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 thiobarbituri c 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. Analyt ical recoveries of MDA added to serum were 94%-101%; within- and betwe en-run CVs were 2.4-3.6% and 4.6-5.5%; and the detection limit for TBA RS in serum was 0.10 mu mol/L. Optimized conditions included: (a) coll ection of either serum or heparinized plasma, (b) preservation from in vitro autoxidation by glutathione and EDTA, and (c) storage at -20 de grees C up to 35 days. The mean (+/-SD) TBARS concentration in 47 heal thy adults was 1.01 (0.21) mu mol/L; no sex-related difference was obs erved. Higher concentrations were measured in patients with renal insu fficiency undergoing hemodialysis and in patients with insulin-depende nt diabetes, chronic pancreatitis, or liver cirrhosis.