Simple plasma work-up for a fast chromatographic analysis of homocysteine, cysteine, methionine and aromatic amino acids

Hušek, Petr; Matucha, Petr; Vránková, Alice; Šimek, Petr

doi:10.1016/s1570-0232(03)00104-1

Cited by 43 publications

(42 citation statements)

References 26 publications

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“…Acid soluble supernatant was collected and used for high-pressure liquid chromatography-linked tandem mass spectrometry (LC/MS/MS) analysis. Metabolites (see Table S1, available on the Blood website; see the Supplemental Materials link at the top of the online article) were detected using LC/MS/MS following derivatization with isopropylchloroformate, based on methods previously published by Husek et al, 52 with the following modifications. An aliquot of culture media and cell pellets (1 ϫ 10 6 cells) were mixed with equal volume of 10% PCA containing 1 mM DTPA.…”

Section: High-pressure Liquid Chromatography-linked Tandem Mass Spectmentioning

confidence: 99%

Erythrocyte glutamine depletion, altered redox environment, and pulmonary hypertension in sickle cell disease

Morris¹,

Suh²,

Hagar³

et al. 2008

Blood

167

143

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Erythrocyte glutathione depletion has been linked to hemolysis and oxidative stress. Glutamine plays an additional antioxidant role through preservation of intracellular nicotinamide adenine dinucleotide phosphate (NADPH) levels, required for glutathione recycling. Decreased nitric oxide (NO) bioavailability, which occurs in the setting of increased hemolysis and oxidative stress, contributes to the pathogenesis of pulmonary hypertension (PH) in sickle cell disease (SCD). We hypothesized that altered glutathione and glutamine metabolism play a role in this process. Total glutathione (and its precursors) and glutamine were assayed in plasma and erythrocytes of 40 SCD patients and 9 healthy volunteers. Erythrocyte total glutathione and glutamine levels were significantly lower in SCD patients than in healthy volunteers. Glutamine depletion was independently associated with PH, defined as a tricuspid regurgitant jet velocity (TRV) of at least 2.5 m/s. The ratio of erythrocyte glutamine:glutamate correlated inversely to TRV (r ‫؍‬ ؊0.62, P < .001), plasma arginase concentration (r ‫؍‬ ؊0.45, P ‫؍‬ .002), and plasma-free hemoglobin level (r ‫؍‬ ؊0.41, P ‫؍‬ .01), linking erythrocyte glutamine depletion to dysregulation of the arginine-NO pathway and increased hemolytic rate. Decreased erythrocyte glutathione and glutamine levels contribute to alterations in the erythrocyte redox environment, which may compromise erythrocyte integrity, contribute to hemolysis, and play a role in the pathogenesis of PH of SCD. IntroductionThe erythrocyte redox environment may contribute to the increased oxidative stress, hemolysis, and decreased nitric oxide (NO) bioavailability observed in pulmonary hypertension (PH), a common complication of hemolytic disorders. Reduced glutathione (gamma-glutamyl-cysteinyl-glycine; GSH) is the most abundant low-molecular weight thiol 1 and the principal thiol redox buffer in erythrocytes. 2,3 The red blood cell contributes up to 10% of whole-body GSH synthesis in humans. [4][5][6] In addition to its role as a critical antioxidant, GSH possesses diverse biological functions involved in detoxification, cell proliferation and apoptosis, redox signaling, gene expression, protein glutathionylation, cytokine production, the immune response, mitochondrial function, and integrity as well as NO metabolism. 7,8 Glutathione is synthesized from glutamate, cysteine, and glycine via reactions catalyzed by 2 cytosolic enzymes, gammaglutamylcysteine ligase and GSH synthetase. The intracellular GSH concentration is the final result of a dynamic balance between the rate of GSH synthesis and the combined rate of intracellular GSH consumption and efflux. GSH is readily oxidized to glutathione disulfide (GSSG) by free radicals and reactive oxygen and nitrogen species. GSSG efflux from cells contributes to a net loss of intracellular GSH. 1 Due to its high intracellular concentrations, GSH variations in oxidation states can significantly modify the redox environment of red blood cells. Within the erythrocyte, GSH may...

