Our aims were to analyze the redox status of thiols and to identify mixed disulfides and targets of redox signaling in pancreas in experimental acute pancreatitis as a model of acute inflammation associated with glutathione depletion. Glutathione depletion in pancreas in acute pancreatitis is not associated with any increase in oxidized glutathione levels or protein glutathionylation. Cystine and homocystine levels as well as protein cysteinylation and γ-glutamyl cysteinylation markedly rose in pancreas after induction of pancreatitis. Protein cysteinylation was undetectable in pancreas under basal conditions. Targets of disulfide stress were identified by Western blotting, diagonal electrophoresis, and proteomic methods. Cysteinylated albumin was detected. Redox-sensitive PP2A and tyrosine protein phosphatase activities diminished in pancreatitis and this loss was abrogated by N-acetylcysteine. According to our findings, disulfide stress may be considered a specific type of oxidative stress in acute inflammation associated with protein cysteinylation and γ-glutamylcysteinylation and oxidation of the pair cysteine/cystine, but without glutathione oxidation or changes in protein glutathionylation. Two types of targets of disulfide stress were identified: redox buffers, such as ribonuclease inhibitor or albumin, and redox-signaling thiols, which include thioredoxin 1, APE1/Ref1, Keap1, tyrosine and serine/threonine phosphatases, and protein disulfide isomerase. These targets exhibit great relevance in DNA repair, cell proliferation, apoptosis, endoplasmic reticulum stress, and inflammatory response. Disulfide stress would be a specific mechanism of redox signaling independent of glutathione redox status involved in inflammation.     

Formation of albumin dimers induced by exposure to peroxides in human plasma: a possible biomarker for oxidative stress

Human serum albumin (HSA) has one free thiol residue at Cys-34 that is likely oxidized by various reactive oxygen species (ROS). We attempted to identify the oxidation product of Cys-34 of HSA following exposure of plasma to ROS. Oxidation induced by tert-butyl hydroperoxide (t-BuOOH) of this free cysteine residue in HSA was observed in detail. Analysis of oxidized albumin in a partially purified fraction obtained by affinity column chromatography clearly revealed the formation of albumin disulfide dimers following t-BuOOH exposure. Albumin disulfide dimer formation was observed in normal plasma following treatment with various peroxides, as well as in untreated plasma from patients on hemodialysis using SDS-PAGE and Western blot analysis. The present results indicate that albumin dimers are oxidative products derived from peroxides, and that their presence in plasma might be a marker of oxidative stress as secondary metabolites of peroxidation.     

Astrocytes provide cysteine to neurons by releasing glutathione

Cysteine is the rate-limiting precursor of glutathione synthesis. Evidence suggests that astrocytes can provide cysteine and/or glutathione to neurons. However, it is still unclear how cysteine is released and what the mechanisms of cysteine maintenance by astrocytes entail. In this report, we analyzed cysteine, glutathione, and related compounds in astrocyte conditioned medium using HPLC methods. In addition to cysteine and glutathione, cysteine-glutathione disulfide was found in the conditioned medium. In cystine-free conditioned medium, however, only glutathione was detected. These results suggest that glutathione is released by astrocytes directly and that cysteine is generated from the extracellular thiol/disulfide exchange reaction of cystine and glutathione: glutathione + cystine<-->cysteine + cysteine-glutathione disulfide. Conditioned medium from neuron-enriched cultures was also assayed in the same way as astrocyte conditioned medium, and no cysteine or glutathione was detected. This shows that neurons cannot themselves provide thiols but instead rely on astrocytes. We analyzed cysteine and related compounds in rat CSF and in plasma of the carotid artery and internal jugular vein. Our results indicate that cystine is transported from blood to the CNS and that the thiol/disulfide exchange reaction occurs in the brain in vivo. Cysteine and glutathione are unstable and oxidized to their disulfide forms under aerobic conditions. Therefore, constant release of glutathione by astrocytes is essential to maintain stable levels of thiols in the CNS.     

Determination of the in vivo redox status of cysteine, cysteinylglycine, homocysteine, and glutathione in human plasma.

An assay that measures the reduced, oxidized, and protein-bound forms of cysteine, cysteinylglycine, homocysteine, and glutathione in human plasma is described. Oxidized and protein-bound thiols are converted to their reduced counterparts by the use of NaBH4, and, following derivatization with monobromobimane (mBrB), the thiol-bimane adducts are quantified by reversed-phase ion-pair liquid chromatography and fluorescence detection. The presence of 50 microM dithioerythritol provides linearity of the standard curves at very low thiol concentrations. Selective determination of the oxidized forms was accomplished by blocking free sulfhydryl groups with N-ethylmaleimide (NEM) and excess NEM is inactivated by the subsequent addition of NaBH4. The reduced forms of the thiols in plasma were trapped with minimal oxidation by derivatizing blood samples at the time of collection. This was attained by drawing blood directly into tubes containing isotonic solutions of mBrB or NEM. The assay is sufficiently sensitive (less than 2 pmol) to detect the various forms of the four thiol compounds in human plasma. The analytical recovery of cysteine, cysteinylglycine, homocysteine, and glutathione was close to 100%, and the within-day precision corresponded to a coefficient of variation of 7, 8, 6, and 7%, respectively. The assay has been used to determine the various forms of the four thiol compounds in human plasma.