Reaction conditions affecting the relationship between thiobarbituric acidreactivity and lipid peroxides in human plasma

The thiobarbituric acid (TBA) reactivity of human plasma was studied to eva luate its adequacy in quantifying lipid peroxidation as an index of systemi c oxidative stress. Two spectrophotometric TBA tests based on the use of ei ther phosphoric acid (pH 2.0, method A) or trichloroacetic plus hydrochlori c acid (pH 0.9, method B) were employed with and without sodium sulfate (SS ) to inhibit sialic acid (SA) reactivity with TBA. To correct for backgroun d absorption, the absorbance values at 572 nm were subtracted from those at 532 nm, which represent the absorption maximum of the TBA:MDA adduct. Meth od B gave values of TBA-reactive substances (TBARS) 2-fold higher than thos e detected with method A. SS lowered TBARS by about 50% with both methods, indicating a significant involvement of SA in plasma TBA reactivity. Standa rd SA, at a physiologically relevant concentration of 1.5 mM, reacted with TBA, creating interference problems, which were substantially eliminated by SS plus correction for background absorbance. When method B was carried ou t in the lipid and protein fraction of plasma, SS inhibited by 65% TBARS fo rmation only in the latter. Protein TBARS may be largely ascribed to SA-con taining glycoproteins and, to a minor extent, protein-bound MDA. Indeed, ED TA did not affect protein TBARS assessed in the presence of SS. TBA reactiv ity of whole plasma and of its lipid fraction was instead inhibited by EDTA , suggesting that lipoperoxides (and possibly monofunctional lipoperoxidati on aldehydes) are involved as MDA precursors in the TBA test. Pretreatment of plasma with KI, a specific reductant of hydroperoxides, decreased TBARS by about 27%. Moreover, aspirin administration to humans to inhibit prostag landin endoperoxide generation reduced plasma TBARS by 40%. In conclusion, reaction conditions affect the relationship between TBA reactivity and lipi d peroxidation in human plasma. After correction for the interfering effect s of SA in the TBA test, 40% of plasma TBARS appears related to in vivo gen erated prostaglandin endoperoxides and only about 60% to lipoperoxidation p roducts. Thus, the TBA test is not totally specific to oxidant-driven lipid peroxidation in human plasma. (C) 2001 Elsevier Science Inc.