BBRC Biochemical and Biophysical Research Communications 338 (2005) 1966–1972 www.elsevier.com/locate/ybbrc
OCTN3 is a mammalian peroxisomal membrane carnitine transporter q Anne-Marie Lamhonwah a,c, Cameron A. Ackerley b, Aina Tilups b, Vernon D. Edwards b, Ronald J. Wanders d, Ingrid Tein a,c,* a
c
Division of Neurology, Department of Pediatrics, The Hospital for Sick Children, Toronto, Canada b Department of Pathology, The Hospital for Sick Children, Toronto, Canada Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ont., Canada M5G 1X8 d Department of Pediatrics, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands Received 23 October 2005 Available online 10 November 2005
Abstract Carnitine is a zwitterion essential for the b-oxidation of fatty acids. The role of the carnitine system is to maintain homeostasis in the acyl-CoA pools of the cell, keeping the acyl-CoA/CoA pool constant even under conditions of very high acyl-CoA turnover, thereby providing cells with a critical source of free CoA. Carnitine derivatives can be moved across intracellular barriers providing a shuttle mechanism between mitochondria, peroxisomes, and microsomes. We now demonstrate expression and colocalization of mOctn3, the intermediate-affinity carnitine transporter (Km 20 lM), and catalase in murine liver peroxisomes by TEM using immunogold labelled anti-mOctn3 and anti-catalase antibodies. We further demonstrate expression of hOCTN3 in control human cultured skin fibroblasts both by Western blotting and immunostaining analysis using our specific anti-mOctn3 antibody. In contrast with two peroxisomal biogenesis disorders, we show reduced expression of hOCTN3 in human PEX 1 deficient Zellweger fibroblasts in which the uptake of peroxisomal matrix enzymes is impaired but the biosynthesis of peroxisomal membrane proteins is normal, versus a complete absence of hOCTN3 in human PEX 19 deficient Zellweger fibroblasts in which both the uptake of peroxisomal matrix enzymes as well as peroxisomal membranes are deficient. This supports the localization of hOCTN3 to the peroxisomal membrane. Given the impermeability of the peroxisomal membrane and the key role of carnitine in the transport of different chain-shortened products out of peroxisomes, there appears to be a critical need for the intermediate-affinity carnitine/organic cation transporter, OCTN3, on peroxisomal membranes now shown to be expressed in both human and murine peroxisomes. This Octn3 localization is in keeping with the essential role of carnitine in peroxisomal lipid metabolism. Ó 2005 Elsevier Inc. All rights reserved. Keywords: Organic cation; Carnitine transporter; Peroxisomal carnitine transporter OCTN3; PEX 1 and PEX 19 deficient fibroblasts
L-Carnitine is a small, highly polar zwitterion that plays an important role in the transport of long-chain fatty acids across the inner mitochondrial membrane for b-oxidation and ATP generation [1]. The transport of carnitine into the cell is mediated by a high-affinity sodium-dependent
q
Abbreviations: CACT, carnitine acylcarnitine translocase; CoA, coenzyme A; CPT, carnitine palmitoyltransferase; ER, endoplasmic reticulum; GFP, green fluorescent protein; OCT, organic cation transporter; SCD, systemic carnitine deficiency; TEA, tetraethylammonium; YFP, yellow fluorescent protein. * Corresponding author. Fax: +1 416 813 6334. E-mail address:
[email protected] (I. Tein). 0006-291X/$ - see front matter Ó 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.10.170
plasmalemmal carnitine transporter, OCTN2, which has recently been cloned [2,3]. The hOCTN2 cDNA codes for a protein of 557 amino acids and a predicted molecular mass of 63 kDa. We and others have described mutations in the SLC22A5 gene encoding OCTN2 in patients with primary systemic carnitine deficiency (SCD) [4]. SCD presents with progressive, infantile-onset hypertrophic cardiomyopathy, recurrent hypoglycemic hypoketotic encephalopathy, weakness, failure to thrive, and microvesicular steatosis in muscle, heart, and liver, and impaired renal reabsorption of carnitine [5,6]. This formerly lethal autosomal recessive disorder is highly treatable with reversal of the cardiomyopathy, provided there is early interven-
