Analysis of biological thiols: derivatization with monobromotrimethylammoniobimane and characterization by electrophoresis and chromatography

A new method for analysis of biological thiols based upon their conversion to fluorescent derivatives by reaction with monobromotrimethylammoniobimane (qBBr) is described. The derivatives are separated by chromatography and by electrophoresis on cellulose thinlayer chromatography plates. The use of two-dimensional mapping makes it possible to differentiate between a wide variety of biological thiols including N-acetylcysteine, CoA, cysteine, cysteinylglycine, cysteamine, ergothioneine, glutathione, γ-glutamylcysteine, homocysteine, mercaptopyrimidine, pantetheine, 4′-phosphopanetheine, thiosulfate, and thiouracil. For applications to biological samples thiols were isolated from crude extracts by binding to a mercuriagarose gel. Following removal from the gel with dithiothreitol, the thiols were derivatized with qBBr. The methods were tested by showing that glutathione is the major thiol in human red blood cells, that glutathione and ergothioneine are the major thiols in Neurospora crassa conidia, and that Bacillus cereus vegetative cells lack glutathione but contain cysteine, pantetheine, and an unidentified thiol in significant amounts.     

Quantitation of reduced and total glutathione at the femtomole level by high-performance liquid chromatography with fluorescence detection: application to red blood cells and cultured fibroblasts

A new rapid and highly sensitive HPLC method with ortho-phthalaldehyde (OPA) pre-column derivatization has been developed for determination of reduced glutathione (GSH) and total glutathione (GSHt) in human red blood cells and cultured fibroblasts. OPA derivatives are separated on a reversed-phase HPLC column with an acetonitrile–sodium acetate gradient system and detected fluorimetrically. An internal standard (glutathione ethyl ester) is added to facilitate quantitation. Total glutathione is determined after reduction of disulfide groups with dithiothreitol; the oxidized glutathione (GSSG) concentration is calculated by subtraction of the GSH level from the GSHt level. The assay shows high sensitivity (50 fmol per injection, the lowest reported), good precision (C.V. <5.0%), an analytical recovery of GSH and GSSG close to 100%, and linearity (r>0.999). This HPLC technique is very simple and rapid. Its wide applicability and high sensitivity make it a convenient and reliable method for glutathione determination in various biological samples.     

The Mercaptopyruvate Sulfurtransferase of Trichomonas vaginalis Links Cysteine Catabolism to the Production of Thioredoxin Persulfide

Trichomonas vaginalis is a protozoan parasite of humans that is able to synthesize cysteine de novo using cysteine synthase but does not produce glutathione. In this study, high pressure liquid chromatography analysis confirmed that cysteine is the major intracellular redox buffer by showing that T. vaginalis contains high levels of cysteine (∼600 μm) comprising more than 70% of the total thiols detected. To investigate possible mechanisms for the regulation of cysteine levels in T. vaginalis, we have characterized enzymes of the mercaptopyruvate pathway. This consists of an aspartate aminotransferase (TvAspAT1), which transaminates cysteine to form 3-mercaptopyruvate (3-MP), and mercaptopyruvate sulfurtransferase (TvMST), which transfers the sulfur of 3-MP to a nucleophilic acceptor, generating pyruvate. TvMST has high activity with 3-MP as a sulfur donor and can use several thiol compounds as sulfur acceptor substrates. Our analysis indicated that TvMST has a k_cat/K_m for reduced thioredoxin of 6.2 × 10⁷ m⁻¹ s⁻¹, more than 100-fold higher than that observed for β-mercaptoethanol and cysteine, suggesting that thioredoxin is a preferred substrate for TvMST. Thiol trapping and mass spectrometry provided direct evidence for the formation of thioredoxin persulfide as a product of this reaction. The thioredoxin persulfide could serve a biological function such as the transfer of the persulfide to a target protein or the sequestered release of sulfide for biosynthesis. Changes in MST activity of T. vaginalis in response to variation in the supply of exogenous cysteine are suggestive of a role for the mercaptopyruvate pathway in the removal of excess intracellular cysteine, redox homeostasis, and antioxidant defense.     

