Immediate stabilization of human blood for delayed quantification of endogenous thiols and disulfides

Abstract

Endogenous thiols undergo rapid and reversible oxidation to disulfides when exposed to oxidants and are, therefore, suitable biomarkers of oxidative stress. However, accurate analysis of thiols in blood is frequently compromised by their artifactual oxidation during sample manipulation, which spuriously elevates the disulfide levels. Here, we describe a validated pre-analytical procedure that prevents both artifactual oxidation of thiols during sample manipulation and their oxidative decay for months in biosamples that are stored at -80°C. Addition of N-ethylmaleimide to blood samples from healthy donors was used to stabilize whole blood, red blood cells, platelets and plasma disulfides, whereas addition of citrate buffer followed by dilution of plasma with H2O was used to stabilize plasma thiols. The concentrations of thiols and disulfides were stable in all biosamples for at least 6 months when analyzed by UV/Vis HPLC at regular intervals. Only 3 ml of blood were needed to perform the analyses of thiols and disulfides in the different blood fractions. This pre-analytical procedure is reliable for use in both animal and human prospective studies. Its ease of implementation makes the method suitable for application to multicenter studies where blood samples are collected by different sites and personnel and are shipped to specific specialized laboratories.

Keywords: Blood; Disulfides; Plasma; Platelets; Red blood cells; Thiols.

Figure 1. Stability of GSH and GSSG in whole blood

Blood was treated with NEM immediately after the collection. After 1 min, aliquots of sample were stored at −80°C. At the indicated times samples were deproteinized by addition of TCA and then GSH and GSSG were measured on the clear supernatant by HPLC and GSH recycling method, respectively. n= 5.

Figure 2. Stability of thiols and disulfides in red blood cells

Blood was treated with NEM immediately after the collection. After 1 min, it was divided into 0.5 ml aliquots, washed by saline twice, and stored at −80°C. At the indicated times aliquots of samples were deproteinized by addition of TCA and GSH and GSSG were measured on the clear supernatant by HPLC and GSH recycling method, respectively. The rest of sample was used for HPLC analysis of cytoplasmic GSSP and for membrane S-glutathionylated proteins (MGP) n= 5. Values of GSH are expressed as nmoles/mg Hb; values of GSSG, GSSP, MGP are expressed as pmoles/mg Hb.

Figure 3. Stability of thiols and disulfides in platelets

Blood was treated with NEM immediately after the collection. After 1 minute, PRP was separated, divided into 0.5 ml aliquots and then deprived of plasma. At the indicated times sample was treated with TCA. GSH and GSSG were measured on the clear supernatant by HPLC and GSH recycling method, respectively. The protein pellet was used for measurement of GSSP by HPLC. n= 5.

Figure 4. Stability of thiols and disulfides in plasma

Blood was treated with citrate buffer, plasma was then separated, treated again with citrate buffer, diluted with H₂O and stored at −80°C for thiol analyses. Disulfides were measured on plasma-NEM obtained by RBC purification and stored at −80°C. At the indicated times protein thiols were measured by colorimetric conjugation with DTNB, LMM-SH (panel A), LMM-SS (panel B) and RSSP (panel C) were measured by HPLC analysis with fluorometric detection. n= 5.

Figure 4. Stability of thiols and disulfides in plasma

Blood was treated with citrate buffer, plasma was then separated, treated again with citrate buffer, diluted with H₂O and stored at −80°C for thiol analyses. Disulfides were measured on plasma-NEM obtained by RBC purification and stored at −80°C. At the indicated times protein thiols were measured by colorimetric conjugation with DTNB, LMM-SH (panel A), LMM-SS (panel B) and RSSP (panel C) were measured by HPLC analysis with fluorometric detection. n= 5.

Scheme 1. Suggested procedure for thiol and disulfide analysis in whole blood, red blood cells, plasma and platelets

Whole blood, red blood cell and platelet thiols and disulfides are stabilized by treatment of blood with NEM immediately after the collection. Red blood cells and platelets are then separated and either analyzed or stored at −80°C until analysis. The plasma samples obtained by purification of RBCs, and therefore already treated with NEM, are used for disulfide analyses. Plasma thiols are stabilized by treatment of whole blood with citrate buffer solution. Once plasma is separated, it can be either freshly measured or further diluted with H₂O and citrate buffer before its storage at −80°C.