The use of pholasin as a probe for the determination of plasma total antioxidant capacity.

BACKGROUND AND OBJECTIVES: Total antioxidant capacity (TAC) reflects the capacity of plasma, or other body fluid, to resist oxidation. The aim of the present study was to assess the prognostic value of Pholasin as a probe for the determination of plasma TAC. DESIGN AND METHODS: Plasma samples either oxidised using the free radical generator 2'-azobis-(2-amidinopropane) hydrochloride (AAPH) at 60 degrees C for 180 min or obtained from diabetic (type 1 and type 2) patients (n = 61 and 124, respectively) with or without polyneuropathy (PN) and/or cardiovascular autonomic neuropathy (CAN) and control subjects (n = 70) were analysed for TAC status. TAC was assessed using two versions of Analysis By Emitted Light (ABEL) tests including quenching of Pholasin chemiluminescence (QPC) with peroxynitrite (ONOO(-)-QPC) and with superoxide anion (O2(-)-QPC).
RESULTS: The utilisation of AAPH to induce peroxyl radical mediated plasma oxidation produced a significant decrease in TAC as assessed by ONOO(-)-QPC and O2(-)-QPC assays in a time-dependent manner. Type 1 and type 2 diabetic patients without PN and/or CAN had lower TAC (ONOO(-)-QPC and O2(-)-QPC) than in control subjects. Further alterations in TAC were noted in the presence of PN and/or CAN. Correlations were found between TAC (ONOO(-)-QPC and O2(-)-QPC) values on duration of diabetes and neurological impairment score-lower limb (NIS-LL) in type 1 diabetic patients.
CONCLUSIONS: This study has established that the ONOO(-)-QPC and O2(-)-QPC versions of the ABEL test fulfil the criteria in terms of simplicity, sensitivity and reliability for the measurement of plasma TAC. These biomarkers may prove useful in studies evaluating the impact of therapeutic drugs or antioxidant interventions aimed at delaying the onset of complications in clinical conditions associated with oxidative stress.