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Carnitine (L-carnitine, L-carnitine fumarate and acetyl-L-carnitine)Note: This page contains notes and research journal abstracts about carnitine. For the article about the use of carnitine for those with PWS, please see here.
Note that muscle biopsy of an infant with PWS showed that "glycogen is markedly increased in some fibers and is seen as free particles [and] there are increased droplets of neutral lipid," while the acylcarnitine profile shows elevated acetylcarnitine (C2). High muscle glycogen results in high pyruvate and therefore high acetyl-CoA and a high acetyl-CoA/CoA ratio that inhibits pyruvate dehydrogenase (PDH) and therefore glucose oxidation. Carnitine is an acetyl group acceptor from acetyl-CoA, thus the high acetylcarnitine in the acylcarnitine profile, a reaction catalyzed by carnitine acetyltransferase (CAT), in a state of high muscle glycogen. However, the high acetylcarnitine results in a decrease in both mitochondrial and cytosolic carnitine available for transporting long-chain fatty acids into the mitochondria, thus impairing fatty acid beta-oxidation (thus perhaps the increased lipids found in the biopsy). Malonyl-CoA is an intermediate in the de novo synthesis of fatty acids (FA) and an inhibitor of carnitine palmitoyltransferase 1 (CPT-1) and thus of fatty acid transport into the mitochondrial and oxidation. Cytosolic citrate is both an activator of acetyl-CoA carboxylase (ACC), the key enzyme governing malonyl-CoA synthesis, and a substrate for the malonyl-CoA precursor, cytosolic acetyl-CoA. High glucose availability at rest has been shown to elevate the cytosolic citrate concentration and consequently the muscle malonyl-CoA concentration, and that is probably the mechanism whereby fat oxidation is inhibited by high glucose availability in resting humans. From Treatment of Mitochondrial Cytopathies (Medscape Pediatrics) Endogenous levo-carnitine (beta-hydroxy-gamma-trimethylammonium butyrate), found in many human tissues, is an amino acid derivative. It is synthesized in the liver and kidney from protein-bound lysine (supplemental oral lysine cannot improve carnitine synthesis) and methionine. It is a water-soluble compound that exhibits biologic activity only when in the levo isoform. Several enzymes and cofactors (iron, ascorbic acid, niacin, and pyridoxine) are involved in its biosynthesis, and only one matrix mitochondrial enzyme is involved in the pathway. Of note, skeletal and heart muscle are unable to synthesize carnitine, and these tissues are therefore dependent on uptake of carnitine from blood. Normal plasma and tissue levels are maintained by both de novo synthesis and exogenous dietary sources. Meat and milk products contain the highest concentrations of dietary carnitine whereas plant products are poor sources. Normal plasma carnitine concentrations are about 25 umol/L in infants and 54 umol/L in adults.[42] The highest concentration of carnitine is found in skeletal muscle (98%), although distribution is shared with heart, kidney, liver, and brain.[43] Carnitine is present in tissues and physiologic fluids as either free carnitine or as the acylcarnitine ester. In normal circumstances, approximately 85 to 90% is present in the free state. The majority of plasma acylcarnitine is represented by acetylcarnitine, which is often nonpathologically elevated in the fasting state. The ratio between acylcarnitine to free carnitine varies with timing of the last meal, composition of that meal, nutritional status, exercise, and disease conditions and is quite sensitive to changes in mitochondrial metabolism. A ratio of 0.25 is considered to be normal, whereas greater than 0.4 is abnormal and is indicative of carnitine insufficiency or insufficient carnitine in light of the metabolic demands.[44] Carnitine is necessary for transporting long-chain fatty acids across the inner mitochondrial membrane for the process of beta-oxidation. This occurs mainly in skeletal muscle, heart, and liver and is carried out by carnitine palmitoyltransferase I (CPT I), acylcarnitine translocase, and CPT II. A second major task of carnitine is to maintain intracellular homeostasis of acyl-CoA. Carnitine transesterifies the acyl-CoA esters that arise during beta-oxidation through the action of carnitine-acyltransferases. The acylcarnitine can then cross the mitochondrial membrane in exchange for free carnitine, thus allowing for restoration of free CoA within the mitochondria. In addition to these major functions, carnitine may also play some role in altering the physiologic properties of cell membranes, such as membrane stabilization.[45] In the setting of inborn errors of metabolism, carnitine serves to detoxify the poisonous metabolic intermediates by forming a less toxic ester. A number of pathologic conditions have been associated with abnormal metabolism of carnitine, the most frequent of which is carnitine deficiency. Carnitine deficiency can be defined as a state where the concentration is not adequate to meet the body's normal carnitine requirement. Systemic carnitine deficiency can be primary but may occur in many disease states, including disorders of oxidative phosphorylation, beta-oxidation, organic acidurias, malnutrition, valproate, and zidovudine use and in those receiving total parenteral nutrition without adequate carnitine replacement. Many metabolic disorders lead to elevated levels of acyl-CoA intermediates, which impair the function of adenine nucleotide translocase, the enzyme that exchanges ADP for ATP across the inner mitochondrial membrane. Carnitine forms an ester linkage with the acyl-CoA, forming the relatively nontoxic acylcarnitine, which is excreted in the urine. Elevated levels of acyl-CoA intermediates over time can lead to a secondary carnitine deficiency. Likewise, a carnitine deficiency itself can result in increased toxicity of the accumulated acyl-CoA compounds. Clinical manifestations of a carnitine-deficient state are varied, including but not limited to cardiomyopathy, acute encephalopathy, myopathy, cognitive delay, central nervous system dysfunction, gastrointestinal dysmotility, and recurrent incidences of metabolic decompensation. Treatment with levo-carnitine should be considered for any person with a primary or secondary carnitine deficiency. The role of carnitine therapy in mitochondrial disease is threefold. As already discussed, carnitine plays a role in reestablishing homeostasis of acyl groups, a process that is aberrant when mitochondrial dysfunction exists, leading to inhibition of respiratory enzymes. In addition, secondary carnitine deficiency exists in the setting of mitochondrial cytopathies; thus, carnitine replacement is essential. Finally, carnitine may provide improved integrity of the mitochondrial membrane, thus adding to membrane stabilization.[46] The typical dose of levo-carnitine is 100 mg per kg per day for children and 2 to 4 grams per day for adults in three divided doses. In the nonacute setting, levo-carnitine is available as a liquid or tablet, but it is also available as an intravenous preparation. The intravenous dose is the same as the oral dose. Prior to initiation of carnitine therapy in any patient, plasma and urine carnitine and acylcarnitine profiles should be obtained. The primary adverse effects include diarrhea and nausea, though carnitine is usually well tolerated at typical doses. It should be noted that oral absorption is variable, and as little as 15% of the oral dose may actually be absorbed. Of 48 patients studied by Campos et al,47 four had both total and free plasma carnitine deficiency (both defined as <30 mmol/L with normals in the low to mid 50s) with carnitine insufficiency (defined as ratio of esterified to free carnitine >0.25 with normal 0.13 ”Ą 0.016), and 17 had isolated carnitine insufficiency. All 21 patients with carnitine deficiency or insufficiency were treated with 50 to 200 mg per kg per day of levo-carnitine. The following improvements were observed following initiation of treatment: 20 of 21 patients with muscle weakness demonstrated subjective improvement in muscle tone, four of eight patients with failure to thrive showed growth acceleration, and eight of eight patients with cardiomyopathy demonstrated improved echocardiographic findings and clinical improvement. The average treatment duration was 11 months (range 1 to 24 months). Plasma carnitine levels 10 days after initiation of treatment were normal or above normal. Many additional reports have demonstrated the beneficial effects of carnitine in the setting of cardiomyopathy due to underlying metabolic etiologies, including mitochondrial abnormalities. From http://www.nationwidelab.com/esrdlab_com/web/content/technology/12.htmNationwide Lab Services The question of deleterious effects of long-term elevated carnitine in continuously supplemented patients remains open. Although the substance has a low order of acute toxicity according to the usual criteria,1 there are reports that long-term high-dose administration tends to promote abnormalities in platelet aggregation.18,19 A paradoxical myasthenia in some dialysis patients on high-dose carnitine supplementation has even been reported, but since DL-carnitine was used as the drug, the effect may well have been due to its content of D(+)-carnitine.20 18. Kalinowski M, Popawski A, Mazerska M, Daniluk A. Effects of L-carnitine on erythropoiesis and blood platelet aggregation in patients with chronic renal failure treated with hemodialysis. Pol Merkuriusz Lek 1999; 6: 76-78. Prim Care Companion J Clin Psychiatry. 2007. No abstract available. Brain Dev. 2007 Jun 16. Organic cation/carnitine transporters transport carnitine, drugs, and xenobiotics (e.g. choline, acetylcarnitine, betaine, valproic acid), and are expressed in muscle, heart, blood vessels, kidney, gut, etc. Objective. To characterize expression patterns of mOctn1, -2 and -3 in murine brain. Methods. We applied our transporter-specific antibodies to mOctn1, -2 and -3, followed by 2(0) antibody and DAB peroxidase detection to serial adult murine brain sections counterstained with hematoxylin. Results. All three transporters showed strong expression in the external plexiform layer of the olfactory bulb and in olfactory nerve, the molecular layer and neuronal processes of input fibres extending vertically in motor cortex, in the dendritic arborization of the cornu ammonis and dendate gyrus (hippocampus), neuronal processes in the arcuate nucleus (hypothalamus), choroid plexus cells, and neuronal cell bodies and dendrites of cranial nerve nuclei V and VII. In the cerebellum, all three transporters were strongly expressed in dendritic processes of Purkinje cells, but Octn1 and -2 were expressed more strongly than Octn3 in Purkinje cell bodies. In spinal cord, Octn1, -2 and -3 were prominent in axons and dendritic end-arborizations of spinal cord neurons in both ascending and descending white matter tracts, whereas Octn3 was also strongly expressed in grey matter, specifically in anterior horn cell bodies. Octn3 was weakly expressed in glomerular layer neuronal cell bodies of olfactory bulb. Conclusions. hOCTN2 deficiency presents with carnitine-responsive cardiomyopathy, myopathy and hypoglycemic, hypoketotic coma with strokes, seizures and delays. In mouse, Octn1, -2 and -3 are expressed in many regions throughout the central nervous system with a pattern suggestive of roles in modulating cerebral bioenergetics and in acetylcholine generation for neurotransmission in olfactory, satiety, limbic, memory, motor and sensory functions. This distribution may play a role in the pattern of neurological injury that occurs in hOCTN2 deficiency during catabolic episodes of hypoglycemic, hypoketotic encephalopathy and which may manifest with cognitive impairment, hypotonia and seizures. Mol Cell Endocrinol. 2007 Mar 15. The transcriptome pattern of metabolic genes in vitamin A deficient (VAD) liver has been compared to the vitamin A-sufficient (VAS) state using the Mouse 32k oligonucleotide (70mer) array. In VAD liver there was a decrease in expression of genes encoding enzymes of mitochondrial fatty acid (FA) oxidation; these genes included fatty acyl CoA ligase, carnitine o-palmitoyl transferase 1, medium chain acyl-CoA dehydrogenase, 3-ketoacyl CoA thiolase, and citrate synthase. Particularly affected was peroxisome metabolism, as genes encoding enzymes of peroxisomal FA oxidation and transport proteins were differentially expressed. These genes included those encoding acyl-CoA oxidase 1, the peroxisomal bifunctional enzyme, peroxisomal thiolase, and carnitine o-octanoyl transferase, the enzyme involved in shuttling FAs from the peroxisome to the mitochondrion. Most genes that were differentially expressed with chronic vitamin A depletion were responsive to treatment with all-trans retinoic acid (RA). Consistent with the decreased expression of genes involved in FA oxidation, we found an increase in hepatic macrocytic lipid accumulation and triglyceride levels. The relevant nuclear receptor gene that was differentially expressed in the VAD liver was that encoding the peroxisome proliferator-activated receptor (PPAR) alpha, the mRNA levels for which were decreased in VAD liver and increased with all-trans RA treatment. Down regulation of the PPAR alpha gene is the likely cause of the altered expression pattern of the above metabolic genes in VAD liver. J Nutr Biochem. 2007 Mar 16. The objective of this study was to determine whether dietary l-carnitine can influence the status of alpha-tocopherol, retinol and selected lipid parameters in aging ovariectomized rats, an animal model for the menopausal state. Fourteen Fisher-344 female rats 18 months old were acclimated for 4 weeks and ovarectomized. Seven rats per treatment were assigned to either a control group fed ad libitum AIN-93M diet or a carnitine group fed the same diet supplemented with l-carnitine. After an 8-week feeding period, blood and selected tissues were taken for analyses. No differences were noted in food intake, body weight, or organ weights due to l-carnitine. Dietary carnitine significantly increased liver alpha-tocopherol and tended to increase plasma alpha-tocopherol (P<.09). No changes in alpha-tocopherol were observed in other tissues including the brain, lungs and retroperitoneal fat. Retinol levels in plasma and tissues were not affected by supplemental l-carnitine. Significant decreases in liver and plasma triglyceride (TG) levels were noted, suggesting increased utilization of fatty acids. No differences were observed in the fatty acid profile of tissues. The results provide evidence that dietary supplementation of l-carnitine enhances the alpha-tocopherol status and improves the utilization of fat leading to lowering of the liver and plasma levels of TG in aging ovariectomized rats. Whether supplemental l-carnitine may be of benefit to postmenopausal women in lowering plasma TG and improving the antioxidant status remains to be studied. J Physiol. 