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tion with high dose oral carnitine [5,6]. This high-affinity carnitine transporter is expressed in muscle, heart, kidney, fibroblasts, etc., and has a Km of 2–6 lM for carnitine [6–9]. A low-affinity carnitine transporter with Km P500 lM is expressed in liver, brain, small intestine, epididymis, etc. [10–12]. An intermediate-affinity carnitine transporter has been suggested by kinetic studies in testis, muscle culture, renal tubular cells, and intestinal epithelial cells [8,13–15]. There thus appears to be a family of carnitine transporters with different affinities for carnitine. A subfamily of the organic cation transporter family, namely the organic cation/carnitine OCTN transporters, Octn1, Octn2, and Octn3, has been isolated and characterized in mice [16]. Organic cation transporters function primarily in the elimination of cationic drugs and other xenobiotics in tissues such as kidney, intestine, liver, and placenta by distinct mechanisms that are either dependent on or independent of a sodium gradient, membrane potential or pH [17]. Human OCTN1 and -2 transport tetraethylammonium (TEA) as well as acylcarnitines [2,18,19], suggesting that OCTNs may be important for the transport of xenobiotics and acylated carnitine as well as for carnitine itself. Tamai et al. [16] found that when murine Octn1, Octn2, and Octn3 cDNAs were transfected into HEK293 cells, the cells exhibited transport activity for carnitine and/or the organic cation TEA. The relative uptake activity ratios of carnitine to TEA were 1.78, 11.3, and 746 for Octn1, -2, -3, respectively, suggesting high specificity of Octn3 for carnitine and a significantly lower carnitine transport activity of Octn1. We previously reported the localization of the high-affinity transporter OCTN2 as a plasmalemmal protein using a GFP-hOCTN2 cDNA construct and demonstrated restoration of functional L-[3H]carnitine uptake in human fibroblast and lymphoblastoid cell lines from patients with OCTN2 deficiency [6,20,21]. Recently, we reported the subcellular localization of mOctn3 in peroxisomes [22]. We expressed a GFP-mOctn3 construct in HepG2 cells and demonstrated its localization in human peroxisomes. This localization was confirmed by Western blot of isolated mouse liver peroxisomes using our highly specific anti-mOctn3 and anti-catalase antibodies. Significantly, the Octn3 antibody detected a human protein of the expected size (63 kDa) in HepG2, confirming the existence of a gene in the human genome capable of encoding this protein. We also studied L-[3H]carnitine uptake in HepG2 transfected with a GFP-mOctn3 construct and showed that the expressed Octn3 protein had an intermediate-affinity Km of 20 lM for carnitine. We hypothesized that the hOCTN3 gene, which has not yet been cloned or annotated in the human reference DNA sequence, is located between OCTN2 and OCTN1 in a segment of DNA that is not properly represented in human genome sequence assemblies at 5q31. This region is syntenic to the locus on mouse chromosome 11 that harbors the murine Octn gene transporter family (Octn1, Octn2, and Octn3).
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To further characterize mOctn3 localization in peroxisomes, we now studied the ultrastructural expression and colocalization of mOctn3 and catalase in murine liver by TEM using immunogold labelled anti-mOctn3 and anticatalase antibodies. In order to further determine the precise localization of OCTN3 within human peroxisomes (e.g., peroxisomal membrane or matrix localization), we studied the expression of hOCTN3, by Western blot analysis and immunostaining with our anti-mOctn3 antibody, in human PEX 1 deficient Zellweger fibroblasts in which the uptake of peroxisomal matrix enzymes is impaired but the biosynthesis of peroxisomal membrane proteins is normal and compared it to the expression of hOCTN3 in PEX 19 deficient Zellweger fibroblasts in which both the uptake of peroxisomal matrix enzymes as well as peroxiomal membranes are deficient. Materials and methods Generation of polyclonal antibodies to mouse Octn1, Octn2, and Octn3. Synthetic peptides to the deduced amino acids at the carboxy-termini of each of the three carnitine transporters were prepared with an additional cysteine residue which could be used in the MAP (multiple antigen peptide system). The sequences of the synthetic peptides for mOctn1, mOctn2, and mOctn3 were NH2-CGKKSTVSVDREESPKVLIT-COOH; NH2-CTR MQKDGEESPTVLKSTAF-COOH and NH2-CKESKGNVSRTS RTSEPKGF-COOH, respectively. The polyclonal antibodies were raised in New Zealand white (NZW) male rabbits. These three generated antibodies specifically differentiated between mOctn1, mOctn2, and mOctn3 as shown in our previous studies [22]. Subcellular localization of mOctn3 using transmission electron microscopy of liver and immunogold labelling. Murine tissues were fixed in 4% paraformaldehyde and 