CARNITINE METABOLISM & RELEVANT HYDROXYLATIONS

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Carnitine is important in normal oxidation of fatty acids for energy. It is provided to mammals from animal protein foods and by de novo synthesis from lysine incorporated into tissue proteins that then is trimethylated. Such proteins are degraded so that tissues including liver, heart, kidney and especially skeletal muscle have free intracellular trimethyllysine that can be converted to carnitine in several enzymatic steps including two hydroxylations catalyzed by Alpha-ketoglutarate-dependent, ascorbate-requiring dioxygenases. The liver is the major organ for final synthesis of carnitine by hydroxylation of Gamma-butyrobetaine. Carnitine levels appear to increase in various ketotic and incipient ketotic states such as diabetes and starvation; this remains to be firmly established, especially whether the increase is due to increased synthesis. We are engaged in a long-term study of metabolism and functions of carnitine, including synthesis, regulation of its synthesis and use, and degradation by intestinal bacteria. Here we propose, by studies using isolated perfused livers of rats, to learn whether increased synthesis occurs when the donor animals have been made diabetic by alloxan or streptozotocin, have been starved, or have received glucagon or the hypolipidemic drug, clofibrate. We then shall study capacities of homogenates of various tissues of normal and above animals to convert trimethyllysine and subsequent intermediates to Gamma-butyrobetaine or, in the liver to carnitine, we shall infer inter-organ dependencies in carnitine biosynthesis. Using isolated perfused livers from ascorbate-deficient guinea pigs, we shall determine whether ascorbate deficiency affects one or both hydroxylases in synthesis of carnitine. Then we shall study the mechanism of hydroxylation of Gamma-butyrobetaine to carnitine at a molecular level, selecting from amoung four proposed mechanisms. For that purpose we shall determine whether the reaction is stereospecific or steroeselective, and measure kinetic isotope effects. Finally, we outline methods to establish pathways of degradation of carnitine to trimethylamine and other products to be identified; the bacteria Ps. putida and Ac. calcoaceticus will be studied. We shall purify the enzyme(s) involved and study mechanisms of action. These organisms may serve as models for further study of truley enteric organisms of the human that could metabolize dietary carnitine and Gamma-butyrobetaine.