The discovery of the genetic defect in the plasma membrane carnitine transporter, OCTN2, first reported in 1988, 1 represents the culmination of a century of research on the role of carnitine in normal physiology and in disease states. During the course of this history, carnitine attracted a great deal of speculation about its possible functions as a vitamin, an essential nutrient, and as a treatment for a variety of metabolic disorders affecting fatty acid oxidation in liver, muscle, and heart. Much of this speculation has not been substantiated, but continues to generate confusion about the role of carnitine deficiency in causing disease and its therapeutic use. This chapter will focus on the role of genetic defects in OCTN2 associated with primary carnitine deficiency. In addition, the chapter will discuss the role of OCTN2 in the mechanism of “secondary carnitine deficiency” associated with several inborn errors of acyl-CoA oxidation.
Carnitine is a small, water soluble molecule whose most important role in humans and other animals is to facilitate the transport of fatty acids across the mitochondrial membrane for production of energy via ß-oxidation. Carnitine was first discovered a century ago by Krimberg in extracts of beef muscle, from which its name derives (Latin caro/carnis for flesh). 2 The chemical formula, C7H15NO3, was soon found and its structure was reported in 1927. Although it was known that large amounts of carnitine were present in muscle tissue, the physiologic function of carnitine remained unknown until Fraenkel and co-workers, in the course of investigating potentially missing B vitamins between 1946 and 1952, showed that carnitine is essential for the growth of a mealworm. 2 They found that Tenebrio molitor mealworm larvae require minute amounts of a water soluble factor, supplied by liver or yeast extracts. Fraenkel suggested this new water soluble factor be named Vitamin BT and subsequently identified it as carnitine. Subsequent work has failed to show that carnitine is a dietary requirement for any other species, including man. This observation is consistent with the fact that most species, other than mealworms, have the capacity for synthesizing carnitine endogenously. Growth of Tenebrio mealworms on extracts of tissue or other biological materials provided the first assay of carnitine content. These assays demonstrated that animal tissues contain very high amounts of carnitine, while it is virtually absent from fruits and vegetables.
The fact that body fat was increased in Fraenkel’s carnitine-deficient mealworms suggested that carnitine might have some role in fat metabolism. In 1955, Fraenkel’s group showed that carnitine could be reversibly acetylated with acetyl-CoA and Fritz reported that carnitine stimulated fatty acid oxidation in liver slices. These observations led to the recognition that carnitine is required to transport fatty acids into mitochondria for oxidation (Fig. 101.1-1). Subsequently, McGarry and Foster showed that, although carnitine participates in the rate-controlling step in fatty acid oxidation (CPT-1), the levels of carnitine do not themselves regulate the rate of fatty acid oxidation. Instead, this process is controlled by the cytosolic concentration of malonyl-CoA, which is not only the first intermediate in fatty acid synthesis, but also is an allosteric inhibitor of CPT-1. 3 Studies of carnitine metabolism demonstrated that it was not catabolized in animals and could be supplied from both dietary sources (meats, milk, eggs, etc.) and endogenous synthesis. The synthetic pathway was elucidated between 1960 and 1980 by several groups, showing that carnitine is synthesized endogenously from trimethyllysine residues released during protein degradation. (Fig. 101.1-2) 4 The free trimethyllysine can be converted to butyrobetaine in multiple tissues and the butyrobetaine is then released to be finally converted to carnitine in liver and kidney (Fig. 101.1-2). Detailed investigations of the enzymes of carnitine biosynthesis have been recently reported by Vaz and Wanders. 5 No human disorders of carnitine biosynthesis or nutritional deficiency have been described to date.
The carnitine cycle in fatty acid oxidation. FATP: Fatty Acid Transporter Protein; FA: Fatty Acid; CPT-1: Carnitine Palmitoyl Transferase-1; CPT-2: Carnitine Palmitoyl Transferase-2; CACT: Carnitine Acyl Carnitine Translocase. Modified from ref. 55
Pathway of carnitine biosynthesis. Modified from ref 44
Between 1965 and 1980, based on the information that carnitine was essential for fatty acid oxidation, many investigators attempted to identify disorders of fat oxidation that could be either caused by, or ameliorated by carnitine therapy. Particular attention was focused on Jamaican vomiting sickness, which is caused by a toxic amino acid in unripe Ackee fruit (Blighia Sapida), Hypoglycin A. It was proposed that Jamaican vomiting sickness was due to sequestration of free Co-enzyme A through the formation of the CoA ester of methylene-cyclopropane-acetic acid (MCPA), the toxic metabolite of Hypoglycin A. 6 In addition, it was hypothesized by Bressler and colleagues that carnitine could reverse the toxic effects of Hypoglycin A by forming acyl-carnitine esters with MCPA and restoring free CoA. This explanation was shown not to be correct by Sherratt and, subsequently, by Tanaka, who demonstrated that MCPA-CoA acts as a “suicide substrate” to irreversibly inhibit several enzymes of fatty acid ß-oxidation, including medium-chain acyl-CoA dehydrogenase, and of amino acid oxidation, including isovaleryl-CoA dehydrogenase. 6
In the 1970s, several investigators identified patients with disorders associated with low carnitine concentrations in tissues or plasma and either attacks of coma and hypoglycemia (“systemic carnitine deficiency”) or muscle weakness and cardiomyopathy (“myopathic carnitine deficiency”). 7 In addition, patients were identified with reduced carnitine levels in plasma or tissue in association with other genetic defects, termed “secondary carnitine deficiency” to contrast with “primary carnitine deficiency” (usually taken as equivalent to “primary systemic carnitine deficiency”). Engel and Rebouche carried out intensive studies of a group of patients with “systemic carnitine deficiency” in the 1970s and 1980s, showing a reduction in renal threshold for carnitine reabsorption, implying that carnitine transport might be very important in regulating body carnitine stores. 8 Interestingly, most of these cases with reduced renal threshold as the basis for their carnitine deficiency probably had “secondary carnitine deficiency” due to medium-chain acyl-CoA dehydrogenase deficiency, or related fatty acid oxidation disorders. (See end of chapter for discussion of the role of the carnitine transporter in secondary carnitine deficiency).
The first cases of “primary carnitine deficiency” associated with genetic defects in the plasma membrane carnitine transporter were described during the late 1980’s. 1,9– 13 These included a child with fasting, hypoketotic hypoglycemia that could be completely corrected by treatment with high-dose oral carnitine.1 Fibroblasts from this child showed impaired uptake of radio-labeled carnitine. Subsequent cases of the disorder were identified that presented with either cardiomyopathy or progressive muscle weakness.9 In addition, several previously described cases of both carnitine-responsive cardiomyopathy and episodic hypoglycemia were subsequently shown to also be due to defects in the plasma membrane transporter.11,14
In the 1990s, a spontaneous animal model of the defect in the plasma membrane carnitine transporter was described, the recessive JVS (for juvenile visceral steatosis) mouse. 15 In 1999, Nezu, et al, identified the SLC22A5 gene as the locus encoding the plasma membrane OCTN2 carnitine transporter in man (and the homologous Slc22a5 gene in jvs mice); they also demonstrated recessive inactivating mutations of this gene in patients with deficiency of the carnitine transporter. 16 Recently, several adult women with deficiency of OCTN2 have been identified who had not previously shown any evidence of the disorder, considerably expanding the clinical phenotype and suggesting that manifestations may be easily missed in some cases. 17