0.1% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4. Portions of liver were minced into mm3 pieces and fixed for an additional 4 h. Following a thorough rinse in phosphate buffer, tissues were infused with 2.3 M sucrose for several hours prior to freezing in liquid nitrogen. Some of the samples were then freeze substituted at 85 °C for 48 h in a solution of absolute methanol containing 0.5% uranyl acetate. The samples were warmed slowly to 20 °C. Following several rinses in cold methanol they were infiltrated in Lowicryl HM20 (Marivac Services, Halifax NS) and embedded and polymerized in the cold with UV. Ultrathin sections of liver, approximately 90 nm thick, were cut on a diamond knife and mounted on formvar-coated nickel grids. Grids were then rinsed with PBS containing 0.5% BSA prior to incubation with antibody 629 (rabbit anti-mOctn3 antibody) for 1 h. The grids were then washed several times with PBS/BSA prior to incubation with goat anti rabbit IgG/10 nm gold complexes (Amersham Life Sciences, Illinois, USA) for an additional hour. Following a thorough rinse in PBS followed by distilled water, grids were stained with an aqueous saturated solution of uranyl acetate and lead citrate. In double labelled samples, the same procedures were followed except that following labelling with antibody 629 and 10 nm particles, some of the liver samples were incubated with a murine antibody against catalase (Sigma, Oakville, Canada) for 1 h, rinsing in PBS/BSA, and incubation with goat anti-mouse IgG 3 nm complexes (Amersham Life Sciences, Illinois, USA). Controls included the omission of either one of the primary antibodies or the antibody gold complexes. All of the grids were examined in a JEOL JEM 1230 transmission electron microscope (JEOL USA, Peabody, MA) and images were acquired with a digital camera (AMT, Danvers, MA). Expression of OCTN3 in control human fibroblasts and Zellweger patient fibroblasts by Western blot analysis. Total protein lysates were isolated from cultured skin fibroblasts (control, Pex1-, and Pex19-deficient cells). Electrophoresis of protein lysates (100 lg per cell line) was done on a 4–12% polyacrylamide gel, followed by protein transfer onto a BioTrace
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PVDF membrane (Pall). The blot was probed with a rabbit affinity-purified anti-mOctn3 antibody [22] and HRP-protein A (Bio-Rad) as secondary antibody. ECL chemiluminescent kit (GE Healthcare) and Kodak BioMax MR film were used in the detection system. Expression of OCTN3 in control human fibroblasts and Zellweger patient fibroblasts (Pex1 and Pex19 deficient cells) by immunostaining. Fibroblasts were seeded on glass coverslips and grown overnight in a-MEM + 10% fetal bovine serum at 37 °C and 5% CO2. Cells were fixed in 4% paraformaldehyde in PBS for 15 min at room temperature. After two washings with PBS, cells were permeabilized in 0.2% Triton X-100 in PBS and then incubated in PBS containing 10% carnation milk for 1 h at room temperature. Incubation of cells with rabbit affinity-purified antimOctn3 (1/1000 dilution) was done for 2 h at room temperature, followed by incubation with secondary antibody (Cy3- goat anti-rabbit IgG (Jackson Immunochemicals) at a dilution of 1/200) for 1 h. Cells were washed in PBS and then analyzed using a confocal microscope (Zeiss Axiovert) and LSM software.
Results and discussion In mouse liver, antibody 629 (anti-mouse Octn3 antibody) was found primarily in the peroxisomes and in the cisternae of the rough endoplasmic reticulum (Fig. 1). The peroxisomal localization was confirmed by co-localization experiments with an antibody against catalase. Peroxisomes, many of which had distinguishable crystalline inclusions, were found to contain both catalase and antibody 629. Western blot analysis of the control human cultured skin fibroblasts using our anti-mOctn3 antibody demonstrated strong cross-reactivity, consistent with the expression of peroxisomal hOCTN3 (Fig. 2). Western blot analysis with our anti-mOctn3 antibody of the PEX 1 deficient Zellweger fibroblasts demonstrated presence of a band which was somewhat reduced in intensity, suggesting
Fig. 1. Transmission electron microscopy of murine liver with immunogold labelled anti-murine Octn3 antibodies (629). This panel demonstrates specific colocalization of mOctn3 and catalase to murine liver peroxisomes using gold-labelled anti-mOctn3 antibody (629) (10 nm gold particles as shown by arrows) and gold-labelled anti-catalase antibody (3 nm gold particles). Note the intense labelling of the peroxisomes (arrows). Bar equals 500 nm.