Mechanistic Details of Glutathione Biosynthesis Revealed by Crystal Structures of Saccharomyces cerevisiae Glutamate Cysteine Ligase

Glutathione is a thiol-disulfide exchange peptide critical for buffering oxidative or chemical stress, and an essential cofactor in several biosynthesis and detoxification pathways. The rate-limiting step in its de novo biosynthesis is catalyzed by glutamate cysteine ligase, a broadly expressed enzyme for which limited structural information is available in higher eukaryotic species. Structural data are critical to the understanding of clinical glutathione deficiency, as well as rational design of enzyme modulators that could impact human disease progression. Here, we have determined the structures of Saccharomyces cerevisiae glutamate cysteine ligase (ScGCL) in the presence of glutamate and MgCl₂ (2.1 Å; R = 18.2%, R_free = 21.9%), and in complex with glutamate, MgCl₂, and ADP (2.7 Å; R = 19.0%, R_free = 24.2%). Inspection of these structures reveals an unusual binding pocket for the α-carboxylate of the glutamate substrate and an ATP-independent Mg²⁺ coordination site, clarifying the Mg²⁺ dependence of the enzymatic reaction. The ScGCL structures were further used to generate a credible homology model of the catalytic subunit of human glutamate cysteine ligase (hGCLC). Examination of the hGCLC model suggests that post-translational modifications of cysteine residues may be involved in the regulation of enzymatic activity, and elucidates the molecular basis of glutathione deficiency associated with patient hGCLC mutations.     

Determination of intracellular glutathione in human skeletal muscle by reversed-phase high-performance liquid chromatography

A chromatographic method for the specific determination of cellular low molecular mass thiols has been applied to human muscle tissue. The method is based on the derivatisation of thiols using monobromobimane, which is a specific reagent for the sulphydryl group. The glutathione and cysteine bimane adducts were separated by reversed-phase HPLC, whilst quantitation of the cysteine and glutathione adducts was achieved by fluorescence spectroscopy. The method was found to yield a quantitative recovery of glutathione (ca. 96%), to be sensitive (down to 20 pmol glutathione/per injection) and reveal a low intra-individual coefficient of variation (C.V. < 5%) of the glutathione concentrations in human skeletal muscle. The concentrations of reduced and total glutathione were 1320 ± 37 μmol/kg wet weight (mean ± S.E.M.) and 1525 ± 66 μmol/kg wet weight, respectively. The method was also applied to tissues from nine healthy volunteers to determine if fluctuations in glutathione level occurred over a 24-h period. No diurnal variation of glutathione level in human skeletal muscle was observed.     

Simultaneous analysis of diphenylmethoxyacetic acid,a metabolite of diphenhydramine,and its deuterium-labeled stable isotope analog in ovine plasma and urine

