Gas chromatographic–mass spectrometric analysis of stable isotopes of cysteine and glutathione in biological samples

Capitan, Pierre; Malmezat, T.; Breuillé, Denis; Obled, Christiane

doi:10.1016/s0378-4347(99)00273-x

Cited by 72 publications

(58 citation statements)

References 13 publications

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“…Analytical procedures. Erythrocyte free cysteine was analyzed using a modification of the method described by Capitan et al (20). Blood was collected into ice-cold EDTA glass tubes and immediately centrifuged at 5,000g for 20 min at 4°C to separate the red cell pellet from plasma.…”

Section: Methodsmentioning

confidence: 99%

“…The ethereal phase was collected and evaporated under nitrogen at 50°C, and samples were incubated for 10 min at 80°C with 250 l of 1 mol/l HCl in methanol to methylate cysteine's carboxylic group. Samples were evaporated under nitrogen at room temperature, and the dry residue was dissolved in 400 l ethylacetate and analyzed using an HP 5971 gas chromatograph mass spectrometer as described (20). Ions at m/z ϭ 220 and 222, representing natural and 2 H 2 -cysteine, respectively, were selectively monitored to assess 2 H 2 -cysteine enrichments; ions at m/z ϭ 160 and 147, representing natural cysteine and penicillamine, respectively, were used to assess cysteine concentrations.…”

Section: Methodsmentioning

confidence: 99%

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Evidence for Accelerated Rates of Glutathione Utilization and Glutathione Depletion in Adolescents With Poorly Controlled Type 1 Diabetes

et al. 2005

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Depletion of glutathione, an important antioxidant present in red cells, has been reported in type 1 diabetes, but the mechanism of this depletion has not been fully characterized. Glutathione depletion can occur through decreased synthesis, increased utilization, or a combination of both. To address this issue, 5-h infusions of L-[3,3-2 H 2 ]cysteine were performed in 16 diabetic adolescents divided into a well-controlled and a poorly controlled group and in eight healthy nondiabetic teenagers as control subjects (HbA 1c 6.3 ؎ 0.2, 10.5 ؎ 0.6, and 4.8 ؎ 0.1%, respectively). Glutathione fractional synthesis rate was determined from 2 H 2 -cysteine incorporation into blood glutathione. We observed that 1) erythrocyte cysteine concentration was 41% lower in poorly controlled patients compared with well-controlled patients (P ‫؍‬ 0.009); 2) erythrocyte glutathione concentration was ϳ29% and ϳ36% lower in well-controlled and poorly controlled patients compared with healthy volunteers; and 3) the fractional synthesis rate of glutathione, although similar in well-controlled and healthy subjects (83 ؎ 14 vs. 82 ؎ 11% per day), was substantially higher in the poorly controlled group (141 ؎ 23% per day, P ‫؍‬ 0.038). These findings suggest that in diabetic adolescents, poor control is associated with a significant depletion of blood glutathione and cysteine, due to increased rates of glutathione utilization. This weakened antioxidant defense may play a role in the pathogenesis of diabetes complications. Diabetes

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Evidence for Accelerated Rates of Glutathione Utilization and Glutathione Depletion in Adolescents With Poorly Controlled Type 1 Diabetes

et al. 2005

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“…However, it is difficult to draw meaningful comparisons based on just a single subject. Furthermore, it would now be highly desirable to carry out nonprimed tracer studies that would allow the fitting of data to a more formal model for purpose of estimating GSH-FSR (24), particularly because of the tendency for the approach used by us and others (11,21,23) to underestimate the FSR. For example, if the data of Fig.…”

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confidence: 99%

Blood glutathione synthesis rates in healthy adults receiving a sulfur amino acid-free diet

