Carnitine (3-hydroxy-4-N-trimethylaminobutyric acid) is a metabolite essential for the transport of long-chain fatty

metabolite essential for the transport of long-chain fatty acids from the cytosol into the mitochondrial matrix and is an important player in energy production via b-oxidation. Therefore, carnitine depletion causes a failure of ATP production and an accumulation of triglycerides in tissues such as the liver, skeletal muscle and heart. Animal tissues contain relatively large amounts of carnitine with the highest concentrations in heart and skeletal muscle. Although animals obtain carnitine primarily from the diet, carnitine is also synthesized by most mammals but is not degraded in the body. Carnitine homeostasis in mammals is maintained by a modest rate of endogenous synthesis, absorption from dietary sources, efficient reabsorption in the kidney and mechanisms present in most tissues that establish and maintain substantial concentration gradients between intracellular and extracellular carnitine pools. Carnitine is synthesized ultimately from the amino acids lysine and methionine. In some proteins (histones, myosin, calmodulin, and actin), lysine residues are trimethylated on the 4-amino group by specific methyltransferases that use S-adenosyl-L-methionine as the methyl donor (Fig. 1). After lysosomal degradation of these proteins, free 6-Ntrimethyllysine (TML) becomes available for carnitine biosynthesis. Four enzymatic steps are required to synthesize carnitine, and the first and last steps are catalyzed by 6-Ntrimethyllysine dioxygenase (TMLD, EC 1.14.11.8) and gbutyrobetaine dioxygenase (g-BBD, EC 1.14.11.1), respectively. TMLD hydroxylates TML on the 3-position to yield 3hydroxy-TML (HTML), and g-BBD hydroxylates g-butyrobetaine (g-BB) on the 3-position to yield carnitine. Both TMLD and g-BBD are dioxygenases; hydroxylation of their substrates is coupled to the conversion of 2-oxoglutarate and molecular oxygen to succinate and carbon dioxide. In addition, the TMLD protein shows high homology to the g-BBD protein, although they appear to belong to separate subfamilies of the 2-oxoglutarate-dependent dioxygenases. TMLD is associated predominantly with mitochondria, whereas g-BBD is localized in the cytosol. Although g-BBD activity has been detected in kidneys from humans, cats, cows, hamsters, rabbits and Rhesus monkeys at equal or higher levels than that in the liver, the activity was not detectable or detected at very low levels in kidneys from Cebus monkeys, sheep, dogs, guinea pigs, mice and rats, in which g-BBD activity predominates in the liver. In 1962, Lindstedts first showed that g-BBD is stimulated considerably by 2-oxoglutarate and that the enzyme requires molecular oxygen, reduced iron (Fe ) and vitamin C (VC, L-ascorbic acid) for enzyme activity. Therefore, many studies reported the enhancement of g-BBD and TMLD activity upon the addition of VC in a dose-dependent manner using tissue extracts or partially purified enzymes. In the absence of VC, however, g-BBD activity was detected by adding glutathione peroxidase and glutathione (GSH) to the reaction mixture, although this test was not performed for TMLD. To ascertain the necessity of VC for g-BBD and TMLD activity in carnitine biosynthesis, researchers used guinea pigs that, like humans, cannot synthesize VC in vivo. Many reports indicated that carnitine levels, especially in tissues September 2008 1673