High-performance liquid chromatographic separation of malondialdehyde-thiobarbituric acid adduct in biological materials (plasma and human cells) using a commercially available reagent.
The assay of malondialdehyde (MDA) is widely used in clinical chemistry laboratories to investigate lipid peroxidation in oxidative pathologies. In the present work, the thiobarbituric acid (TBA) reaction was carried out on plasma, human erythrocytes and fibroblasts. The reagents used were those of the fluorimetry MDA kit manufactured by Sobioda. We have defined the application of this kit to high-performance liquid chromatography. This adaptation satisfied the criteria of good analytical practice. The detection limit was 2.5 pmol per injection. The retention time of the MDA-TBA2 peak (4.96 +/- 0.07 min) led to excellent resolution of the complex. The within-assay (6-12%) and between-assay (11-12%) precisions were satisfactory. The analytical recovery of MDA after spiking samples of human plasma with tetraethoxypropane standards varied from 70 to 100%. The mean lipoperoxide concentration determined in 32 healthy adults (20-40 years) was 1.04 +/- 0.23 mumol l-1 in plasma. Applied to the erythrocytes of fifteen laboratory workers, the method furnished physiological values of 0.59 +/- 0.21 mumol l-1. Concentrations were significantly higher in chronic renal dialysis patients (4.15 +/- 2.35 mumol l-1. The MDA content of fibroblasts cultured in standard medium was 0.38 +/- 0.04 mumol per g of protein and increased (5.78 +/- 1.38 mumol per g of protein) if the cells were grown in an iron-enriched medium. This accurate high-performance liquid chromatographic method for detection of MDA is the first one which can be applied to plasma, red blood cells and cultured cells. This technique will prevent false positives and should make inter-laboratory comparisons possible.
Richard
M J
MJ
Laboratoire de Biochimie C, Centre Hospitalier Regional Universitaire, Grenoble, France.
Guiraud
P
P
Meo
J
J
Favier
A
A
D000328:Adult; D000975:Antioxidants; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004912:Erythrocytes / Q000737:chemistry*; D005347:Fibroblasts / Q000737:chemistry; D006801:Humans; D007202:Indicators and Reagents; D007502:Iron Chelating Agents; D015227:Lipid Peroxidation; D008297:Male; D008315:Malondialdehyde / Q000097:blood / Q000302:isolation & purification*; D010084:Oxidation-Reduction; D013050:Spectrometry, Fluorescence; D013858:Thiobarbiturates / Q000302:isolation & purification*
D000975:Antioxidants; D007202:Indicators and Reagents; D007502:Iron Chelating Agents; D013858:Thiobarbiturates; D008315:Malondialdehyde; C029684:thiobarbituric acid