Micro-method for the determination of glutathione in human blood

Daniela Giustarini, Paolo Fanti, Elena Matteucci, Ranieri Rossi

Research output: Contribution to journalArticlepeer-review

19 Scopus citations

Abstract

A new procedure is described for the visible-range spectrophotometric analysis of glutathione (GSH) in microvolumes of blood (as low as 0.5. μL) collected by fingerstick. Samples are diluted 1 to 300 (v/v) in a stabilizing solution, followed by determination of haemoglobin concentration and by acid deproteination. GSH is then measured in the clear supernatant by colorimetry using DTNB, i.e., 5,5'-dithio-bis(2-nitrobenzoic acid), in aqueous solution at pH 7.8. The DTNB reagent is prepared and kept at pH 6.2 until just prior its addition, thus avoiding spontaneous decomposition of the reagent. The assay is rapid, easy to adapt to large-scale studies and it avoids artefactual oxidation of GSH, a common methodological shortcoming. The method is precise with 1.7 to 3.4% intra-day relative standard deviation (RSD) and 2.2 to 4.2% inter-day RSD, and accurate with -1.4% to 2.3% intra-day relative error (RE) and -2.8% to 1.6% inter-day RE. GSH is recovered by 97.5 to 100% at all tested concentrations. The new colorimetric micro-method was validated by a reliable previously reported HPLC method. The procedure is suitable for minimally invasive investigation of oxidative stress in peripheral blood.

Original languageEnglish (US)
Pages (from-to)191-194
Number of pages4
JournalJournal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences
Volume964
DOIs
StatePublished - Aug 1 2014

Keywords

  • Blood
  • DTNB
  • Fingerstick
  • Glutathione
  • Oxidative stress

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biochemistry
  • Clinical Biochemistry
  • Cell Biology

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