Optimized steps in fluorometric determination of thiobarbituric acid-reactive substances in serum: importance of extraction pH and influence of sample preservation and storage.

A simple, reliable, and reproducible fluorometric method for measuring thiobarbituric acid-reactive substances (TBARS) in serum is proposed, based on the reaction between malondialdehyde (MDA) and thiobarbituric acid. Formation of TBARS was complete at pH 2.4-2.6, but extraction with n-butanol proved complete only at lower pH, i.e., 1.6-1.7. Analytical recoveries of MDA added to serum were 94%-101%; within- and between-run CVs were 2.4-3.6% and 4.6-5.5%; and the detection limit for TBARS in serum was 0.10 mumol/L. Optimized conditions included: (a) collection of either serum or heparinized plasma, (b) preservation from in vitro autoxidation by glutathione and EDTA, and (c) storage at -20 degrees C up to 35 days. The mean (+/- SD) TBARS concentration in 47 healthy adults was 1.01 (0.21) mumol/L; no sex-related difference was observed. Higher concentrations were measured in patients with renal insufficiency undergoing hemodialysis and in patients with insulin-dependent diabetes, chronic pancreatitis, or liver cirrhosis.