Diphenylmethoxyacetic acid (DPMA) is a major metabolite of diphenhydramine in monkeys, dogs, and humans. The metabolic fate of diphenhydramine (DPHM) in sheep is not yet well understood; however, preliminary studies have demonstrated the presence of DPMA in the plasma and urine of sheep following an intravenous bolus of DPHM. Our current studies employ the simultaneous intravenous co-administration of DPHM and the stable isotope analog of DPHM to investigate the pharmacokinetics of DPHM in sheep. In these studies, in order to investigate the pharmacokinetics of the DPMA metabolite, measurement of both unlabeled and stable-isotope labeled DPMA is required. Thus, a stable isotope analog of DPMA ([²H₁₀]DPMA) was synthesized, characterized, and purified for use as an analytical standard. The quantitative method for the gas chromatography—electron-impact mass spectrometry (GC—EI-MS) analysis of DPMA and [²H₁₀]DPMA used a single step liquid-liquid extraction procedure using toluene for sample cleanup. The samples were derivatized with N-methyl-N-(tert.-butyldimethylsilyl) trifluoroacetamide. A 1.0-μl aliquot of the prepared sample was injected into the GC-MS system and quantitated using selected-ion monitoring (SIM). One ion was monitored for each compound, namely, m/z 165 for the internal standard diphenylacetic acid, m/z 183 for DPMA, and m/z 177 for [²H₁₀]DPMA. The ion chromatograms were free from chromatographic peaks co-eluting with the compound of interest. The calibration curve was linear from 2.5 ng/ml (limit of quantitation) to 250.0 ng/ml in both urine and plasma. The intra-day and inter-day variabilities of this assay method were within acceptable limits (below 20% at the limit of quantitation and below 10% at all other concentrations). This method was used to measure the concentration of DPMA and [²H₁₀]DPMA in plasma and urine samples from a ewe in which equimolar amounts of DPHM and [²H₁₀]DPHM were administered by an intravenous bolus dose via the femoral vein. DPMA appeared to persist longer in the plasma and the urine as compared to DPHM. This method is robust and reliable for the quantitation of DPMA and [²H₁₀]DPMA in biological samples obtained from sheep (e.g. plasma and urine).     

Cysteine and glutathione in orange juice

The major part of the sulfhydryl compounds of both Valencia and Navel orange juice was found to exist as cysteine and glutathione. These two compounds were isolated and analyzed as their crystalline S-benzyl derivatives from concentrates prepared by preferential mercuric ion precipitation. Cysteine analysis before and after hydrolysis showed that all of the sulfhydryl of the concentrates was cysteine and glutathione. The glutathione analysis was confirmed by Woodward's glyoxalase method.     

Determination of N-acetylneuraminic acid by gas chromatography-mass spectrometry with a stable isotope as internal standard

N-Acetylneuraminic acid was determined by gas chromatography-mass spectrometry using selected ion-monitoring technique with N-[²H₃]acetylneuraminic acid as an internal standard. M-COOTMS fragments at $\text{m}\text{z}$ 624 of trimethylsilyl derivatives of N-acetylneuraminic acid and at $\text{m}\text{z}$ 627 of that of the internal standard were used as monitoring ions. The standard curve obtained was linear in the range of over 10³, and the lower limit for quantitation was estimated to be a few hundred picograms. This method was used to measure total N-acetylneuraminic acid in the plasma of healthy humans and patients with lung cancer. The total N-acetylneuraminic acid level in the plasma was two to three times higher in the patients than in controls. A few hundred nanoliters of plasma was sufficient for the analysis. The mass fragmentogram of plasma gave a good signal/noise ratio, and measurements were very specific, accurate, and reproducible.     

Sensitive analysis of [ -Pen]enkephalin in rat serum by capillary electrophoresis and laser-induced fluorescence detection

A highly sensitive analytical method based on capillary zone electrophoresis (CZE) coupled with a laser-induced fluorescence (LIF) detector was explored for the analysis of [ -Pen^2,5]enkephalin (DPDPE) in rat serum. DPDPE and the internal standard Phe-Leu-Glu-Glu-Ile (P9396) were extracted from serum samples with C₁₈ solid-phase extraction disk cartridges, followed by derivatization with tetramethylrhodamine-5-isothiocyanate (TRITC) isomer G before introduction onto the capillary column. Complete resolution of DPDPE and the internal standard from other serum components was achieved within 20 min on a 140 cm×50 μm I.D. capillary column with borate buffer (25 mM, pH 8.3). With the current method, it is possible to detect 1.3E-18 mol of DPDPE on column. The results suggest that CZE-LIF is a promising method for the sensitive and specific quantitation of therapeutic peptides in biological matrices.     

