Estimation of acyldihydroxyacetone phosphate and lysophosphatidate in animal tissues.
Chemical and enzymatic methods have been developed to measure small quantities (10(-8) - 10(-10) mol) of acyldihydroxyacetone phosphate in animal tissues. Lipids extracted from tissue samples with acidic CHCl3/methanol were subjected to solvent partitioning at two different pH values for partial purification of this keto-lipid from other lipids. This lipid was then estimated radiometrically either by chemical reduction with NaB3H4 or by enzymatic reduction with [4B-3H]NADPH using a partially purified acyldihydroxyacetone-phosphate reductase (EC 1.1.1.101). Thin-layer chromatography revealed the presence of a number of 3H-labeled lipids in the NaB3H4-reduced product and further purification of the product was necessary to estimate the amount of acyl[2-3H]glycerol 3-phosphate formed. The enzymatic reduction was very specific for acyl/alkyldihydroxyacetone phosphate. The amounts (nmol/g) of these keto-lipids estimated in different tissues by the enzymatic method were 10.06 +/- 0.64 (guinea pig liver), 4.3 +/- 0.15 (rat liver), 2.1 (rat testis), 1.5 (rad kidney) and 1.2 (rat brain). Monoacylglycerol 3-phosphate, i.e., lysophosphatidic acid, which was co-purified with acyldihydroxyacetone phosphate, was found to be present in relatively larger amounts in tissues. The amounts (nmol/g) of this lipid, estimated by enzymatically measuring the amounts of sn-glycerol 3-phosphate released after alkaline methanolysis of the partially purified lipid extracts, were 143 (guinea pig liver), 58 (rat liver), 53 (rat kidney) and 92 (rat brain). Stearic acid (18:0) was found to be the major (65%) fatty acid present in the lysophosphatidate purified from guinea pig liver.
D000818:Animals; D001894:Borohydrides; D004099:Dihydroxyacetone Phosphate / Q000031:analogs & derivatives / Q000032:analysis*; D006168:Guinea Pigs; D007700:Kinetics; D008099:Liver / Q000032:analysis / Q000201:enzymology; D008246:Lysophospholipids; D008930:Mitochondria, Liver / Q000032:analysis; D010712:Phosphatidic Acids / Q000032:analysis*; D011865:Radioisotope Dilution Technique; D012995:Solubility; D013401:Sugar Alcohol Dehydrogenases / Q000302:isolation & purification; D014306:Trioses / Q000032:analysis*; D014316:Tritium
D001894:Borohydrides; D008246:Lysophospholipids; D010712:Phosphatidic Acids; D014306:Trioses; D014316:Tritium; C003037:palmitoyl dihydroxyacetone phosphate; D004099:Dihydroxyacetone Phosphate; C025364:sodium borohydride; D013401:Sugar Alcohol Dehydrogenases; C022312:acylglycerone-phosphate reductase