2007 Mar 1. In skeletal muscle, carnitine plays an essential role in the translocation of long-chain fatty-acids into the mitochondrial matrix for subsequent beta-oxidation, and in the regulation of the mitochondrial acetyl-CoA/CoASH ratio. Interest in these vital metabolic roles of carnitine in skeletal muscle appears to have waned over the past 25 years. However, recent research has shed new light on the importance of carnitine as a regulator of muscle fuel selection. It has been established that muscle free carnitine availability may be limiting to fat oxidation during high intensity sub-maximal exercise. Furthermore, increasing muscle total carnitine content in resting healthy humans (via insulin-mediated stimulation of muscle carnitine transport) reduces muscle glycolysis, increases glycogen storage and is accompanied by an apparent increase in fat oxidation. By increasing muscle pyruvate dehydrogenase complex (PDC) activity and acetylcarnitine content at rest, it has also been established that PDC flux and acetyl group availability limits aerobic ATP re-synthesis at the onset of exercise (the acetyl group deficit). Thus, carnitine plays a vital role in the regulation of muscle fuel metabolism. The demonstration that its availability can be readily manipulated in humans, and impacts on physiological function, will result in renewed business and scientific interest in this compound. Life Sci. 2006 Dec 23. High fructose feeding (60 g/100 g diet) in rodents induces alterations in both glucose and lipid metabolism. The present study was aimed to evaluate whether intraperitoneal carnitine (CA), a transporter of fatty acyl-CoA into the mitochondria, could attenuate derangements in carbohydrate metabolizing enzymes and glucose overproduction in high fructose-diet fed rats. Male Wistar rats of body weight 150-160 g were divided into 4 groups of 6 rats each. Groups 1 and 4 animals received control diet while the groups 2 and 3 rats received high fructose-diet. Groups 3 and 4 animals were treated with CA (300 mg/Kg body weight/day, i.p.) for 30 days. At the end of the experimental period, levels of carnitine, glucose, insulin, lactate, pyruvate, glycerol, triglycerides and free fatty acids in plasma were determined. The activities of carbohydrate metabolizing enzymes and glycogen content in liver and muscle were assayed. Hepatocytes isolated from liver were studied for the gluconeogenic activity in the presence of substrates such as pyruvate, lactate, glycerol, fructose and alanine. Fructose-diet fed animals showed alterations in glucose metabolizing enzymes, increased circulating levels of gluconeogenic substrates and depletion of glycogen in liver and muscle. There was increased glucose output from hepatocytes of animals fed fructose-diet alone with all the gluconeogenic substrates. The abnormalities associated with fructose feeding such as increased gluconeogenesis, reduced glycogen content and other parameters were brought back to near normal levels by CA. Hepatocytes from these animals showed significant inhibition of glucose production from pyruvate (74.3%), lactate (65.4%), glycerol (69.6%), fructose (56.2%) and alanine (63.6%) as compared to CA untreated fructose-fed animals. The benefits observed could be attributed to the effect of CA on fatty acyl-CoA transport. J Clin Endocrinol Metab. 2006 Dec. Context: Carnitine plays an essential role in the integration of fat and carbohydrate oxidation in skeletal muscle, which is impaired in obesity and type 2 diabetes. Objective: The aim of the present study was to investigate the effect of an increase in skeletal muscle total carnitine (TC) content on muscle fuel metabolism. Design: A 5-h iv infusion of saline (control) or l-carnitine was administered while serum insulin was maintained at a physiologically high concentration during two randomized visits. Participants: Seven healthy, nonvegetarian young men (body mass index, 26.1 +/- 1.6 kg/m2) participated in the present study at the University of Nottingham. Main outcome measures: Skeletal muscle pyruvate dehydrogenase complex (PDC) activity and associated muscle metabolites were measured. Results: The combination of hypercarnitinemia (600 micromol/liter) and hyperinsulinemia (160 mU/liter) increased muscle TC content by 15% (P < 0.01) and was associated with decreased pyruvate dehydrogenase complex activity (P < 0.05) and muscle lactate content (P < 0.05) by 30 and 40%, respectively, and an overnight increase in muscle glycogen (P < 0.01) and long-chain acyl-coenzyme A content (P < 0.05) by 30 and 40%, respectively, compared with control. Conclusions: These results suggest that an acute increase in human skeletal muscle TC content results in an inhibition of carbohydrate oxidation in conditions of high carbohydrate availability, possibly due to a carnitine-mediated increase in fat oxidation. These novel findings may have important implications for our understanding of the regulation of muscle fat oxidation, particularly during exercise, when carnitine availability may limit fat oxidation, and in obesity and type 2 diabetes where it is known to be impaired. Regul Pept. 2006 Nov 14. Adiponectin, an adipocyte-derived polypeptide hormone, plays an important role in regulating fatty acid oxidation. beta-oxidation of fatty acids supplies most of the cardiac energy and carnitine palmitoyltransferase (CPT)-1 serves as a key regulator during this process. To characterize the potential effects of adiponectin on CPT-1, we incubated rat neonatal cardiomyocytes with globular adiponectin (gAd). Results showed that gAd promoted the activity and mRNA expression of CPT-1. The underlying signal pathway involved in this modulatory effect was further investigated. Inhibition of AMP-activated protein kinase (AMPK) with adenine 9-beta-d-arabinofuranoside (AraA) completely abrogated gAd-mediated AMPK and acetyl coenzyme A carboxylase (ACC) phosphorylation and suppressed the promotion of CPT-1 activity. gAd also induced the phosphorylation of p38 mitogen-activated protein kinase (MAPK) and peroxisome proliferator-activated receptor (PPAR)-alpha, which was inhibited by AraA. SB202190, a p38MAPK inhibitor, blocked gAd-stimulated PPAR-alpha phosphorylation. When AMPK and/or p38MAPK was inhibited, gAd-enhanced mRNA expression of CPT-1 was partially reduced. In conclusion, our study suggests that the activation of AMPK signaling cascade participates in the promotion effect of gAd on CPT-1. J Clin Pharm. Oct. 2006. Disposition and Metabolite Kinetics of Oral L-carnitine in Humans. Marcus A. Bain, Robert W. Milne, PhD and Allan M. Evans, PhD From the Sansom Institute, School of Pharmacy and Medical Sciences, University of South Australia, Australia. [ Free full text ] The pharmacokinetics of L-carnitine and its metabolites were investigated in 7 healthy subjects following the oral administration of 0, 0.5, 1, and 2 g 3 times a day for 7 days. Mean plasma concentrations of L-carnitine across an 8-hour dose interval increased significantly (P < .05) from a baseline of 54.2 ± 9.3 µM to 80.5 ± 12.5 µM following the 0.5-g dose; there was no further increase at higher doses. There was a significant increase (P < .001) in the renal clearance of L-carnitine indicating saturation of tubular reabsorption. Trimethylamine plasma levels increased proportionately with L-carnitine dose, but there was no change in renal clearance. A significant increase in the plasma concentrations of trimethylamine-N-oxide from baseline was evident only for the 2-g dose of L-carnitine (from 34.5 ± 2.0 to 149 ± 145 µM), and its renal clearance decreased with increasing dose (P < .05). There was no evidence for nonlinearity in the metabolism of trimethylamine to trimethylamine-N-oxide. In conclusion, the pharmacokinetics of oral L-carnitine display nonlinearity above a dose of 0.5 g 3 times a day. JPEN J Parenter Enteral Nutr. 2006 Sep-Oct. Background: Carnitine is an important nutrient in the infant diet. We compared total plasma carnitine concentrations in premature neonates supplemented with carnitine via parenteral and enteral nutrition. Methods: This is a post hoc analysis of plasma total carnitine concentrations and carnitine intake in neonates randomized in a previous study to receive 20 mg/kg/d carnitine supplementation over 8 weeks. Neonates received l-carnitine initially via parenteral nutrition (PN). When neonates were fed enterally, oral supplementation of l-carnitine was given in divided doses with each feeding. Results: Sixteen neonates (27 +/- 2 weeks gestation; 2.9 +/- 1.0 days postnatal age at enrollment; 965.6 +/- 279.1 g birth weight) are included. Concentrations were below reference range (31.1-60.5 nmol/mL) at baseline and exceeded reference range from week 1 through the last study period. Concentrations were not different from week 1 (108 +/- 49) through weeks 4 (87 +/- 34) and 8 (83 +/- 31). Carnitine intakes and concentrations were compared in neonates receiving 100% parenteral carnitine at week 1 (n = 6) and 100% enteral carnitine at week 8 (n = 8). Concentrations at week 1 (100.1 +/- 27.9) were not different (p = .19) from week 8 (78.6 +/- 29.3); an estimate of relative bioavailability was 78.6%. Bioavailability with paired analysis of neonates (n = 5) receiving 100% parenteral carnitine at week 1 and 100% enteral carnitine at week 8 was 83.7% +/- 41.2% (30.1%-140.6%). Conclusions: Parenteral and enteral supplementation of 20 mg/kg/d carnitine results in plasma total carnitine concentrations that exceed the reference range. Concentrations are not different between parenteral to enteral supplementation, suggesting that enteral carnitine is well absorbed when given daily in divided doses with enteral feedings. Yakugaku Zasshi. 2006 Sep. Levocarnitine chloride is used for the therapeutic purpose of levocarnitine deficiency. For infants, however, levocarnitine chloride tablets must be crushed to avoid difficulties associated with swallowing, and also to administer an appropriately low dosage. Since the tablet is extremely hygroscopic and sour, it is dissolved in water containing simple syrup after crushing. In this study we investigated the stability of the drug after dissolution to optimize its preparation for clinical use. It was shown to be stable for at least 90 days after preparation, and microbes did not grow in 1-10% (w/v) solutions (pH 2.0-2.5) regardless of the presence or absence of simple syrup. Furthermore, the autoclaved levocarnitine chloride solution was as stable as the non-autoclaved one. In conclusion, the method employed in our hospital for the preparation of levocarnitine chloride for infants is appropriate and is recommended as a standard medicine supply method among different facilities. Pediatr Hematol Oncol. 2006 Jul-Aug. Carnitine is ingested through animal-derived foods as well as synthesized in vivo. It plays an important role in the energy metabolism of many tissues. Iron acts as a co-factor for the synthesis of carnitine. However, the importance of iron deficiency as a cause of secondary carnitine deficiency is not well established. The aim of this study was to investigate the serum levels of carnitine in children with iron-deficiency anemia compared to those of healthy children and to determine if serum carnitine levels in with or without pica differ. The mean serum carnitine concentration in the iron-deficiency group was significantly lower than that in healthy children (12.44+/- 5.09 and 32.48 +/- 7.92 micromol/L, respectively, p < .001). In the iron-deficient group, serum carnitine levels, ferritin levels, and other hematological parameters were lowest in patients with pica (p < .001). Pearson correlation test indicated a positive correlation between serum carnitine and ferritin levels in iron-deficient patients. Based on the evidence about the effect of low iron on carnitine stores in animal studies, the authors propose that low serum carnitine levels in these children may be secondary to iron-deficiency anemia. However, further large-scale studies are needed to establish the frequency of carnitine deficiency in children with iron-deficiency anemia. J Gerontol A Biol Sci Med Sci. 2006 Jul. The aging process is characterized by a general decline in physiological functions that affects many tissues and increases the risk of death. In the present investigation using various substrates, the respiration rate was observed in young, middle-aged, and aged rats upon administration of carnitine (300 mg/kg body weight) and lipoic acid (100 mg/kg body weight). We observed that the rate of respiration, both State 3 and respiratory control ratio, decreased significantly in aged rats after using various substrates (except succinate). An increase in the State 4 respiration was observed in aged rats when beta-hydroxybutyrate as well as pyruvate and malate were used as substrates, whereas no change in the adenosine diphosphate/oxygen ratio ratio was observed. These changes were brought to normal levels upon cosupplementation of carnitine and lipoic acid. Thus, this study provides evidence for the role of carnitine and lipoic acid in alleviating the age-related decline in mitochondrial respiratory activity. Am J Med Genet C Semin Med Genet. 