Abstract
Free carnitine, acid-soluble total carnitine, and long-chain acylcarnitines in fish tissues were determined by high-performance liquid chromatography (HPLC) equipped with a Cosmosil 5C8 or 5C18-AR column. Free and acid-soluble total carnitine was extracted from such tissues as ordinary muscle, liver, kidney, heart tissue, and plasma of rainbow trout with 6% perchloric acid containing 20mM 2-mercaptoethanol. Acetylcarnitine and short-chain acylcarnitines (C3-C10) in the extract were hydrolyzed to free carnitine by alkaline to analyze acid-soluble total carnitine. Long-chain acylcarnitines (C12<) in the tissues were extracted from the precipitated residue of the free and acid-soluble total carnitine extract and hydrolyzed to free carnitine by alkaline. Coenzyme A (CoASH) was formed stoichiometrically from carnitine and acetylcoenzyme A by carnitine acetyltransferase. The formed CoASH was determined by HPLC. CoASH was successfully isolated from other compounds by each column. The standard curve for carnitine vs the peak area of the CoASH was clearly in proportion from 0.1 μM to 100 μM. Recovery of free carnitine added to the homogenate of ordinary muscle tissue from rainbow trout was 98.9±4.7% (1 μmol/g added) and 99.8±6.2% (0.05 μmol/g added).