Gas chromatographic–mass spectrometric determination of α-ketoisocaproic acid and [2H7]α-ketoisocaproic acid in plasma after derivatization with N-phenyl-1,2-phenylenediamine

A method for determination of α-ketoisocaproic acid (KIC) and [4,5,5,5,6,6,6-²H₇]α-ketoisocaproic acid ([²H₇]KIC) in rat plasma was developed using gas chromatography–mass spectrometry-selected ion monitoring (GC–MS-SIM). [5,5,5-²H₃]α-Ketoisocaproic acid ([²H₃]KIC) was used as an analytical internal standard to account for losses associated with the extraction, derivatization and chromatography. The keto acids were extracted by cation-exchange chromatography using BondElut SCX cartridge and derivatized with N-phenyl-1,2-phenylenediamine to form N-phenylquinoxalinone derivatives. Quantitation was performed by SIM of the respective molecular ions at m/z 278, 281 and 285 for the derivatives of KIC, [²H₃]KIC and [²H₇]KIC on the electron impact method. The limit of detection was found to be 70 fmol per injection (S/N=3) and the limit of quantitation for [²H₇]KIC was around 50 nM in rat plasma. Endogenous KIC concentrations in 50 μl of rat plasma were measured with relative intra- and inter-day precision of 4.0% and 3.3%, respectively. The intra- and inter-day precision for [²H₇]KIC spiked to rat plasma in the range of 0.1 to 10 μM gave good reproducibility with relative standard deviation (RSD) of 6.5% and 5.4%, respectively. The intra- and inter-day relative errors (RE) for [²H₇]KIC were less than 6.4% and 3.8%, respectively. The method was applied to determine the plasma concentration of [²H₇]KIC after an intravenous administration of [²H₇]KIC in rat.     

Determination of glyceryl trinitrate in human plasma by gas chromatography—negative ion chemical ionization—selected ion monitoring

A specific, sensitive and accurate quantitation method for glyceryl trinitrate was developed using gas chromatography—negative ion chemical ionization—selected ion monitoring with dichloromethane as a reagent gas. [¹⁵N₃] and [²H₅, ¹⁵N₃] variants were synthesized from non-labelled or [²H₈]glycerol and [¹⁵N]nitric acid. The former variant was used for preventing adsorption of glyceryl trinitrate onto active sites on column materials and the latter was used as an internal standard for quantitation of glyceryl trinitrate in biological fluids by selected ion monitoring. The quantitation limit of this method is 0.1 ng/ml of human plasma. When glyceryl trinitrate was administered intravenously in the dose of 4 μg/kg to patients receiving hypotensive anesthesia for surgical operation, the plasma levels exhibited a biexponential decay. The mean and standard deviation of half-lives of the α and β phases were found to be about 0.41 ± 0.13 and 5.34 ± 1.60 min, respectively.     

Effect of glutathione on phytochelatin synthesis in tomato cells

Growth of cell suspension cultures of tomato, Lycopersicon esculentum Mill. cv VFNT-Cherry, in the presence of cadmium is inhibited by buthionine sulfoximine, an inhibitor of glutathione synthesis. Cell growth and phytochelatin synthesis are restored to cells treated with buthionine sulfoximine by the addition of glutathione to the medium. Glutathione stimulates the accumulation of phytochelatins in cadmium treated cells, indicating that availability of glutathione can limit synthesis of these peptides. Exogenous glutathione causes a disproportionate increase in the level of smaller phytochelatins, notably [γ-Glu-Cys]₂-Gly. In the presence of buthionine sulfoximine and glutathione, phytochelatins that are produced upon exposure to cadmium incorporate little [³⁵S]cysteine, indicating that these peptides are probably not synthesized by sequential addition of cysteine and glutamate to glutathione.     

l-Cystine inhibits aspartate-β-semialdehyde dehydrogenase by covalently binding to the essential 135Cys of the enzyme