2006 May 15. Carnitine plays an essential role in the transfer of long-chain fatty acids across the inner mitochondrial membrane. This transfer requires enzymes and transporters that accumulate carnitine within the cell (OCTN2 carnitine transporter), conjugate it with long chain fatty acids (carnitine palmitoyl transferase 1, CPT1), transfer the acylcarnitine across the inner plasma membrane (carnitine-acylcarnitine translocase, CACT), and conjugate the fatty acid back to Coenzyme A for subsequent beta oxidation (carnitine palmitoyl transferase 2, CPT2). Deficiency of the OCTN2 carnitine transporter causes primary carnitine deficiency, characterized by increased losses of carnitine in the urine and decreased carnitine accumulation in tissues. Patients can present with hypoketotic hypoglycemia and hepatic encephalopathy, or with skeletal and cardiac myopathy. This disease responds to carnitine supplementation. Defects in the liver isoform of CPT1 present with recurrent attacks of fasting hypoketotic hypoglycemia. The heart and the muscle, which express a genetically distinct form of CPT1, are usually unaffected. These patients can have elevated levels of plasma carnitine. CACT deficiency presents in most cases in the neonatal period with hypoglycemia, hyperammonemia, and cardiomyopathy with arrhythmia leading to cardiac arrest. Plasma carnitine levels are extremely low. Deficiency of CPT2 present more frequently in adults with rhabdomyolysis triggered by prolonged exercise. More severe variants of CPT2 deficiency present in the neonatal period similarly to CACT deficiency associated or not with multiple congenital anomalies. Treatment for deficiency of CPT1, CPT2, and CACT consists in a low-fat diet supplemented with medium chain triglycerides that can be metabolized by mitochondria independently from carnitine, carnitine supplements, and avoidance of fasting and sustained exercise. Neurosci Res. 2006 May. We found reduced locomotor activity (LA) under fasting in systemic carnitine-deficient juvenile visceral steatosis (jvs(-/-)) mice. When food was withdrawn at 8:00 a.m. (lights-off at 7:00 p.m., 12h/cycle), the nocturnal LA of jvs(-/-) mice was much less than the control (jvs(+/+) and jvs(+/-)) mice. LA recovered under carnitine or sucrose administration, but not under medium-chain triglyceride. In addition, fasted jvs(-/-) mice, without any energy supply, were activated by modafinil, a stimulator of the dopamine pathway. These results suggest that the reduced LA is not adequately explained by energy deficit. As the fasted jvs(-/-) mice showed lower body core temperature (BT), we examined the central nervous system regulating LA and BT. We found lower percentage of c-Fos positive orexin [hypocretin] neurons in the lateral hypothalamus and reduced orexin-A concentration in the cerebrospinal fluid of fasted jvs(-/-) mice. Sleep analysis revealed that fasted jvs(-/-) mice had disruption of prolonged wakefulness, with a higher frequency of brief episodes of non-REM sleep during the dark period than fasted jvs(+/+) mice. These results strongly suggest that the reduced LA in fasted jvs(-/-) mice is related to the inhibition of orexin neuronal activity. Stroke. 2006 Feb. Background and purpose: Cerebral ischemic insults disrupt normal respiratory activity in mitochondria. Carnitine plays an essential role in mitochondrial metabolism and in modulating excess acyl-coenzyme A (acyl-CoA) levels. The effects of cerebral ischemia on carnitine metabolism are not well understood, although the newborn may be particularly vulnerable to carnitine deficiency. We used a newborn rat model of hypoxia-ischemia (HI) to test the hypothesis that HI alters acyl-CoA:CoA homeostasis and that this effect can be prevented by treatment with carnitine. Methods: A total of 120 postnatal day 7 rats were subjected to 70 minutes of HI after treatment with 16 mmol/kg intraperitoneal l-carnitine or diluent. Carnitine, acylcarnitines, and excitatory amino acids were measured by mass spectrometry, and carnitine acetyl transferase activity, superoxide, and levels of the mitochondrial phospholipid cardiolipin (CL) were measured at 2- and 24-hour recovery. Results: HI and hypoxia were associated with a significant increase in the ratio of acyl-CoA:CoA, which was prevented by treatment with carnitine. Carnitine treatment also prevented increases in glutamate, glycine, superoxide, and decrease of CL. Conclusions: Carnitine metabolic pathways are compromised in HI and hypoxia. The protective effect of carnitine treatment on HI injury may be attributable to maintaining mitochondrial function. Electromyogr Clin Neurophysiol. 2005 Sep-Oct. Objectives: To determine neuropathy frequency with electromyography (EMG) in asymptomatic diabetic children, and to demonstrate whether the electromyographical abnormalities noted improve after L-carnitine treatment. Patients and Methods: This study was carried out on 51 type 1 diabetes mellitus patients (of whom, 26 were female; average age 12) and 21 healthy children as the control group. Thirty four patients, whose nerve conduction velocity (NCS) was diagnosed as pathological, were treated with L-carnitine (dosage: 2 g/m2/day) for two months and their NCS checked at the end of the treatment period. Results: At least one electrophysiological parameter was abnormal in 38 out of 51 patients (74.6%). At the end of the treatment, Stage 1 a patients (NCS pathologic and neurologic examination normal) demonstrated a 44% improvement in all pathologic NCS parameters and a 50% improvement in sympathetic skin responses (SSR), while in Stage 1 b patients (NCS and neurologic examination pathologic) a matching ratio of improvement was detected in SSR but no definite improvement was noted in the all pathologic NCS parameters. Conclusion: Starting carnitine treatment in the early stages may be more effective in the treatment of sub-clinical neuropathy. A two-month treatment period may not be sufficient in detecting an electrophysiological improvement in cases where neurological deficits had been determined. Ukr Biokhim Zh. 2005 Jul-Aug. Recently reported data clarify our understanding of the molecular aspects of carnitine in medicine. Carnitine is a compound necessary for the transport of acyl-CoA across the inner mitochondrial membrane for their beta-oxidation. Only L-isomer of carnitine is biologically active. The D-isomer may actually compete with L-carnitine for absorption and transport, increasing the risk of carnitine deficiency. By interaction with CoA, carnitine is involved in the intermediary metabolism by modulating free CoA pools in the cell. Detoxification properties and anabolic, antiapoptotic and neuroprotective roles of carnitine is presented. Carnitine deficiency occurs as a primary genetic defect of carnitine transport and secondary to a variety of genetic and acquired disorders. The pathophysiological states associated with carnitine deficiency have been summarized. L-Carnitine is effective for the treatment of primary and secondary carnitine deficiencies. Acetyl-L-carnitine improves cognition in the brain, significantly reversed age-associated decline in mitochondrial membrane potential and improved ambulatory activity. The therapeutic effects of carnitine and acetylcarnitine are discussed. Eur J Hum Genet. 2005 May. The fragile X syndrome is caused by a >200 CGG repeat expansion within the FMR1 gene promoter, with consequent DNA hypermethylation and inactivation of its expression. To further clarify the mechanisms that suppress the activity of the mutant gene and the conditions that may permit its reactivation, we investigated the acetylation and methylation status of three different regions of the FMR1 gene (promoter, exon 1 and exon 16) of three fragile X cell lines, using a chromatin immunoprecipitation (ChIP) assay with antibodies against acetylated-H3/H4 histones and against dimethylated lysine residues K4 and K9 of histone H3 (H3-K4 and H3-K9). We then coupled the ChIP assay with real-time PCR, obtaining absolute quantification of immunoprecipitated chromatin. Basal levels of histone acetylation and H3-K4 methylation were much higher in transcriptionally active wild-type controls than in inactive fragile X cell lines. Treatment of fragile X cell lines with the DNA demethylating drug 5-aza-2-deoxycytidine (5-azadC), known to reactivate the FMR1 gene, induced a decrease of H3-K9 methylation, an increase of H3 and H4 acetylation and an increase of H3-K4 methylation. Treatment with acetyl-L-carnitine (ALC), a compound that reduces the in vitro expression of the FRAXA fragile site without affecting DNA methylation, caused an increase of H3 and H4 acetylation. However, H3-K4 methylation remained extremely low, in accordance with the observation that ALC alone does not reactivate the FMR1 gene. Our experiments indicate that H3-K4 methylation and DNA demethylation are the main epigenetic switches activating the expression of the FMR1 gene, with histone acetylation playing an ancillary role. Tumori. 2005 Mar-Apr. Aims and background: In addition to bone marrow suppression and renal toxicity, neurotoxicity is a commonly occurring side effect of widely used chemotherapeutic agents like taxanes, cisplatin and vinca alkaloids. Neurotoxicity can cause antitumor therapy discontinuation or dose regimen modification. The aim of the present exploratory study was to investigate the activity of acetyl-L-carnitine in reversing peripheral neuropathy in patients with chemotherapy-induced peripheral neuropathy. Methods and study design: Twenty-seven patients (16 males and 11 females) with paclitaxel and/or cisplatin-induced neuropathy (according to WHO recommendations for the grading of acute and subacute toxic effects) were enrolled. Patients received at least one cisplatin- (n = 5) or one paclitaxel- (n = 11) based regimen, or a combination of both (n = 11). Patients with chemotherapy-induced peripheral neuropathy were treated with acetyl-L-carnitine 1 g/die i.v. infusion over 1-2 h for at least 10 days. Results: Twenty-six patients were evaluated for response having completed at least 10 days of acetyl-L-carnitine therapy (median, 14 days; range, 10-20). At least one WHO grade improvement in the peripheral neuropathy severity was shown in 73% of the patients. A case of insomnia related to ALC treatment was reported in one patient. Acetyl-L-carnitine seems to be an effective and well-tolerated agent for the treatment of chemotherapy-induced peripheral neuropathy. Conclusions: Our preliminary results should be confirmed in double-blind, placebo controlled studies. J Neurosci Res. 2005 Feb 15. Efficient functioning of maintenance and repair processes seem to be crucial for both survival and physical quality of life. This is accomplished by a complex network of the so-called longevity assurance processes, under control of several genes termed vitagenes. These include members of the heat shock protein system, and there is now evidence that the heat shock response contributes to establishing a cytoprotective state in a wide variety of human conditions, including inflammation, neurodegenerative disorders, and aging. Among the various heat shock proteins, heme oxygenase-1 has received considerable attention; it has been recently demonstrated that heme oxygenase-1 induction, by generating the vasoactive molecule carbon monoxide and the potent antioxidant bilirubin, could represent a protective system potentially active against brain oxidative injury. Acetyl-L-carnitine is proposed as a therapeutic agent for several neurodegenerative disorders. Accordingly, we report here that treatment of astrocytes with acetyl-L-carnitine induces heme oxygenase-1 in a dose- and time-dependent manner and that this effect was associated with up-regulation of heat shock protein 60 as well as high expression of the redox-sensitive transcription factor Nrf2 in the nuclear fraction of treated cells. In addition, we show that addition of acetyl-L-carnitine to astrocytes, prior to proinflammatory lipopolysaccharide- and interferon-gamma-induced nitrosative stress, prevents changes in mitochondrial respiratory chain complex activity, protein nitrosation and antioxidant status induced by inflammatory cytokine insult. Given the broad cytoprotective properties of the heat shock response, molecules inducing this defense mechanism appear to be possible candidates for novel cytoprotective strategies. Particularly, manipulation of endogenous cellular defense mechanisms via acetyl-L-carnitine may represent an innovative approach to therapeutic intervention in diseases causing tissue damage, such as neurodegeneration. We hypothesize that maintenance or recovery of the activity of vitagenes may delay the aging process and decrease the risk of age-related diseases. Ann N Y Acad Sci. 2004 Nov. Mitochondrial oxidation of long-chain fatty acids provides an important source of energy for the heart as well as for skeletal muscle during prolonged aerobic work and for hepatic ketogenesis during long-term fasting. The carnitine shuttle is responsible for transferring long-chain fatty acids across the barrier of the inner mitochondrial membrane to gain access to the enzymes of beta-oxidation. The shuttle consists of three enzymes (carnitine palmitoyltransferase 1, carnitine acylcarnitine translocase, carnitine palmitoyl-transferase 2) and a small, soluble molecule, carnitine, to transport fatty acids as their long-chain fatty acylcarnitine esters. Carnitine is provided in the diet (animal protein) and also synthesized at low rates from trimethyl-lysine residues generated during protein catabolism. Carnitine turnover rates (300-500 micromol/day) are <1% of body stores; 98% of carnitine stores are intracellular (total carnitine levels are 40-50 microM in plasma vs. 2-3 mM in tissue). Carnitine is removed by urinary excretion after reabsorption of 98% of the filtered load; the renal carnitine threshold determines plasma concentrations and total body carnitine stores. Because of its key role in fatty acid oxidation, there has long been interest in the possibility that carnitine might be of benefit in genetic or acquired disorders of energy production to improve fatty acid oxidation, to remove accumulated toxic fatty acyl-CoA metabolites, or to restore the balance between free and acyl-CoA. Two disorders have been described in children where the supply of carnitine becomes limiting for fatty acid oxidation: (1) A recessive defect of the muscle/kidney sodium-dependent, plasma membrane carnitine symporter, which presents in infancy with cardiomyopathy or hypoketotic hypoglycemia; treatment with oral carnitine is required for survival. (2) Chronic administration of pivalate-conjugated antibiotics in which excretion of pivaloyl-carnitine can lead to carnitine depletion; tissue levels may become low enough to limit fatty acid oxidation, although no cases of illness due to carnitine deficiency have been described. There is speculation that carnitine supplements might be beneficial in other settings (such as genetic acyl-CoA oxidation defects-"secondary carnitine deficiency", chronic ischemia, hyperalimentation, nutritional carnitine deficiency), but efficacy has not been documented. The formation of abnormal acylcarnitines has been helpful in expanded newborn screening programs using tandem mass-spectrometry of blood spot acylcarnitine profiles to detect genetic fatty acid oxidation defects in neonates. Carnitine-deficient diets (vegetarian) do not have much effect on carnitine pools in adults. A modest 50% reduction in carnitine levels is associated with hyperalimentation in newborn infants, but is of doubtful significance. The above considerations indicate that carnitine does not become rate-limiting unless extremely low; testing the benefits of nutritional supplements may require invasive endurance studies of fasting ketogenesis or muscle and cardiovascular work. Psychopharmacology (Berl). 