Lyons

Rauh-Pfeiffer

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

147

112

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The availability of cysteine is thought to be the rate limiting factor for synthesis of the tripeptide glutathione (GSH), based on studies in rodents. GSH status is compromised in various disease states and by certain medications leading to increased morbidity and poor survival. To determine the possible importance of dietary cyst(e)ine availability for whole blood glutathione synthesis in humans, we developed a convenient mass spectrometric method for measurement of the isotopic enrichment of intact GSH and then applied it in a controlled metabolic study. Seven healthy male subjects received during two separate 10-day periods an L-amino acid based diet supplying an adequate amino acid intake or a sulfur amino acid (SAA) (methionine and cysteine) free mixture (SAA-free). On day 10, L-[1-13 C]cysteine was given as a primed, constant i.v. infusion (3mol⅐kg ؊1 ⅐h ؊1 ) for 6 h, and incorporation of label into whole blood GSH determined by GC͞MS selected ion monitoring. The fractional synthesis rate (mean ؎ SD; day -1 ) of whole blood GSH was 0.65 ؎ 0.13 for the adequate diet and 0.49 ؎ 0.13 for the SAA-free diet (P < 0.01). Whole blood GSH was 1,142 ؎ 243 and 1,216 ؎ 162 M for the adequate and SAA-free periods (P > 0.05), and the absolute rate of GSH synthesis was 747 ؎ 216 and 579 ؎ 135 mol⅐liter ؊1 ⅐day ؊1 , respectively (P < 0.05). Thus, a restricted dietary supply of SAA slows the rate of whole blood GSH synthesis and diminishes turnover, with maintenance of the GSH concentration in healthy subjects.

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“…In the GC-MS technique, GSH was performed in N,S-ethoxycarbonyl methyl ester derivatives. This is a very sensitive method; however, it is time consuming and expensive because of the many specific reagents (Humbert et al 2001;Capitan et al 1999;Küster et al 2008). What is more, many variables may disrupt the analysis process, such as an incorrect pH (Camera and Picardo 2002;Monostori et al 2009) or the temperature of GC-MS measurement (Iwasaki et al 2009).…”

Section: Short Review Of a Few Techniques Often Used To Determine Glumentioning

confidence: 99%

The Use of Different MS Techniques to Determine Glutathione Levels in Marine Tissues

2012

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Several techniques were used, mainly mass spectrometry connected with gas chromatography, matrixassisted laser desorption with ionization time-of-flight and mass spectrometry connected with liquid chromatography. A major problem was encountered in determining glutathione, which can be conditioned by the pH and selected reducing agents. The GSH form can be oxidized through derivatization, and a small glutathione amount in the biological samples may hinder the determination process. Another problem is the existence of a metal ion in the tested organism; therefore, often a reagent with a chelating function is added to the sample and the mobile phase in liquid chromatography is applied with appropriate polarity for GSH and GSSG. We determined the concentrations of total, reduced, and oxidized glutathione in the liver, hepatopancreas, muscle, and gonad tissues of brown shrimp (Crangon crangon) and fish (Psetta maxima and Clupea harengus membras). The highest concentrations of tGSH were recorded in the shrimp hepatopancreas (7.21 ± 0.011 μmol g −1 wet weight), in herring liver (2.85±0.025 μmol g −1 ), and in turbot liver (1.86±0.063 μmol g −1). In turn, the highest concentrations were reported for GSSG in the muscle of shrimp (0.140±0.000204 μmol g ). We also investigated seasonal changes in the concentrations of glutathione in the muscle of C. crangon shrimp in the annual cycle. The lowest values of total glutathione were recorded during spring and autumn, which could be correlated with the increase in lipid peroxidation and oxidative stress.

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Gas chromatographic–mass spectrometric analysis of stable isotopes of cysteine and glutathione in biological samples

Cited by 72 publications

References 13 publications

Evidence for Accelerated Rates of Glutathione Utilization and Glutathione Depletion in Adolescents With Poorly Controlled Type 1 Diabetes

Evidence for Accelerated Rates of Glutathione Utilization and Glutathione Depletion in Adolescents With Poorly Controlled Type 1 Diabetes

Blood glutathione synthesis rates in healthy adults receiving a sulfur amino acid-free diet

The Use of Different MS Techniques to Determine Glutathione Levels in Marine Tissues

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