32] Synthesis of radioactive (−)-carnitine from γ-aminobutyrate

Abstract

This chapter discusses the Synthesis of Radioactive (—)-carnitine from γ-aminobutyrat. γ-Aminobutyrate is methylated to γ-butyrobetaine using radioactive methyl iodide under conditions giving high yields with respect to both these precursors. γ-Butyrobetaine is then quantitatively converted to (—)-carnitine using a 50–60% ammonium sulfate fraction of rat liver supernatant as the source of γ-butyrobetaine hydroxylase. The product is purified by column chromatography on a cation exchanger and is then desalted by passing through a column of ion-retardation resin. A 50–60% ammonium sulfate fraction of rat liver supernatant is employed and is obtained as follows: thirty percent (w/v) homogenate of liver from adult rat is prepared in 210 m M mannitol, 70 m M sucrose, 0.1 mM EDTA, 10 m M Tris–HCl (pH 7.4), 0.1 m M dithiothreitol using a Potter–Elvehjem homogenizer and centrifuged at 17,000 g for 10 min. All operations are carried out at 0–4°. The assay procedure involves the methylation of γ-aminobutyrate to γ-butyrobetaine, conversion of γ-butyrobetaine to (—)-carnitine, and characterization of radioactive (—)-carnitine. The ability of γ-butyrobetaine hydroxylase to hydroxylate γ-butyrobetaine stereospecifically ensures a high stereochemical purity of the product synthesized by the method presented in the chapter; conversely in the procedures that employ a β -hydroxy intermediate as the methyl group acceptor, the stereochemical purity of the methylated product is limited by that of the methyl group acceptor employed. Methyl-labeled radioactive (—)-carnitine can also be prepared by the method which involves demethylation of commercial (—)-carnitine to obtain (—)dimethylamino- β -hydroxybutyrate which is then methylated back to (—)-carnitine using radioactive methyl iodide.