2004 Nov. The attention deficit/hyperactivity disorder (ADHD) can affect human infants and adolescents. One important feature of this disorder is behavioural impulsivity. This study assessed the ability of chronic acetyl-L-carnitine (ALC, saline or 100 mg/kg SC, plus 50 mg/kg orally) to reduce impulsivity in a validated animal model for ADHD. Food-restricted rats were tested during adolescence (postnatal days, pnd, 30-45) in operant chambers with two nose-poking holes, one delivering one food pellet immediately, and the other five pellets after a delay. Delay length was increased over days (from 0 to 80 s). Individual differences in the preference-delay curve emerged, with the identification of two distinct subpopulations, i.e. one with a nearly horizontal curve and another with a very steep ("impulsive") slope. The impulsivity profile was slightly but consistently reduced by chronic ALC administration. Consistent results were also obtained with methylphenidate (MPH, saline or 3 mg/kg IP twice daily). Impulsive rats exhibited a lower metabolite/serotonin (5HIAA/5HT) ratio in the medial frontal cortex (MFC) and lower noradrenaline (NA) levels in the MFC and cingulate cortex (CC) when compared with the other subgroup. The ALC treatment increased NA levels in the CC and the 5HIAA/5HT ratio in both CC and MFC. Present data suggest that ALC, a drug devoid of psychostimulant properties, may have some beneficial effects in the treatment of ADHD children. Ann N Y Acad Sci. 2004 Nov. In mammals, the carnitine pool consists of nonesterified L-carnitine and many acylcarnitine esters. Of these esters, acetyl-L-carnitine is quantitatively and functionally the most significant. Carnitine homeostasis is maintained by absorption from diet, a modest rate of synthesis, and efficient renal reabsorption. Dietary L-carnitine is absorbed by active and passive transfer across enterocyte membranes. Bioavailability of dietary L-carnitine is 54-87% and is dependent on the amount of L-carnitine in the meal. Absorption of L-carnitine dietary supplements (0.5-6 g) is primarily passive; bioavailability is 14-18% of dose. Unabsorbed L-carnitine is mostly degraded by microorganisms in the large intestine. Circulating L-carnitine is distributed to two kinetically defined compartments: one large and slow-turnover (presumably muscle), and another relatively small and rapid-turnover (presumably liver, kidney, and other tissues). At normal dietary L-carnitine intake, whole-body turnover time in humans is 38-119 h. In vitro experiments suggest that acetyl-L-carnitine is partially hydrolyzed in enterocytes during absorption. In vivo, circulating acetyl-L-carnitine concentration was increased 43% after oral acetyl-L-carnitine supplements of 2 g/day, indicating that acetyl-L-carnitine is absorbed at least partially without hydrolysis. After single-dose intravenous administration (0.5 g), acetyl-L-carnitine is rapidly, but not completely hydrolyzed, and acetyl-L-carnitine and L-carnitine concentrations return to baseline within 12 h. At normal circulating l-carnitine concentrations, renal l-carnitine reabsorption is highly efficient (90-99% of filtered load; clearance, 1-3 mL/min), but displays saturation kinetics. Thus, as circulating L-carnitine concentration increases (as after high-dose intravenous or oral administration of L-carnitine), efficiency of reabsorption decreases and clearance increases, resulting in rapid decline of circulating L-carnitine concentration to baseline. Elimination kinetics for acetyl-L-carnitine are similar to those for L-carnitine. There is evidence for renal tubular secretion of both L-carnitine and acetyl-L-carnitine. Future research should address the correlation of supplement dosage, changes and maintenance of tissue L-carnitine and acetyl-L-carnitine concentrations, and metabolic and functional changes and outcomes. Ann N Y Acad Sci. 2004 Nov. L-carnitine and acetyl-L-carnitine (ALC) are both used to improve mitochondrial function. Although it has been argued that ALC is better than l-carnitine in absorption and activity, there has been no experiment to compare the two compounds at the same dose. In the present experiment, the effects of ALC and L-carnitine on the levels of free, acyl, and total L-carnitine in plasma and brain, rat ambulatory activity, and biomarkers of oxidative stress are investigated. Aged rats (23 months old) were given ALC or L-carnitine at 0.15% in drinking water for 4 weeks. L-carnitine and ALC were similar in elevating carnitine levels in plasma and brain. Both increased ambulatory activity similarly. However, ALC decreased the lipid peroxidation (malondialdehyde, MDA) in the old rat brain, while L-carnitine did not. ALC decreased the extent of oxidized nucleotides (oxo8dG/oxo8G) immunostaining in the hippocampal CA1 and cortex, while L-carnitine did not. ALC decreased nitrotyrosine immunostaining in the hippocampal CA1 and white matter, while L-carnitine did not. In conclusion, ALC and L-carnitine were similar in increasing ambulatory activity in old rats and elevating carnitine levels in blood and brain. However, ALC was effective, unlike L-carnitine, in decreasing oxidative damage, including MDA, oxo8dG/oxo8G, and nitrotyrosine, in old rat brain. These data suggest that ALC may be a better dietary supplement than L-carnitine. Cochrane Database Syst Rev. 2004 Oct 18. Background: Apnea of prematurity is a common problem in preterm infants in the neonatal intensive care setting (NICU), often delaying their discharge home or transfer to a step down unit. Premature infants are at increased risk of carnitine deficiency. Carnitine supplementation has been used for both prevention and treatment of apnea. Objectives: To determine whether treatment with carnitine will reduce the frequency of apnea, the duration of ventilation and the duration of hospital stay in preterm infants with recurrent apnea. Search strategy: Computerised searches were carried out by two reviewers independently. Searches were made of MEDLINE (1966 to May 2004), EMBASE (1980 to May 2004), CINAHL (1982-2004 June 2004,1st week), the Cochrane Central Register of Controlled Trials (CENTRAL, The Cochrane Library, Issue 2, 2004), abstracts of annual meetings of the Society for Pediatric Research (1995-2004), and contacts were made with the subject experts. Selection criteria: Only randomized or quasi-randomized treatment trials of preterm infants with a diagnosis of recurrent apnea of prematurity were considered. Trials were included if they involved treatment with carnitine compared to placebo or no treatment, and measured at least one of the following outcomes: failure of resolution of apneas, the duration of ventilation and the duration of hospital stay. Data collection and analysis: Two reviewers evaluated the papers for inclusion criteria and quality. Corresponding authors were contacted for further information where needed. Main results: No eligible trials were identified. Reviewers' conclusions: Despite the plausible rationale for the treatment of apnea of prematurity with carnitine, there are insufficient data to support its use for this indication. Further studies are needed to determine the role of this treatment in clinical practice. Mol Aspects Med. 2004 Oct-Dec. L-Carnitine (L-C) is a naturally occurring quaternary ammonium compound endogenous in all mammalian species and is a vital cofactor for the mitochondrial oxidation of fatty acids. Fatty acids are utilized as an energy substrate in all tissues, and although glucose is the main energetic substrate in adult brain, fatty acids have also been shown to be utilized by brain as an energy substrate. L-C also participates in the control of the mitochondrial acyl-CoA/CoA ratio, peroxisomal oxidation of fatty acids, and the production of ketone bodies. Due to their intrinsic interaction with the bioenergetic processes, they play an important role in diseases associated with metabolic compromise, especially mitochondrial-related disorders. A deficiency of carnitine is known to have major deleterious effects on the CNS. Several syndromes of secondary carnitine deficiency have been described that may result from defects in intermediary metabolism and alterations principally involving mitochondrial oxidative pathways. Mitochondrial superoxide formation resulting from disturbed electron transfer within the respiratory chain may affect the activities of respiratory chain complexes I, II, III, IV, and V and underlie some CNS pathologies. This mitochondrial dysfunction may be ameliorated by L-C and its esters. In addition to its metabolic role, L-C and its esters such as acetyl-L-carnitine (ALC) poses unique neuroprotective, neuromodulatory, and neurotrophic properties which may play an important role in counteracting various disease processes. Neural dysfunction and metabolic imbalances underlie many diseases, and the inclusion of metabolic modifiers may provide an alternative and early intervention approach, which may limit further developmental damage, cognitive loss, and improve long-term therapeutic outcomes. The neurophysiological and neuroprotective actions of L-C and ALC on cellular processes in the central and peripheral nervous system show such effects. Indeed, many studies have shown improvement in processes, such as memory and learning, and are discussed in this review. Mol Aspects Med. 2004 Oct-Dec. The carnitine-acylcarnitine translocase (CACT) is one of the components of the carnitine cycle. The carnitine cycle is necessary to shuttle long-chain fatty acids from the cytosol into the intramitochondrial space where mitochondrial beta-oxidation of fatty acids takes place. The oxidation of fatty acids yields acetyl-coenzyme A (CoA) units, which may either be degraded to CO(2) and H(2)O in the citric acid cycle to produce ATP or converted into ketone bodies which occurs in liver and kidneys. Metabolic consequences of a defective CACT are hypoketotic hypoglycaemia under fasting conditions, hyperammonemia, elevated creatine kinase and transaminases, dicarboxylic aciduria, very low free carnitine and an abnormal acylcarnitine profile with marked elevation of the long-chain acylcarnitines. Clinical signs and symptoms in CACT deficient patients, are a combination of energy depletion and endogenous toxicity. The predominantly affected organs are brain, heart and skeletal muscle, and liver, leading to neurological abnormalities, cardiomyopathy and arrythmias, skeletal muscle damage and liver dysfunction. Most patients become symptomatic in the neonatal period with a rapidly progressive deterioration and a high mortality rate. However, presentations at a later age with a milder phenotype have also been reported. The therapeutic approach is the same as in other long-chain fatty acid disorders and includes intravenous glucose (+/- insulin) administration to maximally inhibit lipolysis and subsequent fatty acid oxidation during the acute deterioration, along with other measures such as ammonia detoxification, depending on the clinical features. Long-term strategy consists of avoidance of fasting with frequent meals and a special diet with restriction of long-chain fatty acids. Due to the extremely low free carnitine concentrations, carnitine supplementation is often needed. Acylcarnitine profiling in plasma is the assay of choice for the diagnosis at a metabolite level. However, since the acylcarnitine profile observed in CACT-deficient patients is identical to that in CPT2-deficient patients, definitive identification of CACT-deficiency in a certain patient requires determination of the activity of CACT. Subsequently, mutational analysis of the CACT gene can be performed. So far, 9 different mutations have been identified in the CACT gene. AIDS. 2004 Jul 23. Nucleoside analogue reverse transcriptase inhibitors (NRTI) disrupt neuronal mitochondrial DNA synthesis, impairing energy metabolism and resulting in a distal symmetrical polyneuropathy (DSP), an antiretroviral toxic neuropathy (ATN) that causes significant morbidity in HIV disease. Serum acetyl-l-carnitine (ALCAR) levels are decreased in neuropathy associated with NRTI therapy. ALCAR enhances neurotrophic support of sensory neurons and promotes energy metabolism, potentially causing nerve regeneration and symptom relief. Objective: To assess the efficacy of oral ALCAR (1500 mg twice daily) for up to 33 months in an open cohort of 21 HIV-positive patients with established ATN. Methods: Skin biopsies were excised from the leg before ALCAR treatment, at 6-12 month intervals thereafter and from HIV-negative non-neuropathic controls. Fibre types in epidermal, dermal and sweat gland innervation were quantified immunohistochemically. Results: After 6 month's treatment, mean immunostaining area for small sensory fibres increased (epidermis 100%, P = 0.006; dermis 133%, P < 0.05) by more than that for all fibre types (epidermis 16%, P = 0.04; dermis 49%, P < 0.05; sweat glands 60%, P < 0.001) or for sympathetic fibres (sweat glands 41%, P < 0.0003). Compared with controls, epidermal, dermal and sweat gland innervation reached 92%, 80% and 69%, respectively, after 6 month's treatment. Innervation improvements continued (epidermis and dermis) or stabilized (sweat glands) after 24 month's treatment. Neuropathic grade improved in 76% of patients and remained unchanged in 19%. HIV RNA load, CD4 and CD8 cell counts did not alter significantly throughout the study. Conclusions: ALCAR treatment improves symptoms, causes peripheral nerve regeneration and is proposed as a pathogenesis-based treatment for DSP. Mol Genet Metab. 