ANALYSIS OF ALDEHYDIC LIPID-PEROXIDATION PRODUCTS BY TLC/DENSITOMETRY

We have explored the use of thin-layer chromatography (TLC)/densitometry in both the reflectance and fluorescence mode for quantitation of specific products of lipid peroxidation. Aldehydic peroxidation products were generated by exposure of arachidonic acid to iron and ascorbic acid for 24 hr. Several methods for the quantitative analysis of peroxidation products by TLC/densitometry were compared using two different aldehydespecific derivatizing reagents, namely dinitrophenylhydrazine (DNPH) and cyclohexanedione (CHD). DNPH hydrazones of the arachidonic acid-peroxidation products, upon TLC separation on silica gel, revealed prominent alkanal and hydroxyalkenal bands. Reverse phase high performance liquid chromatography confirmed that the primary alkanal component was hexanal, while the primary hydroxyalkenal was 4-hydroxynonenal. Semiquantitative methods for the direct analysis of these products by TLC/densitometry were worked out based on the use of external hydrazone standards. TLC/densitometry (fluorescence mode) was used to measure CHD adducts of aldehydes by forming the derivatives in the presence of decanal (used as an internal standard) and separating the derivatives by reverse phase TLC. Hexanal-CHD was detectable upon application of 0.5 nanomoles while 4-hydroxynonenal showed a lower response and was detectable with 10 nanomoles. Using appropriate response factors, hexanal and 4-hydroxynonenal were measured in the aldehyde sample from arachidonic acid and results were similar to those obtained by the DNPH method. Similar approaches were used to analyze the peroxidation products of docosahexaenoic acid (24-hr exposure) and of rat liver microsomes exposed to iron for 30 min. The DHA peroxidation products contained extremely low levels of alkanals, while polar aldehydes and hydroxyalkenals were prominent. Formation of alkanals, osazones, hydroxyalkenals and phospholipid aldehydes from iron-exposed microsomes was also demonstrated. Uses and limitations of these methods of aldehyde measurement are discussed.