Briefly, plasma stabilized and deproteinized with 60 g/L metaphosphoric acid
was centrifuged, and an aliquot of the supernatant was injected on a Cl8 reversed-phase analytical column.
The GSH and GSSG calibrators were prepared quantitatively, 40 [micro]mol/L each, in 10 g/L metaphosphoric acid.
Immediately after blood sampling, samples were put on ice and treated by metaphosphoric acid, an approach described as among the most reliable for glutathione storage (2, 20).
Perchloric acid was chosen because metaphosphoric acid
was too weak and very unstable, and residual sulfosalicylate remaining after neutralization by alkali tended to hinder the following enzymatic process.
Commonly used acids include metaphosphoric acid and trichloroacetic acid [reviewed by Washko et al.
When deproteinization was required, one-half volume of 200 g/L trichloroacetic acid solution, 3 mol/L perchloric acid solution, or 200 g/L metaphosphoric acid solution was added to one volume of plasma, and the mixture was stirred vigorously.
Furthermore, the method was applied to the deproteinized plasma samples by acids including metaphosphoric acid, trichloroacetic acid, and perchloric acid.
Cupric ions did not act as a catalyst at all, whereas ferric ions accelerated the oxidation of AsA to DHA in trichloroacetic acid and perchloric acid solutions but not in metaphosphoric acid solution.
Furthermore, one volume of 5 g/L transferrin (human holo-transferrin, Sigma) solution containing 100 [micro]mol/L AsA was mixed with one-half volume of 200 g/L metaphosphoric acid, 200 g/L trichloroacetic acid, or 3 mol/L perchloric acid.
Albumin (human, fraction V), 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), nitroblue tetrazolium, sodium dodecyl sulfate (SDS), SOD (from human erythrocytes), 1,1,3,3-tetraethoxypropane, thiobarbituric acid (TBA), and xanthine were purchased from Sigma; phenylhydrazine hydrochloride, metaphosphoric acid
(MPA), phosphotungstic acid, and sodium azide were from Riedel; n-butanol, Tris, CUS[O.
Metaphosphoric acid (MPA) was purchased from Aldrich Chemical Co.
1) Nonstandard abbreviations: AA, ascorbic acid; DHAA, dehydroascorbic acid; DTT, dithiothreitol; OPDA, o-phenylenediamine; and MPA, metaphosphoric acid .