2004 Apr. Marked progress has been made over the past 15 years in defining the specific biochemical defects and underlying molecular mechanisms of oxidative phosphorylation disorders, but limited information is currently available on the development and evaluation of effective treatment approaches. Metabolic therapies that have been reported to produce a positive effect include coenzyme Q(10) (ubiquinone), other antioxidants such as ascorbic acid and vitamin E, riboflavin, thiamine, niacin, vitamin K (phylloquinone and menadione), and carnitine. The goal of these therapies is to increase mitochondrial ATP production, and to slow or arrest the progression of clinical symptoms. In the present study, we demonstrate for the first time that there is a significant increase in ATP synthetic capacity in lymphocytes from patients undergoing cofactor treatment. We also examined in vitro cofactor supplementation in control lymphocytes in order to determine the effect of the individual components of the cofactor treatment on ATP synthesis. A dose-dependent increase in ATP synthesis with CoQ(10) incubation was demonstrated, which supports the proposal that CoQ(10) may have a beneficial effect in the treatment of oxidative phosphorylation (OXPHOS) disorders. Psychosom Med. 2004 Mar-Apr. Objectives: We compared the effects of acetylcarnitine, propionylcarnitine and both compounds on the symptoms of chronic fatigue syndrome (CFS). Methods: In an open, randomized fashion we compared 2 g/d acetyl-L-carnitine, 2 g/d propionyl-L-carnitine, and its combination in 3 groups of 30 CFS patients during 24 weeks. Effects were rated by clinical global impression of change. Secondary endpoints were the Multidimensional Fatigue Inventory, McGill Pain Questionnaire, and the Stroop attention concentration test. Scores were assessed 8 weeks before treatment; at randomization; after 8, 16, and 24 weeks of treatment; and 2 weeks later. Results: Clinical global impression of change after treatment showed considerable improvement in 59% of the patients in the acetylcarnitine group and 63% in the propionylcarnitine group, but less in the acetylcarnitine plus propionylcarnitine group (37%). Acetylcarnitine significantly improved mental fatigue (p =.015) and propionylcarnitine improved general fatigue (p =.004). Attention concentration improved in all groups, whereas pain complaints did not decrease in any group. Two weeks after treatment, worsening of fatigue was experienced by 52%, 50%, and 37% in the acetylcarnitine, propionylcarnitine, and combined group, respectively. In the acetylcarnitine group, but not in the other groups, the changes in plasma carnitine levels correlated with clinical improvement. Conclusions: Acetylcarnitine and propionylcarnitine showed beneficial effect on fatigue and attention concentration. Less improvement was found by the combined treatment. Acetylcarnitine had main effect on mental fatigue and propionylcarnitine on general fatigue. Excerpts from the full text article: Introduction Estimations of the prevalence of chronic fatigue syndrome (CFS) range from 1 to 4 (1,2). Although the cause of CFS is not known, sets of criteria for diagnosis of the condition have been published. The most widely accepted criteria were formulated in the consensus meeting supervised by the Centers for Disease Control and Prevention (CDC) (3). Patients with CFS are defined by the presence of clinically evaluated, unexplained persistent chronic fatigue of new or definite onset (not lifelong) resulting in substantial reduction in previous levels of activities, and the occurrence of 4 or more of the following symptoms (all of which must have persisted for 6 months and must not have predated the fatigue): self-reported severe impairment in short-term memory or concentration, sore throat, tender lymph nodes, muscle pain, multijoint pain without swelling or redness, headache of a new type, unrefreshing sleep, and postexertional malaise lasting more than 24 hours. Exclusions include a clear underlying organic cause, substance misuse, and severe psychiatric disorder such as psychotic depression. Less severe psychiatric disorders such as major depression without Diagnostic and Statistical Manual (DSM)-IV-defined melancholic features or anxiety disorders are not exclusionary diagnoses and are frequently comorbid with CFS (4). Kuratsune et al. (5) and Plioplys and Plioplys (6) reported a decrease in plasma acylcarnitine in CFS, but this was not confirmed by others (79). The first report on carnitine treatment in CFS was by Grau et al. (10), who found no effect. Plioplys and Plioplys (11) reported significant improvement of CFS symptoms after 2 months of 3 g daily orally administered L-carnitine. In a preliminary open label study, we treated 150 CFS patients with 1 g oral L-carnitine bid. After 6 months, 104 patients (69%) reported marked improvement by clinical global impression of change (CGI) ["I feel (very) much better"]. Another 18 CFS patients were included in a randomized double-blind study. Six were treated with oral acetyl-L-carnitine 1 g/d plus L-carnitine 1 g/d (low dose), 6 received twice the dosage (high dose), and 6 received placebos. After 6 months, marked improvement was reported by 4 patients in the low dosage group, by none in the high dosage group, and by 1 in the placebo group (unpublished results). Carnitine is essential for the mitochondrial oxidation of long-chain fatty acids (12), because it acts as a carrier of acyl groups across the inner mitochondrial membrane to the matrix for ß-oxidation. Later, many more functions were detected in detoxification of acyl-CoA and acylcarnitine export from the cell, increasing free CoA/acyl-CoA in the cell affecting lipid, protein, and carbohydrate metabolism [review (13)], membrane synthesis and repair (14), and ac(et)ylation of amino acids, histones, and proteins. The latter process is involved in posttranslational modification, membrane binding, and G-protein signaling. Carnitine promoted perfusion in ischemia (15), decreased oxidative stress (16,17), and attenuated damaging effects of mitochondrial inhibitors and uncoupler in neurons (18). Mitochondria were not the only organelles participating in the actions of the carnitine system; also peroxisomes, endo- and sarcoplasmic reticulum, and nuclear membrane metabolized carnitine (19). Moreover, every human cell (including the erythrocyte) contains carnitine palmitoyltransferase activity, and carnitine and acylcarnitines were detected in every cell compartment and body fluid (13). Many biochemical, pharmacological, and clinical studies have investigated the action of carnitine and its esters acetylcarnitine and propionylcarnitine. Acetylcarnitine is a universal mitochondrial energy source and acetyl donor. It promoted energy metabolism and neurotransmission in the aged or neurotoxin-treated or ischemic reperfused rodent brain [reviews (18,20)]. Promising results were obtained in human degenerative brain diseases [review (21)]. Propionylcarnitine is also an energy source, stimulated the Krebs cycle as a precursor of succinyl-CoA, decreased oxidative stress in various systems, and improved cardiac dysfunction in rodent models (20,23). It was found to be effective in human cardiovascular disorders [reviews (24,25)]. Acetyl- and propionylcarnitine appeared generally somewhat more effective than L-carnitine. Subjects and methods We compared the effect of acetyl-L-carnitine (ALC) and propionyl-L-carnitine (PLC) on complaints of patients with CFS. We chose to conduct an open exploratory study with a discussion with the participants about the experienced change in their clinical condition, supported by objective questionnaires. The sample size was based on the results of the 2 preliminary studies mentioned above, indicating improvement in 50% of the patients in the low dose groups and less than 20% in the high dosage group. The sample size was not based on differences between the ALC and the PLC groups because data were not available. The present trial included 90 patients with CFS according to the CDC criteria, in an open randomized study. Patients were recruited from the polyclinic at the CFS Research Centre Amsterdam. Excluded were patients with an evident underlying organic cause, substance misuse, and severe psychiatric disorder such as psychotic depression. Presence of exclusion criteria according to the CDC (3,4) was revealed by structured interview, physical examination, and extensive laboratory tests. [...] After randomization, patients received 2 g acetyl-L-carnitine per day in the ALC group, 2 g propionyl-L-carnitine in the PLC group, and 2 g ACL plus 2 g PLC in the ALC+PLC group. The medication was taken after breakfast and dinner for 24 weeks. The dropouts were analyzed during the whole trial, and their data were included in the results. The trial profile is presented in Figure 1. Assessed for eligibility were 114 patients, and 90 were enrolled in the study. Table 1 shows the gender distribution (78% females), the average age (3742 years), the duration of CFS (median 3 years or more), and the plasma carnitine levels at randomization, that were (about) the same in the ALC, the PLC, and the ALC+PLC group. The plasma carnitine and carnitine esters levels showed no abnormalities and were similar to those in controls (8). Eight patients stopped because of side effects - 3 in the ALC group, 2 in the PLC group, and 3 in the ALC+PLC group. They experienced an overstimulated feeling and sleeplessness. Another 8 patients stopped, because they did not experience any effect from the treatment - 4 in the ALC group, 1 in the PLC group, and 3 in the ALC+PLC group. Two patients stopped for reasons unrelated to the treatment. [...] Results Table 2 summarizes the CGIs after 8 weeks of no treatment and after 8, 16, and 24 weeks of treatment. Improvement was reported after 24 weeks of treatment by 59% of the ALC group, 63% of the PLC group, and 37% of the ALC+PLC group, whereas 10% in the ALC group, 3% in the PLC group, and 16% in the ALC+PLC group felt worse. After 24 weeks of therapy, the medication was stopped and all patients returned 2 weeks later for follow-up. Worsening of CFS in the 2 weeks was reported by 52% in the ALC group, 50% in the PLC group, and by 37% in the ALC+PLC group. No patients improved (Table 2). In the ALC group, 4 of the 17 patients who improved at 24 weeks experienced no change at follow-up, 2 weeks later. In the PLC group, improvement continued in 7 patients and in the ALC+PLC group in 3 patients (not shown). In the ALC group, 2 patients had not improved at 24 weeks, but worsened at follow-up. This occurred in 3 patients in the PLC group and 2 patients in the ALC+PLC group (not shown). Table 3 summarizes the general fatigue, physical fatigue, and mental fatigue scores. Significant improvement of general fatigue score was found in the PLC (p = .004) and in the ALC+PLC group (p = .000), and that of mental fatigue in the ALC group (p = .015). The physical fatigue score was not significantly improved in the PLC group (p = .069). Table 4 show the scores of the attention concentration test and the pain test. The score of the Stroop test is presented as median and quartiles because the distribution of the data in the PLC group at randomization showed a not-normal distribution. The attention concentration score improved significantly in all groups (Table 4b). The pain score was unexpectedly low in the ALC group (Table 4a). None of the treatments had significant effect on the pain scores (Table 4b). The CGI was correlated with the improvement in the general fatigue, physical and mental fatigue score in all the groups (r 0.362, p < .05), and with the attention concentration score in the ALC (r = 0.476, p = .010) and the ALC+PLC (r = 0.489, p = .006), but not in the PLC group. The CGI was not correlated with the pain score in any of the groups. The levels of plasma L-carnitine increased significantly in all groups of patients, but somewhat less in the responders. Surprisingly, carnitine levels in the group receiving ALC plus PLC became not considerably higher than in the other 2 groups. The levels of the carnitine esters increased in all groups, but remained low compared with L-carnitine (Table 5). The increase of carnitine levels in women was not different from that in the male participants. The change of the plasma L-carnitine concentration in the ALC group was inversely related to clinical improvement, and the least increase of carnitine was related to most improvement. This was not found in the PLC and ALC+PLC group. Change of plasma carnitine was related to improvement of general and mental fatigue in the ALC group, but not in the PLC and ALC+PLC group. Change of plasma carnitine was not related to change in attention-concentration or pain score. Plasma ALC and PLC were not related to clinical outcome (correlations not shown). Discussion The first aim of the study was to compare the different influences of acetylcarnitine and propionylcarnitine on symptoms of CFS. The second goal was to obtain an indication of the percentage of responders. Therefore, the study was designed as an observation period of 8 weeks, followed by a randomized trial and an observation period of 2 weeks. In the first observation period the scores for fatigue, concentration, and pain of the patients proved to be stable. The medication was well accepted by the patients, with a dropout number of 8 because of side effects. The nature of the side effects - overstimulation and sleeplessness - is different from those reported in most other studies, describing trimethylamine formation or diarrhea in a few patients. In advance, we had expected that ALC would have most effect on attention concentration, PLC on physical fatigue, and ALC+PLC on both. The CGI improved in the ALC and the PLC