Fig. 2. Western blot analysis of hOCTN3 in normal control human skin fibroblasts and in PEX 1 and PEX 19 deficient fibroblasts using antimurine Octn3 antibody. The blot demonstrates strong cross-reactivity of the anti-murine Octn3 antibody with hOCTN3 in normal control cultured human skin fibroblasts, somewhat reduced reactivity in the PEX 1 deficient Zellweger fibroblasts, and no cross-reactivity with the PEX 19 deficient Zellweger fibroblasts.
reduced numbers of peroxisomes and complete absence of cross-reactivity in the PEX 19 fibroblasts, suggesting complete absence of peroxisomes. Immunostaining of the control human cultured skin fibroblast using our anti-mOctn3 antibody and a Cy3-labelled goat anti-rabbit antibody and viewed under fluorescence microscope demonstrated intracellular localization of hOCTN3 protein (Fig. 3). Immunostaining of the PEX 1 deficient Zellweger fibroblasts demonstrated the presence of hOCTN3 but in reduced amounts, consistent with a reduced number of peroxisomes in contrast to the PEX 19 deficient fibroblasts which showed complete absence of hOCTN3 protein expression, suggesting absence of peroxisomes and consistent with our findings on Western blot analysis. The cell-wide mammalian carnitine system consists of 8 different carnitine acyltransferases (carnitine acetyltransferase, carnitine octanoyltransferase, carnitine palmitoyltransferases I and II, and carnitine acylcarnitine translocase) characterized by kinetic, physical, and localization studies which link at least four intracellular pools of acyl-CoA (cytosolic, mitochondrial, peroxisomal, and endoplasmic reticulum) that supply a multitude of lipid metabolic pathways [23]. The role of the carnitine system is to maintain homeostasis in the acyl-CoA pools of the cell, keeping the acyl-CoA/CoA pool constant even under conditions of very high acyl-CoA turnover and providing cells with a critical source of free coenzyme A (CoA).
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Fig. 3. Immunostaining of hOCTN3 in normal control human skin fibroblasts and in PEX 1 deficient fibroblasts using anti-murine Octn3 antibody and Cy3 goat anti-rabbit antibody with fluorescence microscopy. Note the strong staining of the control fibroblasts in (A) and the present but somewhat reduced staining of the PEX 1 deficient fibroblasts in (B). There was absence of immunostaining of the PEX 19 deficient fibroblasts (data not shown).
Carnitine derivatives can be moved across intracellular barriers thereby providing a shuttle mechanism between mitochondria, peroxisomes, and microsomes for complex lipid-synthetic and -breakdown pathways [23]. Carnitine also has a detoxifying role of trapping potentially toxic acyl-CoA metabolites that may increase during acute metabolic crises [1,12]. In the mitochondria, carnitine serves as an essential cofactor for long-chain fatty acid oxidation by transferring long-chain fatty acids as acylcarnitine esters across the inner mitochondrial membrane for ensuant intramitochondrial b-oxidation. The inner mitochondrial membrane is impermeable to CoA and its derivatives, namely fatty acyl-CoA thioesters formed at the outer mitochondrial membrane. Thus, long-chain acyl-CoA is first converted into its acylcarnitine form, e.g., palmitoylcarnitine, with release of free CoA. This is accomplished by using carnitine as a cofactor by the reversible enzyme carnitine palmitoyltransferase I (CPT I), located on the inner side of the outer mitochondrial membrane [24]. Palmitoylcarnitine is then translocated across the inner mitochondrial membrane by carnitine:acylcarnitine translocase (CACT) [25]. In the matrix, palmitoylcarnitine in the presence of free CoA is then converted back to palmitoyl-CoA and carnitine by CPT II, situated on the inner side of the inner mitochondrial membrane. The recently cloned, sodium-dependent human high-affinity plasmalemmal carnitine transporter, OCTN2 [2,3], has a Km concentration requirement of 2–5 lM for Cn [6] which is easily met by the normal serum carnitine concen-