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Section: High-pressure Liquid Chromatography-linked Tandem Mass Spectmentioning

confidence: 99%

Erythrocyte glutamine depletion, altered redox environment, and pulmonary hypertension in sickle cell disease

Morris¹,

Suh²,

Hagar³

et al. 2008

Blood

167

143

View full text Add to dashboard Cite

show abstract

“…Intracellular and extracellular GSH and GSSG were detected using LC/MS/MS following derivatization with isopropylchloroformate based on methods previously published by Husek et al (34) with the following modifications (Suh, et al, manuscript in preparation). An aliquot of culture media and cell pellets (1 × 10 6 cells) were mixed with equal volume of 10 % perchloric acid (PCA) containing 1 mM DTPA.…”

Section: Lc/ms/ms Detection Of Gsh Gssg Cysteine and Cystinementioning

confidence: 99%

Organelle redox of CF and CFTR-corrected airway epithelia

Schwarzer¹,

Illek²,

Suh³

et al. 2007

Free Radical Biology and Medicine

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In cystic fibrosis reduced CFTR function may alter redox properties of airway epithelial cells. Redox-sensitive GFP (roGFP1) and imaging microscopy were used to measure redox potentials of cytosol, ER, mitochondria and cell surface of cystic fibrosis nasal epithelial cells and CFTRcorrected cells. We also measured glutathione and cysteine thiol redox states in cell lysates and apical fluids to provide coverage over a range of redox potentials and environments that might be affected by CFTR. As measured with roGFP1, redox potentials at the cell surface (~ -207 ±8 mV) and in the ER (~ -217 ±1 mV) and rates of regulation of the apical fluid and ER lumen following DTT treatment were similar for CF and CFTR-corrected cells. CF and CFTR-corrected cells had similar redox potentials in mitochondria (-344 ±9 mV) and cytosol (-322 ±7 mV). Oxidation of carboxy-dichlorodihydrofluoresceindiacetate and of apical Amplex Red occurred at equal rates in CF and CFTR-corrected cells. Glutathione and cysteine redox couples in cell lysates and apical fluid were equal in CF and CFTR-corrected cells. These quantitative estimates of organelle redox potentials combined with apical and cell measurements using small molecule couples confirmed there were no differences in redox properties of CF and CFTR-corrected cells.

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“…Trimethylsilyl (TMS) derivatization agents, such as bis(trimethylsilyl) trifluoroacetamide (BSTFA) and N-methyl-N-trimethylsilyltrifluoroacetamide (MSTFA) containing 1% trimethylchlorosilane (TMCS), have been frequently applied in GC-MS-based metabolic profiling studies [16,17]; however, TMS derivatization only proceeds in nonaqueous media, and the entire reaction procedure is very labor-intensive and time-consuming [15]. As an effective alternative to TMS, ethyl chloroformate (ECF) is a suitable derivative agent for a broad array of low molecular weight metabolites (generally, MW <1000 Da) including organic acids, amines, amino acids, and aminoalcohols that are present in biological matrices [18][19][20]. Recently, we successfully applied an optimized two-step ECF derivatization protocol to GC-MSbased urinary metabonomic studies.…”

Section: Introductionmentioning

confidence: 99%

GC-MS with ethyl chloroformate derivatization for comprehensive analysis of metabolites in serum and its application to human uremia

et al. 2008

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Abstract:An optimized method based on GC-MS with ethyl chloroformate derivatization has been developed for the comprehensive analysis of endogenous metabolites in serum. Twenty-two reference standards and serum samples were used to validate the proposed method. The correlation coefficient was higher than 0.9900 for each of the standards, and the LOD varied from 125 to 300 pg on-column. The analytical equipment exhibited good repeatability (RSD<10%) for all of the standards. Both the repeatability and the within-48-h stability of the analytical method were satisfactory (RSD<10%) for the 18 metabolites identified in the serum samples. Mean recovery was acceptable for the 18 metabolites, ranging from 70% to 120% with RSDs of less than 10%. Using the optimized protocol and a subsequent multivariate statistical technique, complete differentiation was achieved between the metabolic profile of uremic patients and that of age-and sex-matched normal subjects. Significantly decreased levels of valine, leucine, and isoleucine and increased levels of myristic acid and linoleic acid were observed in the patient group. This work demonstrated that this method is suitable for serumbased metabolic profiling studies.

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Simple plasma work-up for a fast chromatographic analysis of homocysteine, cysteine, methionine and aromatic amino acids

Cited by 43 publications

References 26 publications

Erythrocyte glutamine depletion, altered redox environment, and pulmonary hypertension in sickle cell disease

Erythrocyte glutamine depletion, altered redox environment, and pulmonary hypertension in sickle cell disease

Organelle redox of CF and CFTR-corrected airway epithelia

GC-MS with ethyl chloroformate derivatization for comprehensive analysis of metabolites in serum and its application to human uremia

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