group, but not significantly in the ALC+PLC group, likely because of the higher total carnitine ester dose and indicating an inversed U-form doseresponse curve. An indication for the same effect was found in the preliminary double-blind pilot study. The effect of ALC on mental fatigue and attention concentration was significant. The PLC group showed most improvement in general and physical fatigue and slightly less in attention concentration. The ALC+PLC group improved very well on general fatigue and also, but not significantly, on physical and mental fatigue. The pain score was not influenced in a significant way by any medication. The CGI as an overall impression correlated significantly with the change of the 3 separate fatigue scores, thus validating the concept (not shown). In the present study, all patients had normal plasma free carnitine levels, and a normal spectrum of the acylcarnitine species, excluding primary and secondary carnitine deficiency (Table 1). In the second observation period, 50% of patients deteriorated in the ALC and PLC groups. For most the relapse was hardly acceptable, because they had adapted their activity to the improvement. The improved patients in the ALC+PLC group (37%) all deteriorated in the second observation period. The different effects of acetylcarnitine and propionylcarnitine could be explained by a different transport of the esters from plasma to the tissues and the brain. If so, the observation that the concentration of carnitine in the plasma in the ALC group increased more in patients with less therapeutic effect, indicates lower transport. It is conceivable that ALC and PLC are not transported as such, because the plasma ALC and PLC levels were relatively low and showed no correlation with improvement. The precise mode of action of the carnitine esters in alleviating the CFS symptoms is not clear. In this study, acetylcarnitine apparently had a more "central" action and propionylcarnitine a more "peripheral" action, which is in line with other human studies. It is generally thought that acetylcarnitine can easily pass the bloodbrain barrier, and that propionylcarnitine preferentially enters the heart. The fate of double-labeled acetylcarnitine has been studied by Kuratsune et al. (32). They showed that only the acetyl moiety is rapidly taken up by the brain, leaving the carnitine moiety in the circulation. This was in line with older studies [reviewed in (13)] establishing that carnitine import in the brain is a slow process with low affinity. Recently, however, with immortalized RBE4 cells, an in vitro model of the bloodbrain barrier, it was shown that these cells expressed the high-affinity carnitine transporter OCTN2, and transported carnitine, acetylcarnitine, and propionylcarnitine (33). When this is the case, a preferential uptake of acetylcarnitine by brain and a preferential uptake of propionylcarnitine by heart are less likely, because OCTN2 is also present in muscle and several other cells. Perhaps another messenger contributes to, or is involved in, the action of the carnitine esters, such as purines (34) or cortisol. Biopharm Drug Dispos. 2003 Nov. OCTN2 (SLC22A5), an organic cation/carnitine transporter, is widely distributed throughout the body, including the brain. In the present study, the involvement of OCTN2 in acetyl-L-carnitine (ALCAR) permeation across the blood-brain barrier (BBB) was examined using a microdialysis method in mouse. OCTN2 function was examined by comparison of wild-type mice with jvs mice, which express defective OCTN2 and are considered a model for primary systemic carnitine deficiency. Zero-net-flux method analysis indicated higher in vivo recovery of ALCAR and lower physiological ALCAR concentration in thalamus extracellular fluid (ECF) in jvs mice compared with wild-type mice. Externally added ALCAR showed significantly slower initial uptake across the BBB in jvs mouse. These results indicated that OCTN2 is functionally involved in ALCAR transfer across the BBB. Total radioactivity in ECF after i.v. administration of radiolabelled ALCAR remained constant for the rest of the experimental period. Accordingly, our results indicate that ALCAR is transported from blood to brain ECF by OCTN2 at least in part, and its concentration in brain ECF is regulated by other events such as protein binding and anabolic reactions in the brain, as well as by transport across the BBB. Pediatr Res. 2003 Nov. Perinatal hypoxia-ischemia remains a significant cause of neonatal mortality and neurodevelopmental disability. Numerous lines of evidence indicate that cerebral ischemic insults disrupt normal respiratory activity in mitochondria. Carnitine (3-hydroxy-4-N-trimethylammonium-butyrate) has an essential role in fatty acid transport in the mitochondrion and in modulating potentially toxic acyl-CoA levels in the mitochondrial matrix. There are no naturally occurring esterases available to reduce the accumulation of acyl-CoA but this process can be overcome by exogenous carnitine. We used a newborn rat model of perinatal hypoxia-ischemia to test the hypothesis that treatment with l-carnitine would reduce the neuropathologic injury resulting from hypoxia-ischemia in the developing brain. We found that treatment with l-carnitine during hypoxia-ischemia reduces neurologic injury in the immature rat after both a 7- and 28-d recovery period. We saw no neuroprotective effect when l-carnitine was administered after hypoxia-ischemia. Treatment with d-carnitine [synthetic form with reversed "twist"] resulted in an increase in mortality during hypoxia-ischemia. Carnitine is easy to administer, has low toxicity, and is routinely used in neonates as well as children with epilepsy, cardiomyopathy, and inborn errors of metabolism. l-Carnitine merits further investigation as a treatment modality for the asphyxiated newborn or as prophylaxis for the at-risk fetus or newborn. J Am Diet Assoc. 2003 Aug. Mitochondrial disorders are degenerative diseases characterized by a decrease in the ability of mitochondria to supply cellular energy requirements. Substantial progress has been made in defining the specific biochemical defects and underlying molecular mechanisms, but limited information is available about the development and evaluation of effective treatment approaches. The goal of nutritional cofactor therapy is to increase mitochondrial adenosine 5'-triphosphate production and slow or arrest the progression of clinical symptoms. Accumulation of toxic metabolites and reduction of electron transfer activity have prompted the use of antioxidants, electron transfer mediators (which bypass the defective site), and enzyme cofactors. Metabolic therapies that have been reported to produce a positive effect include Coenzyme Q(10) (ubiquinone); other antioxidants such as ascorbic acid, vitamin E, and lipoic acid; riboflavin; thiamin; niacin; vitamin K (phylloquinone and menadione); creatine; and carnitine. A literature review of the use of these supplements in mitochondrial disorders is presented. J Med Genet. 2003 Jun. Selections from the full text article: Fragile X syndrome (FXS) is a triplet repeat disorder caused by a large expansion of the CGG repeat in the 5'-untranslated region (UTR) of the fragile X mental retardation (FMR1) gene.1,2 Full mutation alleles are almost always associated with extensive hypermethylation of the repeat and of the upstream CpG island, which correlates with gene silencing and absence of the FMR1 protein. Cognitive function ranging from severe mental retardation to learning disabilities are found in affected people of both sexes. Many mildly affected people show "mosaic" methylation at the FMR1 promoter.3,4 Unusual alleles carrying a completely or partially unmethylated full mutation have been described.5,6 It was shown that in male patients with FXS with unmethylated alleles in the full mutation range, the FMR1 mRNA level is higher than in normal controls. This finding shows that upregulation of the FMR1 gene occurs in cells with unmethylated full mutation alleles and that the CGG triplet expansion does not suppress transcription directly.7 Thus, abnormal hypermethylation of the FMR1 promoter suppresses gene transcription. This hypothesis is also supported by the ability of 5-azadeoxycytidine (5-azadC) to restore the FMR1 gene expression in lymphoblastoid cell lines from patients with non-mosaic full mutation FXS by inducing DNA demethylation.8,9 The silencing of the hypermethylated FMR1 gene is consistent with a model in which methylation is coupled with the histone acetylation state. It has been found that the 5' end of the FMR1 gene of patients with FXS is associated with deacetylated histones H3 and H410 and that the treatment of fragile X cells with 5-azadC results in the reassociation of acetylated histones with the FMR1 promoter and transcriptional reactivation. This finding suggests that both methylation and histone deacetylation are linked to transcriptional inactivity.10,11 In fact, it has been shown that fragile X cell lines treated with histone hyperacetylating drugs can markedly potentiate the effect of 5-azadC on FMR1 gene expression.12 However, when used alone, such drugs induce only a modest reactivation of the FMR1 gene.12 The same pattern of dominance of DNA methylation over histone acetylation has also been reported for other genes, the promoter of which resides in a CpG island.13 Key points We report the effect of acetyl-L-carnitine and L-carnitine on the methylation of the FMR1 promoter in long term cultures. The methylation status of the FMR1 promoter region containing 52 CpG sites was analysed in lymphoblastoid cell lines derived from healthy subjects and patients with FXS by a sensitive bisulphite based technique. We also analysed the 23 CpG sites in exon 1 of the SNRPN gene. CpG sites in control cultures from healthy subjects remained unmethylated in all the experimental conditions described. No changes were seen in the SNRPN gene. The promoter region of the untreated fragile X cell lines remained generally hypermethylated although the methylation level of individual CpGs was variable. Both acetyl-L-carnitine and L-carnitine induced a modest though evident decrease of the FMR1 promoter hypermethylation in two of the three fragile X cell lines. Our data suggest that long term treatment with the two carnitines has a mild but detectable effect against methylation of the FMR1 alleles. Changes of the methylation patterns over a five year period of alleles from five brothers variably affected by FXS indicate that methylation of individual CpG cytosines is strikingly variable in hypermethylated genotypes obtained from an individual patient.14 A reduced frequency of hypermethylated alleles occurred in the leucocytes of the two mildly affected brothers. These findings suggest that maintenance of cytosine methylation is a dynamic process that favours unmethylated alleles. It is conceivable that some compounds can be identified that may modulate this process and achieve gene reactivation. Carnitine is a well known naturally occurring compound with an essential role in intermediary metabolism, mainly at the mitochondrial level. Acetyl-L-carnitine (-trimethyl-ß-acetyl-butyrrobetaine) is the carnitine ester naturally present in the central nervous system, differently distributed in the various areas.15 The enzyme carnitine acetyltransferase catalyses both the formation of acetyl-L-carnitine from carnitine and acetyl-coenzyme A (acetyl-CoA) and the reversible reaction. The modulation of the intracellular concentration of free CoA and acetyl-CoA is recognised to be a common mechanism for the various physiological activities of acetyl-L-carnitine,16,17 such as the acetylation of H4 histones.18 The chemical structure of acetyl-L-carnitine is similar to that of the acetylating agent butyrate. It has been shown that acetyl-L-carnitine, as well as butyrate, inhibits cytogenetic expression of the fragile X site in cultured lymphocytes of patients,19 suggesting that the interaction of these substances with the chromatin structure at the fragile site was present. Carnitine was also shown to suppress position effect variegation in Drosophila,20 another indication of a direct effect on chromatin. Finally, recent evidence suggests that acetyl-L-carnitine, the physiological form of carnitine, acts as a histone hyperacetylating agent at the FMR1 locus in fragile X cells.21 Because transcription of the FMR1 gene in fully mutated patients was obtained by treatment with butyrate,12 we decided to evaluate the effect of acetyl-L-carnitine and L-carnitine in lymphoblastoid cultures from patients with FXS. In the present study we investigated the effect on the CpG island methylation present in the FMR1 promoter after treatment with L-carnitine and acetyl-L-carnitine. We assessed the methylation status at 52 CpG sites of the FMR1 promoter using bisulphite treated, polymerase chain reaction (PCR) amplified genomic DNA obtained from lymphoblastoid cell cultures from healthy subjects and patients with FXS with CGG repeat expansions of different lengths. Furthermore, we controlled the effect of the two compounds on the methylation status of the putative promoter and exon 1 region of the gene called small nuclear ribonucleoprotein polypeptide N (SNRPN). This gene is reported to be involved in Prader-Willi syndrome and Angelman syndrome.22,23 It was chosen because in normal subjects only one allele is methylated, so each of the 23 CpGs present in this region will result in 50% methylated. In this way we could evaluate the effects of L-carnitine and acetyl-L-carnitine on another DNA region and our experimental approach at the same time. [...] Results In this study we investigated the effect of long term treatment with L-carnitine and acetyl-L-carnitine on the methylation status of the CpG island in the promoter region of the FMR1 gene by the bisulphite sequencing technique and on the transcription of the fully mutated FMR1 gene. Lymphoblastoid cell lines from patients with FXS and from healthy subjects were analysed during cell culture progression in the absence or presence either of L-carnitine or acetyl-L-carnitine. The fragile X cell cultures analysed (L, M, and F) were characterised by different CGG repeat expansions, which remained unchanged throughout the