Lipoperoxides in plasma as measured by liquid-chromatographic separation of malondialdehyde-thiobarbituric acid adduct.

TLDR

The assay measures plasma lipoperoxides by hydrolyzing samples in dilute H₃PO₄ at 100 °C, complexing the resulting malondialdehyde with thiobarbituric acid, precipitating proteins with methanol, separating the protein‑free extract on a C18 column, and quantifying the MDA‑TBA adduct spectrophotometrically at 532 nm. The method detects as little as 0.15 µmol MDA L⁻¹, exhibits 8–13 % run‑to‑run CV, achieves 98 % recovery, measures average lipoperoxide concentrations of 0.60 µmol L⁻¹ in healthy humans and 1.4 µmol L⁻¹ in control rats, and shows 1.5–2.3 × higher levels in rats 1–3 days after subcutaneous NiCl₂ at 250–750 µmol kg⁻¹.

Abstract

This assay of plasma lipoperoxides involves hydrolysis in dilute H3PO4 at 100 degrees C; complexation of malondialdehyde (MDA), a hydrolysis product, with thiobarbituric acid (TBA); methanol precipitation of plasma proteins; fractionation of the protein-free extract on a C18 column; and spectrophotometric quantification of the MDA-TBA adduct at 532 nm. The detection limit was 0.15 mumol of MDA per liter of plasma. Run-to-run precision (CV) averaged 8 to 13%. Analytical recovery of MDA after addition of tetraethoxypropane standards to 21 specimens of human or rat plasma averaged 98% (SD 7%). Lipoperoxide concentrations (as MDA) averaged 0.60 (SD 0.13) mumol/L in plasma specimens from 41 healthy persons and 1.4 (SD 0.3) mumol/L in plasma specimens from 12 control rats. Mean lipoperoxide concentrations were 1.5 to 2.3 times as great in plasma sampled from rats one to three days after subcutaneous administration of NiCl2 at dosages (250 to 750 mumol per kilogram body wt) previously shown to induce lipid peroxidation in lung, liver, and kidney.