Measurement of biological thiols and disulfides by high-performance liquid chromatography and electrochemical detection of silver mercaptide formation

Kuninori, Toyo; Nishiyama, Junko

doi:10.1016/0003-2697(91)90349-x

Cited by 32 publications

(15 citation statements)

References 20 publications

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“…By high-performance capillary electrophoresis analysis, no GSSG was detectable. Another study that analyzed GSH and GSSG levels of normal human red blood cells by HPLC and electrochemical detection reported a ratio of 297:1 (30). The uncertainty of the least squares fitting methodology is ϳ5% and hence the detectability of GSSG in excess GSH by using XAS is 5:100.…”

Section: Resultsmentioning

confidence: 99%

“…Several procedures for the determination of GSH and GSSG from different biological sources have been reported in the literature, but all require extraction procedures for the determination of glutathione in biological materials. An in situ determination is not possible with chemical (25) and enzymatic (26)(27)(28) methods, HPLC (29)(30)(31)(32)(33)(34)(35)(36)(37)(38), flow cytometry (39)(40)(41) and, more recently, capillary electrophoresis (42,43). GSH and GSSG can be detected by using spectrophotometric (44,45), spectrofluorimetric (46)(47)(48)(49)(50)(51)(52), and electrochemical methods (30,34,37,(53)(54)(55)(56)(57)(58) with levels of sensitivity ranging from nanomolar (33,35) to picomolar (31,32).…”

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confidence: 99%

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Sulfur K-edge x-ray absorption spectroscopy: A spectroscopic tool to examine the redox state of S-containing metabolites in vivo

Rompel

Cinco

Latimer

et al. 1998

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

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The sulfur K-edge x-ray absorption spectra for the amino acids cysteine and methionine and their corresponding oxidized forms cystine and methionine sulfoxide are presented. Distinct differences in the shape of the edge and the inf lection point energy for cysteine and cystine are observed. For methionine sulfoxide the inf lection point energy is 2.8 eV higher compared with methionine. Glutathione, the most abundant thiol in animal cells, also has been investigated. The x-ray absorption near-edge structure spectrum of reduced glutathione resembles that of cysteine, whereas the spectrum of oxidized glutathione resembles that of cystine. The characteristic differences between the thiol and disulfide spectra enable one to determine the redox status (thiol to disulfide ratio) in intact biological systems, such as unbroken cells, where glutathione and cyst(e)ine are the two major sulfurcontaining components. The sulfur K-edge spectra for whole human blood, plasma, and erythrocytes are shown. The erythrocyte sulfur K-edge spectrum is similar to that of fully reduced glutathione. Simulation of the plasma spectrum indicated 32% thiol and 68% disulfide sulfur. The whole blood spectrum can be simulated by a combination of 46% disulfide and 54% thiol sulfur.This paper describes an application of sulfur K-edge x-ray absorption spectroscopy (XAS) to determine the ratio of thiol to disulfide in biological systems. Sulfur is an important element in chemistry and biology, and often it is desirable to assay thiols and disulfide and their ratio in biological samples. There are no spectroscopic techniques available that can distinguish between thiol and disulfide sulfur or sulfur in higher formal oxidation states. This is not surprising, because there are no significant optical markers for sulfur. Also, only one sulfur isotope of Ͻ1% natural abundance has a nuclear magnetic moment; but this isotope possesses an unfavorable nuclear spin with a small magnetic moment and a large quadrupole moment, making NMR unattractive for sulfur analysis.Sulfur K-edge XAS has been shown to exhibit a strong correlation between oxidation state and inflection point energy (IPE) and to have a rich x-ray absorption near-edge structure (XANES) that is related to chemical structure (1-4) and can be used to distinguish between thiol and disulfide sulfur.The two sulfur-containing amino acids found in all cells are cysteine and methionine. Cysteine can be oxidized to the disulfide form cystine. Additionally there are ''mixed'' disulfides (cysteine bound to proteins by formation of a disulfide linkage). Methionine can be reversibly oxidized by HO ⅐ , 1

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Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

Sulfur K-edge x-ray absorption spectroscopy: A spectroscopic tool to examine the redox state of S-containing metabolites in vivo

Rompel

Cinco

Latimer

et al. 1998

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

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“…Another study that analyzed GSH and GSSG levels of normal human red blood cells by HPLC and electrochemical detection reported a ratio of 297:1. 211 The uncertainty of the least squares fitting methodology is ~ 5% and hence the detectability of GSSG in excess GSH using XAS is 5:100. …”