Aspartate-β-semialdehyde dehydrogenase (ASADH) from Escherichia coli is inhibited by l- and d-cystine, and by other cystine derivatives. Enzyme inhibition is quantitatively reversed by addition of dithiothreitol (DTT), dithioerythrytol, β-mercaptoethanol, di-mercaptopropanol or glutathione to the cystine-inactivated enzyme. Cystine labeling of the enzyme is a pH dependent process and is optimal at pH values ranging from 7.0 to 7.5. Both the cysteine incorporation profile and the inactivation curve of the enzyme as a function of pH suggest that a group(s) with pK_a of 8.5 could be involved in cystine binding. Stoichiometry of the inactivation reaction indicates that one cysteine residue from the enzyme subunit is reactive against cystine, as found by direct incorporation of radioactive cystine into the enzyme and by free-thiol titration of the enzyme with 5,5′-dithiobis-2-nitrobenzoic acid (DTNB) before and after the cystine treatment. One mole of cysteine is released from each mol of cystine after reaction with the enzyme. ASA, NADP and NADPH did not prevent cystine inhibition. The [³⁵S]cysteine-labelled enzyme can be visualized after electrophoresis in polyacrylamide gels and further detection by autoradiography. After pepsin treatment of the [³⁵S]cysteine-inactivated enzyme, a main radioactive peptide was isolated by HPLC. The amino acid sequence of this peptide was determined as FVGGN(Cys)₂TVSL, thus demonstrating that the essential ¹³⁵Cys is the amino acid residue modified by the treatment with cystine.     

Some chemical properties of carboxymethyl derivatives of amino acids

Tritium-labeled carboxymethyl derivatives of various functional groups in proteins show ready exchange of the tritium label when exposed to standard protein hydrolysis conditions (6 n HCl, 21 h, 110 °C). While N_?-[³H]carboxymethyl lysine does not show any tritium exchange, the two N-[³H]carboxymethyl histidine derivatives lose their tritium with a half time of about 15 h, and S-[³H]carboxymethyl cysteine loses its tritium with a half time of about 1.5 h. The tritium exchange of S-[³H]carboxymethyl methionine was so fast that the derivative could not be prepared with any of the tritium label intact. The rate of exchange for this compound was consequently determined by following the disappearance of the methylene NMR signal when S-carboxymethyl methionine was dissolved in D₂O. The half time for the exchange was about 12 min. Mechanisms involving either a sulfonium ion or enolization of the protonated conjugate carboxylic acid appear to give a satisfactory explanation of the relative stability of the different derivatives. The practical use of the differential rate of hydrogen exchange as a means of establishing rapidly and with small quantities of material the site of carboxymethylation in unknown proteins is suggested.     

Loss of the mitochondrial lipid cardiolipin leads to decreased glutathione synthesis

Previous studies demonstrated that loss of CL in the yeast mutant crd1Δ leads to perturbation of mitochondrial iron‑sulfur (FeS) cluster biogenesis, resulting in decreased activity of mitochondrial and cytosolic Fe-S-requiring enzymes, including aconitase and sulfite reductase. In the current study, we show that crd1Δ cells exhibit decreased levels of glutamate and cysteine and are deficient in the essential antioxidant, glutathione, a tripeptide of glutamate, cysteine, and glycine. Glutathione is the most abundant non-protein thiol essential for maintaining intracellular redox potential in almost all eukaryotes, including yeast. Consistent with glutathione deficiency, the growth defect of crd1Δ cells at elevated temperature was rescued by supplementation of glutathione or glutamate and cysteine. Sensitivity to the oxidants iron (FeSO₄) and hydrogen peroxide (H₂O₂), was rescued by supplementation of glutathione. The decreased intracellular glutathione concentration in crd1Δ was restored by supplementation of glutamate and cysteine, but not by overexpressing YAP1, an activator of expression of glutathione biosynthetic enzymes. These findings show for the first time that CL plays a critical role in regulating intracellular glutathione metabolism.     