long term culturing as detected by Southern blot analysis. [...] The methylation status of the 52 tested CpG sites in cell cultures from patients with FXS is depicted in fig 2. The bisulphite sequencing of the FMR1 promoter was performed for each culture at the start of the cell culturing, after long term culturing, and after long term culturing in the presence of L-carnitine or acetyl-L-carnitine. Each column has been calculated as the ratio between the height of the thymine electropherogram peak and the sum of heights of cytosine and thymine peaks at the individual CpG site. Therefore, the black part of each column reflects the percentage of demethylation seen for each CpG site. A few individual CpG dinucleotides were partially unmethylated in all the starting cultures. In the F cell line the number of the sites that showed a partial demethylation increased from two to four in the control culture and to five in cells treated with L-carnitine and acetyl-L-carnitine. In this culture we found a striking spontaneous demethylation of sites 28 and 29. In the M cell line the CpG sites that are partially unmethylated increased from six and eight in the starting and untreated cultures, to 10 and 14 in cells treated with L-carnitine and acetyl-L-carnitine, respectively. In the L cell line the number of unmethylated CpG sites increased from four in the starting culture to six in the untreated culture, and to 13 in cells treated with L-carnitine and to 11 in cells treated with acetyl-L-carnitine. Only sites 28 and 42 were partially unmethylated in all experimental conditions. In particular, position 28 was unmethylated in up to 70% of the cells in the starting culture and in cultures treated with the two compounds. The mean methylation value for each culture, averaged on the 52 CpG sites, is summarised in table 1. The overall hypermethylated status of the L and M cell lines did not change after long term culturing with any added compound, but we found a decrease of mean methylation both with L-carnitine and acetyl-L-carnitine. The methylation degree of the F cell line did not change in long term cultures in the presence of the two compounds, although it showed a tendency to decrease methylation as well. The marked reduction of the mean methylation value in the untreated long term culture is the result of the unexpected high demethylation of only two sites (70% for site 28 and 60% for site 29). None of the 52 cytosines analysed in control cultures from healthy subjects was methylated in all the experimental conditions described confirming that methylation over time of the normal FMR1 gene is a very unlikely event. To control for the effect of our treatments on a non-pathologically methylated sequence, the human SNRPN sequence in the putative promoter, exon 1, and 5' region of intron 1 was also analysed. The methylation pattern of all individual 23 CpG sites in the DNA fragment analysed was evaluated. As expected, a 50% methylation was found in both control and fragile X derived starting cultures. The quite striking similarity between related electropherograms enables us to infer that not even a minimal change occurred after long term culturing either with or without the two carnitines (data not shown). The RT-PCR designed for detecting transcriptional reactivation of the FMR1 gene in the three tested fragile X cell lines was negative before treatment, as expected, but remained negative even after long term culturing with both acetyl-L-carnitine and L-carnitine; meanwhile FMR1 gene expression was detected in all cultures from normal subjects. Discussion The clinical features of fragile X syndrome are the result of the hampered transcriptional activity of the FMR1 promoter secondary to its hypermethylated status. Attempts to reactivate gene expression in cells with a CGG expansion in the full mutation range have been successful. This was obtained by reducing DNA methylation with 5-azadC,8,9 and, although to a lesser extent, by inducing histone hyperacetylation with drugs such as trichostatin A, 4-phenylbutyrate, and sodium butyrate.12 A general view recently emerged that histones regulate the access of proteins to the DNA and post-transcriptional histone modifications such as lysine acetylation or methylation mediate the epigenetic effects of DNA methylation patterns.26 It has become apparent that histone deacetylation follows DNA hypermethylation in a cascade process leading to chromatin inactivation11 and it was shown by chromatin immunoprecipitation that the inactive fully mutated FMR1 gene is associated with relatively hypoacetylated histones.10 It has been shown that USF1, USF2, and -Pal/Nrf-1 are the major transcription factors that bind the FMR1 promoter at the E box and -Pal/Nrf-1 DNA binding sites, respectively,25 as indicated in fig 1. The binding of -Pal/Nrf-1 is abolished by methylation, whereas the binding of USF1 and USF2 is only reduced.25 Thus, DNA methylation can have a direct, as well as an indirect, effect on the transcription of the FMR1 gene and seems to be dominant over histone acetylation, that is, FMR1 reactivation cannot take place efficiently only with histone hyperacetylating drugs, if DNA is not previously demethylated, as suggested by Chiurazzi et al.12 However, data obtained from studies on Neurospora crassa27 suggested that, at least in some instances, histone hyperacetylation may eventually cause DNA demethylation,11 just as DNA demethylation would induce histone reacetylation (and gene reactivation). We hypothesised that a long term treatment with histone hyperacetylating drugs may therefore be effective in reactivating FMR1 expression in fragile X cell cultures. Unfortunately, butyrate cannot be added for too long in culture because it readily causes cell cycle arrest, and previous experiments with sodium butyrate or 4-phenylbutyrate alone had to be limited to short treatments of 24-48 hours.12 Previous results from our groups have shown that acetyl-L-carnitine increases levels of H4 acetylation,17 also in the FMR1 gene itself.21 Therefore we were interested in exploring the DNA methylation status of the FMR1 promoter after long term treatment with acetyl-L-carnitine and L-carnitine. In the present study, no change in the CGG repeat expansion was detected by Southern blotting and long term treatment with the two carnitines that were well tolerated by cells that possibly seemed to benefit in growth and viability. However, no reactivation of FMR1 gene transcription has been shown in treated fragile X cell lines compared with the untreated ones. Experiments with RT-PCR were negative at the start of treatment in cell lines F, M, and L, but remained negative after the three month treatment with the two compounds. We found that the overall methylation status of the FMR1 promoter in fragile X lymphoblastoid cell lines is quite stable after culturing for about 100 cell duplications without any treatment. However, as previously reported,13 we found that methylation of individual CpG cytosine is variable in hypermethylated cell lines. On the other hand, we found a modest though measurable reduction of the hypermethylated status of the promoter in two of the three fragile X cell lines (L and M) grown with L-carnitine or acetyl-L-carnitine. It is noteworthy that the two compounds were less efficient in the F cell line, which harbours an expansion of more than 2.5 kb suggesting that acetyl-L-carnitine and L-carnitine may be somewhat effective in reversing the hypermethylation present in full mutations with smaller CGG expansions. The SNRPN gene is monoallelically expressed and has been used as an internal control for testing the potential demethylating effect of carnitines on another locus as well as for checking the efficiency of our experimental approach. The SNRPN gene is expressed from the paternal allele and hypermethylation is present only in the maternal allele. We consistently obtained a 1:1 ratio between the cytosine and thymine peaks at each of the 23 CpG sites investigated, before any treatment, confirming that our bisulphite sequencing approach was working well. Treatment with carnitines did not change the hemimethylated status of the SNRPN promoter region. These data suggest that carnitines do not affect the methylation status of the cell itself but may be effective in the abnormal hypermethylation of the FMR1 gene to move the methylation equilibrium towards the unmethylated status. Overall these data show that methylation of individual CpGs in the FMR1 gene is a dynamic process that seems to favour unmethylated alleles (see sites 28 and 29 of F cell lines). This trend seems to be favoured by the carnitines we used. We suppose that L-carnitine and acetyl-L-carnitine, acting on the histone hyperacetylation process, favour demethylation of the FMR1 gene. It is conceivable to suppose that much longer treatments with acetyl-L-carnitine and L-carnitine might further decrease the methylation status. Any future pharmacological attempt at reactivating the FMR1 gene in vivo should therefore contemplate the use of safe DNA demethylating drugs ideally targeted to the FMR1 promoter region. Brain Res. 2003 Apr 4. The transport of L-carnitine (4-N-trimethylamino-3-hydroxybutyric acid), a compound known to be transported by the organic cation transporter/carnitine transporter OCTN2, was studied in immortalized rat brain endothelial cells (RBE4). The cells were found to take up L-carnitine by a sodium-dependent process. This uptake process was saturable with an apparent Michaelis-Menten constant for L-carnitine of 54+/-10 microM and a maximal velocity of 215+/-35 pmol/mg protein/h. Besides L-carnitine, the cells also took up acetyl-L-carnitine and propionyl-L-carnitine in a sodium-dependent manner and TEA in a sodium-independent manner. RT-PCR with primers specific for the rat OCTN2 transporter revealed the existence of OCTN2 mRNA in RBE4 cells. Screening of a cDNA library from RBE4 cells with rat OCTN2 cDNA as a probe identified a positive clone which showed, when expressed in HeLa cells, the functional characteristics of OCTN2. The HeLa cells expressing the RBE4 OCTN2 cDNA showed a sixfold increase in L-carnitine uptake and a fourfold increase in TEA uptake in a sodium-containing buffer. Typical inhibitors for organic cation transporters (e.g. MPP(+) or TEA) showed an inhibitory effect on the transport of L-carnitine and TEA into the transfected cells. Similarly, unlabeled L-carnitine inhibited the transport of [3H]-L-carnitine and [14C]TEA in transfected HeLa cells. It is concluded that RBE4 cells, a widely used in vitro model of the blood-brain barrier (BBB), express the organic cation/carnitine transporter OCTN2. Bratisl Lek Listy. 2003. Medium chain acyl-CoA dehydrogenase (MCAD) deficiency is the most common disorder of fatty acid beta-oxidation and presents acutely with hypoglycemia, or a Reye-like illness with low free carnitine, often provoked by an infection or an excessive period of fasting. After acute attack these children are for the most time asymptomatic and may have normal plasma free carnitine concentrations. We observed a regularity in time course of serum free carnitine concentration during two attacks of Reye-like illness in patient with MCAD deficiency. Molecular investigation confirmed that the patient was homozygote for A985G mutation. Free carnitine was measured by enzymatic UV-test. First attack of severe hypoglycemia and Reye-like symptoms started at the age of 15 months and the second at the age of 25 months. In both episodes, treatment with intravenous glucose was given immediately, but without carnitine supplementation. Between the attacks patient was on a normal diet. In both attacks, low serum free carnitine concentration from the time of acute attack continually decreased for up to 8-13 days and then normalized at about 25 days after attack. We think that the time course of serum free carnitine may help in knowledge about carnitine depletion in MCAD deficiency. This is the first observation of this pattern during an acute attack and needs to be confirmed by other patients with MCAD deficiency. Neurosci Lett. 2002 Sep 6. We evaluated the role of acetyl-L-carnitine (ALCAR) in protecting primary motoneuron cultures exposed to excitotoxic agents or serum-brain derived neurotrophic factor (BDNF) deprived. To exclude that ALCAR works as a metabolic source, we compared its effects with those of L-carnitine (L-CAR), that seems to have no neurotrophic effect. A concentration of 10 mM ALCAR, but not L-CAR, significantly reduced the toxic effect of 50 microM N-methyl-D-aspartate (NMDA, % viability: NMDA 45.4+/-2.80, NMDA+ALCAR 90.8+/-11.8; P<0.01) and of 5 microM kainate in cultured motoneurons (% viability: kainate 40.66+/-10.73; kainate+ALCAR 63.80+/-13.88; P<0.05). The effect was due to a shift to the right of the dose-response curve for kainate (EC50 for kainate 5.99+/-1.012 microM; kainate+ALCAR 8.62+/-1.13 microM; P<0.05). ALCAR, but not L-CAR, significantly protected against BDNF and serum-deprivation reducing the apoptotic cell death (% viability respect to control: without BDNF/serum 61.8+/-13.3: without BDNF/serum+ALCAR 111.8+/-13.9; P<0.01). Immunocytochemistry showed an increase in choline acethyltransferase and tyrosine kinaseB receptors in motoneurons treated with ALCAR but not with L-CAR. These results suggest that ALCAR treatment improves the motoneurons activity, acting as a neurotrophic factor. Prostaglandins Leukot Essent Fatty Acids. 