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trations of 50 lM [26]. We previously demonstrated the expression of the GFP-OCTN2 fusion protein in the plasma membrane of OCTN2 mutant cultured skin fibroblasts and lymphoblasts [21]. In contrast, in the present study, we have demonstrated expression of an intermediate affinity carnitine transporter, OCTN3, in the peroxisomal membrane rather than the plasma membrane. This peroxisomal localization would thus appear to be physiologically appropriate, given Km concentration requirements for carnitine of 20 lM which would be easily fulfilled by the normal tissue carnitine concentrations in muscle, heart, and liver[26], but less so by the significantly lower serum carnitine concentrations. The total carnitine concentrations in control and ischemic perfused rat heart cytosol are 2.56 and 2.37 mM, respectively [27]. Since acylcarnitine esters account for at least one-half of total carnitine under most metabolic conditions, a Km of 20 lM would be consistent with the ability of peroxisomal OCTN3 to function under most physiologic conditions. In addition to its crucial role in mitochondrial b-oxidation, carnitine is also important in peroxisomal b-oxidation. Carnitine shuttles chain-shortened products produced by the b-oxidation of hexacosanoic acid (C26:0) and pristanic acid out of peroxisomes to mitochondria as carnitine esters [1,28]. In addition, carnitine has been shown to shuttle propionyl-CoA as a carnitine ester from peroxisomes to mitochondria [29]. Further evidence for the involvement of the carnitine system in peroxisomal b-oxidation includes the documented presence of carnitine acyltransferases [30] and carnitine translocase [31] in peroxisomes. In addition, the peroxisomal membrane in Saccharomyces cerevisiae is impermeable to acetyl-CoA under in vivo conditions, and oleate oxidation in yeast lacking citrate synthase is dependent upon carnitine and carnitine acetyltransferase to remove the acetyl-CoA produced [32]. Furthermore, carnitine-dependent inhibition of the peroxisomal matrix enzyme, 3-keto-thiolase, by 2-bromo-palmitoyl-CoA suggests that the long-chain acylcarnitine, not acyl-CoA, crosses peroxisomal membranes [33]. This strongly suggests that the former concept that peroxisomal b-oxidation is not dependent on carnitine is incorrect. Originally it was thought that peroxisomes were freely permeable to low-molecular mass compounds [34] such as CoA, carnitine, ATP, and NADH, all below 1000 Da. This was subsequently thought to be caused by a nonselective pore-forming protein [35]. However, more recent studies in the yeast S. cerevisiae [32] have clearly established that under in vivo conditions the peroxisomal membrane does form a permeability barrier, implying the existence of metabolite carriers in the peroxisomal membrane. This is further supported by the finding of a pH gradient across the peroxisomal membrane [36] that can be dissipated by uncouplers. It is likely that the fragile peroxisomal membrane could leak out during isolation procedures which would help to explain the earlier findings [37]. If the peroxisomal lipid bilayer is indeed impermeable under in vivo
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conditions, then specific transporters have to be present to allow metabolite transport [28]. At least four presumed peroxisomal transport proteins have been identified in S. cerevisiae [28]. The situation appears to be less clear in higher eukaryotes. However, recent studies have identified four human peroxisomal half ABC transporters including ALDP, ALDR, PMP70, and PMP69 [38]. ALDP appears to catalyze the uptake of very long-chain fatty acyl-CoAs. The membrane of spinach leaf peroxisomes has also been found to contain an anion-selective channel (porin) which is permeable to a variety of different monovalent inorganic and organic anions and which has a binding site for dicarboxylic anions [39]. Given the essential role of carnitine in the transport of different chain-shortened products out of peroxisomes and the impermeability of the peroxisomal membrane, there would appear to be a critical need for a carnitine/organic cation transporter on the peroxisomal membrane which could be met by the intermediate-affinity OCTN3 protein which we have shown to be expressed in both human and murine peroxisomes. In the current study, we have demonstrated that hOCTN3 is present but expressed in somewhat reduced amounts in the PEX 1 deficient Zellweger fibroblasts compared to normal control human skin fibroblasts but is completely absent in the PEX 19 deficient Zellweger fibroblasts. In PEX 1 deficient patients, the uptake of peroxisomal matrix enzymes is impaired, but the biosynthesis of peroxisomal membrane proteins is normal so that the expression of ALDP and PMP70 is normal. In contrast, in PEX 19 deficient patients, both the