Section: Sulfur-containing Amino Acidsmentioning

confidence: 99%

“…The level of oxidized to reduced glutathione can change significantly upon oxidative stress and provides useful information about the redox and detoxification status of cells and tissues. [202][203][204][205] Several 211,215,218,[232][233][234][235][236] with levels of sensitivity ranging from nmol 214,216 to pmol. 212,213 For spectrophotometric detection glutathione extracts are treated with iodoacetate to block free thiol groups and then with Sanger´s reagent (as one example) to produce a N-dinitrophenyl derivative that is analyzable.…”

Section: Introductionmentioning

confidence: 99%

X-ray absorption spectroscopy on the calcium cofactor to the manganese cluster in photosynthetic oxygen evolution

Cinco¹

1999

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PS II). To further test and verify whetherCa is close to the Mn cluster, we substituted strontium for Ca and probed from the Sr point of view for any nearby Mn. The extended X-ray absorption fine structure (EXAFS) of Sr-reactivated PS II indicates major differences between the intact and NH 2 OH-treated samples. In intact samples, the Fourier transform of the Sr EXAFS shows a Fourier peak that is missing in inactive samples. This peak II is best simulated by two Mn neighbors at a distance of 3.5 Å, confirming the proximity of Ca (Sr) cofactor to the Mn cluster. In addition, polarized Sr EXAFS on oriented Sr-reactivated samples shows this peak II is dichroic: large magnitude at 10° (angle between the PS II membrane normal and the x-ray electric field vector) and small at 80°. Analysis of the dichroism yields the relative angle between the Sr-Mn vector and membrane normal (23° ± 4°), and the isotropic coordination number for these layered samples.ii X-ray absorption spectroscopy has also been employed to assess the degree of similarity between the manganese cluster in PS II and a family of synthetic manganese complexes containing the distorted cubane [Mn 4 O 3 X] core (X = benzoate, acetate, methoxide, hydroxide, azide, fluoride, chloride or bromide). In addition, Mn 4 O 3 Cl complexes containing three or six terminal Cl ligands at three of the Mn were included in this study. The EXAFS method detects the small changes in the core structures as X is varied in this series, and serves to exclude these distorted cubanes of C 3v symmetry as a topological model for the Mn catalytic cluster.The sulfur K-edge x-ray absorption near-edge structure (XANES) spectra for the amino acids cysteine, methionine, their corresponding oxidized forms cystine and methionine sulfoxide, and glutathione show distinct differences between the thiol and disulfide forms. Sulfur XANES is also used to detect changes (within 5%) of the thiol-to-disulfide ratio in whole human blood, plasma, and erythrocytes.iii To my parents, and to my brothers Raoul and Dannyi v

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“…Unfortunately, this is a complicated and time-consuming procedure. The HPLC method permits accurate determination by separation of GSH or GSSG from matrix impurities and has been used as a specific procedure for the quantitation of GSH or GSSG, applying alternative detection systems for monitoring signal responses, such as, precolumn derivatization with UV-visible [12][13][14], fluorescence [15][16][17] detection or electrochemical detection [18][19][20][21]. However, most HPLC methods remain difficult because of the complex sampling procedure or troublesome equipment maintenance.…”

Section: Introductionmentioning

confidence: 99%

Determination of reduced-form glutathione and total glutathione in blood and plasma by high performance liquid chromatography with on-column fluorescence derivatization

1998

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SummaryAn on-column fluorometric derivatization method has been developed for the determination of reduced-form glutathione and total glutathione in blood and plasma by high-performance liquid chromatography. Blood hemolysate or diluted plasma was deproteinized with perchloric acid. For total glutathione determination, any oxidized forms of glutathione in samples were converted to reduced-form glutathione with dithiothreitol prior to protein precipitation. Deproteinized samples were injected without pretreatment. Glutathione was fluorescence-derivatized and separated on a C18-bonded vinyl alcohol polymer column with ortho-phthalaldehyde in sodium borate buffer-acetonitrile, as mobile phase; the ortho-phthataldehyde-glutathione, fluorescent isoindole adduct is measured by its fluorescence response. Due to the high sensitivity, selectivity reproducibility and the simple HPLC system, the method is particularly suitable for routine assay of numerous or small amounts of samples.

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Measurement of biological thiols and disulfides by high-performance liquid chromatography and electrochemical detection of silver mercaptide formation

Cited by 32 publications

References 20 publications

Sulfur K-edge x-ray absorption spectroscopy: A spectroscopic tool to examine the redox state of S-containing metabolites in vivo

Sulfur K-edge x-ray absorption spectroscopy: A spectroscopic tool to examine the redox state of S-containing metabolites in vivo

X-ray absorption spectroscopy on the calcium cofactor to the manganese cluster in photosynthetic oxygen evolution

Determination of reduced-form glutathione and total glutathione in blood and plasma by high performance liquid chromatography with on-column fluorescence derivatization

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