Fluorescent/ultraviolet absorbing ester derivative formation and analysis of eicosanoids by high-pressure liquid chromatography

A method is described for the quantitative analysis of eicosanoids (arachidonic acid metabolites, nee, prostaglandins) by reverse-phase high-pressure liquid chromatography following formation of the ester derivative with p-(9-anthroyloxy)phenacyl bromide. The lower limit of detection of the eicosanoid ester is 280 pg (ultraviolet—254 nm) and approximately 50 pg (fluorescence 249 emission, 413-nm cutoff). We separated the esters of seven common eicosanoids by reverse-phase chromatography with acetonitrile and water. Thromboxane B₂ chromatographs as two species and coelutes with PGF_2α. Separation of all others is adequate, including the three metabolites of prostacyclin (6-keto-PGF_1α, 6-keto-PGE₁, 13,14-dihydro-6,15-diketo-PGF_1α). We obtained good correlation between radioimmunoassay and derivative analysis of standard 6-keto-PGF_1α extracted from lactated Ringer's solution with standard technique, as well as 6-keto-PGF_1α quantitation from tissue culture medium that had contained pulmonary endothelial cells. This method should be applicable to analysis of eicosanoids extracted from biological matrices.     

Regulation of Assimilatory Sulfate Reduction by Herbicide Safeners in Zea mays L

Effects of the herbicide safeners N,N-diallyl-2,2-dichloroacetamide and 4-dichloroacetyl-3,4-dihydro-3-methyl-2H-1,4-benzooxazin (CGA 154281) on the contents in cysteine and glutathione, on the assimilation of ³⁵SO₄²⁻, and on the enzymes of assimilatory sulfate reduction were analyzed in roots and primary leaves of maize (Zea mays) seedlings. Both safeners induced an increase in cysteine and glutathione. In labeling experiments using ³⁵SO₄²⁻, roots of plants cultivated in the presence of safeners contained an increased level of radioactivity in glutathione and cysteine as compared with controls. A significant increase in uptake of sulfate was only detected in the presence of CGA 154281. One millimolar N,N-diallyl-2,2-dichloroacetamide applied to the roots for 6 days increased the activity of adenosine 5′-phosphosulfate sulfotransferase about 20- and threefold in the roots and leaves, respectively, compared with controls. CGA 154281 at 10 micromolar caused a sevenfold increase of this enzyme activity in the roots, but did not affect it significantly in the leaves. A significant increase in ATP-sulfurylase (EC 2.7.7.4) activity was only detected in the roots cultivated in the presence of 10 micromolar CGA 154281. Both safeners had no effect on the activity of sulfite reductase (EC 1.8.7.1) and O-acetyl-l-serine sulfhydrylase (EC 4.2.99.8). The herbicide metolachlor alone or combined with the safeners induced levels of adenosine 5′-phosphosulfate sulfotransferase, which were higher than those of the appropriate controls. Taken together these results show that the herbicide safeners increased both the level of adenosine 5′-phosphosulfate sulfotransferase activity and of the thiols cysteine and glutathione. This indicates that these safeners may be involved in eliminating the previously proposed regulatory mechanism, in which increased concentrations of thiols regulate assimilatory sulfate reduction by decreasing the activities of the enzymes involved.     

Requirement of thiols for activation of coronary arterial guanylate cyclase by glyceryl trinitrate and sodium nitrite possible involvement of S-nitrosothiols

Glyceryl trinitrate specifically required cysteine, whereas NaNO₂ at concentrations less than 10 mM required one of several thiols or ascorbate, to activate soluble guanylate cyclase from bovine coronary artery. However, guanylate cyclase activation by nitroprusside or nitric oxide did not require the addition of thiols or ascorbate. Whereas various thiols enhanced activation by nitropruside, none of the thiols tested enhanced activation by nitric oxide. S-Nitrosocysteine, which is formed when cysteine reacts with either NO₂^? or nitric oxide, was a potent activator of guanylate cyclase. Similarly, micromolar concentrations of the S-nitroso derivatives of penicillamine, GSH and dithiothreitol, prepared by reacting the thiol with nitric oxide, activated guanylate cyclase. Guanylate cyclase activation by S-nitrosothiols resembled that by nitric oxide and nitroprusside in that activation was inhibited by methemoglobin, ferricyanide and methylene blue. Similarly, guanylate cyclase activation by glyceryl trinitrate plus cysteine, and by NaNO₂ plus either a thiol or ascorbate, was inhibited by methemoglobin, ferricyanide and methylene blue. These data suggest that the activation of guanylate cyclase by each of the compounds tested may occur through a common mechanism, perhaps involving nitric oxide. Moreover, these findings suggest that S-nitrosothiols could act as intermediates in the activation of guanylate cyclase by glyceryl trinitrate, NaNO₂ and possibly     