uptake of peroxisomal matrix enzymes as well as the peroxiomal membrane proteins is deficient because PEX 19 plays a key role in the generation of peroxisomal membrane. These findings in the aggregate therefore support the localization of hOCTN3 to the peroxismal membrane. PEX 1 and PEX 19 deficiencies are classified as autosomal recessive peroxisome biogenesis disorders in which there is loss of multiple peroxisomal metabolic functions [40,41]. These disorders include four overlapping clinical phenotypes of varying severity, namely classical Zellweger syndrome, atypical Zellweger syndrome, neonatal adrenoleukodystrophy, and infantile Refsum disease which are characterized by early onset complex developmental and metabolic phenotypes with neurodegeneration, retinopathy, sensorineural hearing loss, and hepatic involvement, and other individual associated features [42]. The PEX 1 gene encodes a 147-kDa member of the AAA protein family (ATPases associated with diverse cellular activities) [43,44]. Mutations in PEX 1 are the most common cause of peroxisome biogenesis disorders and result in severe defects in peroxisomal matrix protein import and destabilization of PEX 5, the receptor for the type-1 peroxisomal targeting signal, even though peroxisomes are present in these cells and capable of importing peroxisomal membrane proteins [44]. Pex 1p acts in the terminal steps of peroxisomal matrix protein import [45]. The Pex 19p is an oleic acid-inducible, farnesylated protein of 39.7 kDa that
is essential for peroxisome biogenesis in S. cerevisiae [41]. Cells lacking Pex 19p are characterized by the absence of morphologically detectable peroxisomes and mislocation of peroxisomal matrix proteins to the cytosol. Pex 19p is required for the proper localization, assembly, and stability of peroxisomal membrane proteins [46,47]. Furthermore, it has been recently shown by YFP-tagged Pex 3 and Pex 19 proteins, and real-time fluorescence microscopy in S. cerevisiae that Pex 19 enriches first at the Pex 3 foci on the endoplasmic reticulum (ER) and then on the budding off maturing peroxisomes. These results demonstrate that peroxisomes are generated from domains in the ER [48]. This may help to explain our ultrastructural localization of mOctn3 not only to peroxisomes but also to the ER in liver. In conclusion, the integrated roles of functionally different but complementary carnitine transporters are essential to the maintenance of cellular carnitine homeostasis. Predicted clinical phenotypes for defects in the OCTN3 transporter may parallel the cardiomyopathy and episodic hypoketotic hypoglycemic encephalopathy phenotypes of OCTN2. Alternatively, given the important expression of the intermediate-affinity carnitine transporter in bowel [11,14,49] as well as the pivotal role of b-oxidation in bowel bioenergetic metabolism [50,51], defects in this transporter may serve as a potential susceptibility gene for inflammatory bowel disease. Presence of undefined bacterial metabolites in colonic lumen causing specific breakdown of fatty acid oxidation in colonic epithelial cells has been proposed as an initiating event in inflammatory bowel disease that leads to an immune response and eicosanoid response perpetuating epithelial cell damage [52]. The hOCTN1 and hOCTN2 genes have been localized to the 5q31 locus. Genetic variation in this 5q31 gene cluster has been shown to confer susceptibility to CrohnÕs disease [53] though analysis, to date, of the known genes in this region has not allowed identification of the causal gene for CrohnÕs disease. Using syntenic comparison with the murine Octn gene family, we have recently predicted that there may be a human OCTN3 gene localized to this same region [22]. Thus, OCTN3 may serve as a candidate gene, which if mutated, could confer susceptibility to CrohnÕs disease. In conclusion, identification of mutations in the gene encoding for OCTN3 may provide important clues in certain hitherto undiagnosed cases of tissue carnitine depletion, hypertrophic cardiomyopathy, lipid storage myopathy, hypoglycemic coma, sudden infant death syndrome or inflammatory bowel disease who, most importantly, may significantly benefit from L-carnitine therapy. Acknowledgments This research is supported in part by Heart and Stroke Foundation of Ontario Grant # T-5424 and by a grant from the PhysiciansÕ Services Incorporated Foundation of Ontario.
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