J. Pilz et al., Measurement of free and bound malondialdehyde in plasma by high-performance liquid chromatography as the 2,4-dinitrophenylhydrazine derivative, J CHROMAT B, 742(2), 2000, pp. 315-325
We established a method for the detection of free and total (free and bound
) malondialdehyde (MDA) in human plasma samples after derivatisation with 2
,4-dinitrophenylhydrazine (DNPH). Free MDA was prepared by perchloric acid
deproteinisation whereas an alkaline hydrolysation step for 30 min at 60 de
grees C was introduced prior to protein precipitation for the determination
of total MDA. Derivatisation was accomplished in 10 min at room temperatur
e subsequently chromatographed by HPLC on a reversed-phase 3 mu m C-18 colu
mn with UV detection (310 nm). The detection limit was 25 pmol/ml for free
and 0.3 nmol/ml for total MDA. The recovery of MDA added to different human
plasma samples was 93.6% (n=11; RSD 7.1%) for the hydrolysation procedure.
In samples from 12 healthy volunteers who underwent a hypoxic treatment (1
3% O-2 for 6 h) we estimated a baseline value of total MDA of 2.16 nmol/ml
(SD 0.29) (ambient air) with a significant increase to 2.92 (nmol/ml, SD 0.
57; P=0.01) after the end of this physiological oxidative stress challenge.
Plasma values of free MDA in these samples were close to our detection lim
it. The presented technique can easily performed with an isocratic HPLC app
aratus and provides highly specific results for MDA as do sophisticated GC-
MS methods. (C) 2000 Elsevier Science B.V. All rights reserved.