High-performance liquid chromatography of coenzyme A esters formed by transesterification of short-chain acylcarnitines: diagnosis of acidemias by urinary analysis.
A protocol for the identification and estimation of short-chain esters of carnitine is described; it is useful for the diagnosis of acidemias. By this method, carnitine esters in urine are converted to coenzyme A esters enzymatically with carnitine acetyltransferase (CAT): short-chain acylcarnitine + CoA cat in equilibrium short-chain acyl-CoA + carnitine. The coenzyme A esters are separated by high-performance liquid chromatography using a radial compression system with a C8 Radial-Pak cartridge and a mobile phase containing 0.025 M tetraethylammonium phosphate in a linear gradient of 1 to 50% methanol. Coenzyme A esters are quantitated by integrator determination of the area under the 254-nm absorption peaks. Enzymatic conversion approaches 100% for acetyl and propionyl esters except in the presence of high levels of free carnitine, which lowers the proportion of ester as acyl-CoA at equilibrium. However, since acidemia patients produce urine low in free carnitine, this problem is minimized. The method is rapid and simple and identifies propionic, methylmalonic, and isovaleric acidemias.
D000138:Acidosis / Q000175:diagnosis / Q000652:urine*; D002331:Carnitine / Q000031:analogs & derivatives* / Q000652:urine; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003065:Coenzyme A / Q000031:analogs & derivatives* / Q000032:analysis; D004951:Esterification; D006801:Humans
D002331:Carnitine; D003065:Coenzyme A