Determination of in vivo protein binding of homocysteine and its relation to free homocysteine in the liver and other tissues of the rat.

Low concentrations (0.5-6 nmol/g) of homocysteine (Hcy) have recently been demonstrated in acid extracts of various tissues of the mouse and rat (Ueland, P.M., Helland, S., Broch, O.-J., and Schanche, J.-S. (1984) J. Biol. Chem. 259, 2360-2364). This is referred to as free Hcy in tissues. This paper describes a method for the determination of protein-bound Hcy, which involves precipitation and washing of tissue protein with ammonium sulfate, release of Hcy from native proteins in the presence of dithioerythritol, and determination of free Hcy by a sensitive radioenzymic assay. Both free and bound Hcy decreased markedly in rat tissues within a few seconds following death of the animal. The amount of protein-bound Hcy was highest in liver, somewhat lower in kidney, brain, heart, lung, and spleen. The ratio between free and bound Hcy was between 1 and 2 in most tissues, except in cerebellum, containing a large excess of free Hcy (free/bound ratio of 18). Free Hcy was almost exclusively localized to the soluble fraction of rat liver, whereas protein-bound Hcy was about equally distributed between this fraction and the microsomes. Isolated rat hepatocytes contained free and protein-bound Hcy in proportions observed in whole liver, but a large amount of Hcy was exported into the extracellular medium. The half-lives, as determined from pulse-chase experiments with [35S] methionine, were 53 s for S-adenosylmethionine, 2 s for S-adenosylhomocysteine and 3 s for Hcy (free and bound regarded as a single pool). Furthermore, isotope equilibrium between these metabolites and between free and bound Hcy throughout the rapid chase period suggests the turnover rates of S-adenosylhomocysteine and Hcy to be production rate limited, and the dissociation rate of the Hcy-protein complex may greatly exceed the turnover rate of Hcy. Thus, the half-lives of Hcy are such that participation of both free and bound Hcy in metabolic regulation is feasible.