GC–MS analysis of S-nitrosothiols after conversion to S-nitroso-N-acetyl cysteine ethyl ester and in-injector nitrosation of ethyl acetate

S-Nitrosothiols from low-molecular-mass and high-molecular-mass thiols, including glutathione, albumin and hemoglobin, are endogenous potent vasodilators and inhibitors of platelet aggregation. By utilizing the S-transnitrosation reaction and by using the lipophilic (pK_L 0.78) and strong nucleophilic synthetic thiol N-acetyl cysteine ethyl ester (NACET) we have developed a GC–MS method for the analysis of S-nitrosothiols and their ¹⁵N- or ²H–¹⁵N-labelled analogs as S-nitroso-N-acetyl cysteine ethyl ester (SNACET) and S¹⁵NACET or d₃-S¹⁵NACET derivatives, respectively, after their extraction with ethyl acetate. Injection of ethyl acetate solutions of S-nitrosothiols produced two main reaction products, compound X and compound Y, within the injector in dependence on its temperature. Quantification was performed by selected-ion monitoring of m/z 46 (i.e., [NO₂]^?) for SNACET and m/z 47 (i.e., [¹⁵NO₂]^?) for S¹⁵NACET/d₃-S¹⁵NACET for compound X, and m/z 157 for SNACET and m/z 160 for d₃-S¹⁵NACET for compound Y. In this article we describe the development, validation and in vitro and in vivo applications of the method to aqueous buffered solutions, human and rabbit plasma. Given the ester functionality of SNACET/S¹⁵NACET/d₃-S¹⁵NACET, stability studies were performed using metal chelators and esterase inhibitors. The method was found to be suitable for the quantitative determination of various S-nitrosothiols including SNACET externally added to human plasma (0–10 μM). Nitrite contamination in ethyl acetate was found to interfere. Our results suggest that the concentration of endogenous S-nitrosothiols in human plasma does not exceed about 200 nM in total. Oral administration of S¹⁵NACET to rabbits (40–63 μmol/kg body weight) resulted in formation of ALB-S¹⁵NO, [¹⁵N]nitrite and [¹⁵N]nitrate in plasma.     

Quantitation of glutathione and its oxidation products in erythrocytes by multiple-label stable-isotope dilution

A multiple-label stable isotope dilution assay for quantifying glutathione (GSH), glutathione disulfide (GSSG), and glutathione sulfonic acid in erythrocytes was developed. As the internal standards, [¹³C₃,¹⁵N]glutathione, [¹³C₄,¹⁵N₂]glutathione disulfide, and [¹³C₃,¹⁵N]glutathione sulfonic acid were used. Analytes and internal standards were detected by LC–MS/MS after derivatization of GSH with iodoacetic acid and dansylation of all compounds under study. The calibration functions for all analytes relative to their respective isotopologic standards revealed slopes close to 1.0 and negligible intercepts. As various labelings of the standards for GSH and GSSG were used, their simultaneous quantitation was possible, although GSH was partly oxidized to its disulfide during analysis. The degree of this artifact formation of GSSG was calculated from the abundance of the mixed disulfide formed from unlabeled GSH and its respective standard. Thus, the detected GSSG amount could be corrected for the artifact amount. In this way, the amount of GSSG in erythrocytes was found to be less than 0.5% of the GSH concentration. Similar to GSSG, the detected amount of glutathione sulfonic acid was found to be formed at least in part during the analytical process, but the degree could not be quantified.