2002 Jul. To determine safety and the efficacy of carnitine treatment in children with attention-deficit hyperactivity disorder (ADHD). The ADHD behavior was observed by parents completing the Child Behavior Checklist (CBCL) and by teachers completing the Conners teacher-rating score, in a randomized, double-blind, placebo-controlled double-crossover trial. In 13/24 boys receiving carnitine, home behavior improved as assessed with the CBCL total score (P < 0.02). In 13/24 boys, school behavior improved as assessed with the Conners teacher-rating score (P < 0.05). Before treatment, the CBCL total and sub-scores were significantly different from those of normal Dutch boys (P < 0.0001). Responders showed a significant improvement of the CBCL total scores compared to baseline (P < 0.0001). In the majority of boys no side effects were seen. At baseline and after carnitine treatment, responders showed higher levels of plasma-free carnitine (P < 0.03) and acetylcarnitine (P < 0.05). Compared to baseline, the carnitine treatment caused in the responsive patients a decrease of 20-65% (8-48 points) as assessed by the CBCL total problem rating scale. Treatment with carnitine significantly decreased the attention problems and aggressive behavior in boys with ADHD. Proc Natl Acad Sci USA. 2002 Feb 19. Accumulation of oxidative damage to mitochondria, protein, and nucleic acid in the brain may lead to neuronal and cognitive dysfunction. The effects on cognitive function, brain mitochondrial structure, and biomarkers of oxidative damage were studied after feeding old rats two mitochondrial metabolites, acetyl-l-carnitine (ALCAR) [0.5% or 0.2% (wt/vol) in drinking water], and/or R-alpha-lipoic acid (LA) [0.2% or 0.1% (wt/wt) in diet]. Spatial memory was assessed by using the Morris water maze; temporal memory was tested by using the peak procedure (a time-discrimination procedure). Dietary supplementation with ALCAR and/or LA improved memory, the combination being the most effective for two different tests of spatial memory (P < 0.05; P < 0.01) and for temporal memory (P < 0.05). Immunohistochemical analysis showed that oxidative damage to nucleic acids (8-hydroxyguanosine and 8-hydroxy-2'-deoxyguanosine) increased with age in the hippocampus, a region important for memory. Oxidative damage to nucleic acids occurred predominantly in RNA. Dietary administration of ALCAR and/or LA significantly reduced the extent of oxidized RNA, the combination being the most effective. Electron microscopic studies in the hippocampus showed that ALCAR and/or LA reversed age-associated mitochondrial structural decay. These results suggest that feeding ALCAR and LA to old rats improves performance on memory tasks by lowering oxidative damage and improving mitochondrial function. Proc Natl Acad Sci USA. 2002 Feb 19. Mitochondrial-supported bioenergetics decline and oxidative stress increases during aging. To address whether the dietary addition of acetyl-l-carnitine [ALCAR, 1.5% (wt/vol) in the drinking water] and/or (R)-alpha-lipoic acid [LA, 0.5% (wt/wt) in the chow] improved these endpoints, young (2-4 mo) and old (24-28 mo) F344 rats were supplemented for up to 1 mo before death and hepatocyte isolation. ALCAR+LA partially reversed the age-related decline in average mitochondrial membrane potential and significantly increased (P = 0.02) hepatocellular O(2) consumption, indicating that mitochondrial-supported cellular metabolism was markedly improved by this feeding regimen. ALCAR+LA also increased ambulatory activity in both young and old rats; moreover, the improvement was significantly greater (P = 0.03) in old versus young animals and also greater when compared with old rats fed ALCAR or LA alone. To determine whether ALCAR+LA also affected indices of oxidative stress, ascorbic acid and markers of lipid peroxidation (malondialdehyde) were monitored. The hepatocellular ascorbate level markedly declined with age (P = 0.003) but was restored to the level seen in young rats when ALCAR+LA was given. The level of malondialdehyde, which was significantly higher (P = 0.0001) in old versus young rats, also declined after ALCAR+LA supplementation and was not significantly different from that of young unsupplemented rats. Feeding ALCAR in combination with LA increased metabolism and lowered oxidative stress more than either compound alone. Excerpts from the full text article: [...] Mitochondria are targets of their own oxidant by-products. The steady-state oxidative damage in mitochondria is high relative to other organelles, and the percentage of oxygen converted to superoxide increases with age (3, 4, 5, 6). This leads to a vicious cycle of increasing mitochondrial damage, which adversely affects cell function (7), and results in a loss of ATP-generating capacity, especially in times of greater energy demand, thereby compromising vital ATP-dependent reactions. Cellular processes affected by mitochondrial decay include detoxification, repair systems, DNA replication, osmotic balance, and higher-order >processes (7), such as cognitive function (7, 8, 9). [...] Several dietary supplements, including the mitochondrial cofactor and antioxidant lipoic acid (LA), increase endogenous antioxidants or mitochondrial bioenergetics (13, 14, 15). Feeding old rats acetyl-l-carnitine (ALCAR), a mitochondrial metabolite, reverses the age-related decline in tissue carnitine levels and improves mitochondrial fatty acid beta-oxidation in the tissues studied (15, 16, 17, 18). ALCAR supplementation also reverses the age-related alterations in fatty acid profiles and loss in cardiolipin levels, an essential phospholipid required for mitochondrial substrate transport (15, 16, 17). We demonstrated that ALCAR supplementation reverses the age-associated decline in metabolic activity in rats, suggesting that ALCAR improves mitochondrial function and increases general metabolic activity (19, 20). ALCAR-induced improvement in metabolic parameters appear to be responsible for improving short-term memory deficits and cognitive function in elderly subjects given ALCAR (21, 22) and in old rats (9). This increased metabolic activity may come at a price, however, because supplementing rats with high levels of ALCAR lowered hepatocellular antioxidant status (19). This ALCAR-induced antioxidant loss was not seen, however, in other organs (T.M.H. and D. Heath, unpublished work) or when lower doses were given (23). We also showed that giving high [1.5% (wt/vol)], but not lower [0.5% (wt/vol)], supplemental doses of ALCAR to old rats increased mitochondrial oxidant flux, suggesting that while high ALCAR supplementation may increase electron flow through the electron transport chain, it also heightens formation of ROS as a consequence. We thus hypothesized that ALCAR supplemented with an antioxidant may have the salutary effect of increasing mitochondrial function and general metabolic activity without a concomitant increase in oxidative stress. We chose LA as a cosupplement for two reasons: (i) it is a naturally occurring cofactor for mitochondrial alpha-keto acid dehydrogenases (24), which may aid in cellular glucose-dependent ATP production (25); and (ii) in its reduced form, LA is a potent antioxidant and also increases intracellular ascorbate and glutathione concentrations (15, 26). Thus, LA and ALCAR may act together to reverse age-related metabolic decline and also reduce indices of oxidative stress. We show that the combined supplementation of ALCAR and LA (ALCAR+LA) reverses age-related metabolic decline, improves hepatocellular ascorbate levels, and lowers oxidant appearance and oxidative damage. Materials and Methods [...] ALCAR Supplementation. Old and young rats were given a 1.5% (wt/vol; pH adjusted to ~6) solution of ALCAR in their drinking water and allowed to drink ad libitum for 1 mo before death and hepatocyte isolation. Both young and old rats typically drank ~20 ml/rat per day (data not shown), which provided a daily ALCAR dose of ~0.75 g/kg body wt per day for old rats and 1.2 g/kg body wt per day for young rats. LA Supplementation. Young and old rats were given LA [0.5% (wt/wt)] mixed into the AIN-93M chow (Dyets, Bethlehem, PA) for 2 weeks before death. Unsupplemented animals were fed Purina rodent chow and water ad libitum. The pellets were made into a mush and fed to some young and old rats for 2 weeks before cell isolation. Both young and old rats typically ate ~15 g/rat per day (data not shown), which provides a daily LA dose of 0.12 g/kg body weight for young rats and 0.075 g/kg body weight for old rats. Results [...] We previously showed significantly lower average mitochondrial membrane potential (delta-¦·) in the majority of hepatocytes from old rats compared with young rats, but a 1-mo feeding regimen of 1.5% (wt/vol) ALCAR reversed this decline in delta-¦· (19). For the present study, we also found a marked age-related decline in this key parameter of mitochondrial function (Fig. 1). Relative to mean fluorescence characteristics seen in hepatocytes from young unsupplemented animals, the average delta-¦· for hepatocytes from old rats was 53.8 +- 8.0% lower (n = 5), representing a significant loss (P = 0.02). Feeding ALCAR+LA to old rats markedly reversed this decline (Fig. 1). Old rats on the ALCAR+LA supplemented diet had an average delta-¦· that was only 22.8 +- 6.0% lower relative to young unsupplemented rats. Thus, dietary supplementation with ALCAR+LA partially restored the loss of mitochondrial delta-¦· although the improvement was not as great as previously observed with ALCAR alone (19). We previously showed in separate reports that ALCAR or LA supplementation increased hepatocellular and myocardial oxygen consumption, indicating that either compound was able to increase cellular metabolism (18, 19). Young and old rats were supplemented with or without ALCAR+LA before cell isolation, and this general parameter of metabolic rate was monitored by using an oxygen electrode. Hepatocellular oxygen consumption declined from 1.03 +- 0.17 (n = 5) to 0.54 +- 0.09 ¦Ģmol/min per 106 cells (n = 5) in young versus old unsupplemented rats, a significant (P = 0.03) decline of 47.6% with age. These results are in agreement with our previous results and suggest that there is an age-related decline in hepatocellular metabolic rate. Oxygen consumption in hepatocytes from old rats treated with ALCAR+LA was 0.82 +- 0.07 ¦Ģmol O2/min per 106 cells versus 0.95 +- 0.05 umol/min per 106 in unsupplemented (n = 5) young rats (P = 0.02). Thus, feeding ALCAR+LA to old rats significantly reversed the age-related decline in hepatocellular oxygen consumption. Ambulatory Activity. To further explore whether ALCAR+LA generally improved metabolic rate on a whole animal basis, we studied ambulatory activity in animals fed with or without ALCAR+LA. Old rats exhibited a 3-fold decline in ambulatory activity in terms of overall movement and the amount of time spent in movement (Table 1). The speed of old animals when in movement was not different from that shown by young animals, suggesting that the age-related decline in activity was not caused by pain or the inability to move, but rather it reflected a general loss of metabolic activity. Animals were then fed ALCAR+LA for 1 mo (in the case of LA 2 weeks) and again tested for ambulatory activity. Results show that ALCAR+LA significantly improved ambulatory activity in young and old animals. For the young animals, the amount of active time and the overall distance traveled increased by ”Ö31% when compared with their activity before ALCAR+LA supplementation. A much greater increase was observed in old rats. Ambulation and overall distance traveled more than doubled from 20 +- 2 s per movement and 177 +- 19 cm/h to 43 +- 3 s per movement and 376 +- 23 cm/h, respectively. This increase, although still not as good, on average, as young untreated rats, nevertheless represented a significant (P = 0.03) improvement versus that of old untreated animals. Thus, ALCAR+LA supplementation not only reverses the age-related decline in oxygen consumption, a cellular parameter of metabolic activity, but also increases ambulatory activity, a general physiological parameter of metabolic activity. Antioxidant Status/Oxidative Stress. We previously observed that feeding 1.5% (wt/vol) ALCAR alone to old rats, although markedly increasing metabolic activity through improved mitochondrial function, also resulted in heightened oxidant production and decreased low molecular weight antioxidant status. This finding was presumably caused by increased formation of ROS/reactive nitrogen species as by-products of heightened metabolic activity. To understand whether feeding ALCAR+LA could ameliorate this potential increase in oxidative stress, we measured ascorbic acid status, overall oxidant production, and markers of oxidative damage in freshly isolated hepatocytes taken from young and old rats fed with or without ALCAR+LA. Hepatocytes from old rats had significantly lower ascorbate levels as compared with young rats (7.29 +- 2.97 versus 3.38 +- 0.67; P = 0.003) (Fig. 2), suggesting that liver antioxidant status may be compromised with age. We observed, as previously, that ALCAR supplementation at 1.5% (wt/vol) resulted in a further and significant decline in ascorbate levels beyond the observed age-related loss in this key antioxidant. However, ALCAR+LA supplementation reversed the ALCAR-induced and age-related loss of ascorbate such that there was no longer a significant difference (P = 0.3) in hepatocellular ascorbate values between ALCAR+LA-treated old rats and that of untreated young animals (Fig. 2). To further investigate whether ALCAR+LA actually affected oxidative stress parameters in old rats, hepatocellular oxidant production was monitored by using 2',7'-dichlorofluorescin oxidation. This cell permeant dye becomes fluorescent when it is oxidized. Thus, general oxidant production can be monitored in cells by measuring the rate of increased fluorescence over time. Cells isolated from young and old rats exhibited a marked difference in fluorescence appearance (Fig. 3). Oxidant production increased over 30.8% with age from 2,942.3 +- 99.3 to 3,835.22 +- 303.6 fluorescence units/min per umol O2 consumed per 106 cells. This finding is in agreement with our previously published results (18) and is consistent with lower antioxidant status and heightened mitochondrial oxidant production during aging. Addition of ALCAR+LA to the diet of old rats caused a significant decline in appearance of oxidants to 2,801.79 +- 308.0 fluorescence units/min per umol O2 consumed per 106 cells, which was not different from untreated or ALCAR+LA-fed young rats. Thus, the combination of ALCAR with LA not only reverses the age-related increase in oxidants, but also the additional oxidants induced by high doses of ALCAR. These results suggest that ALCAR+LA supplementation not only improves metabolic rate and physiological activity, but does so without causing a concomitant increase in oxidants. To further assess whether ALCAR+LA modulated age-related and ALCAR-induced oxidative stress, we also measured steady-state levels of MDA, a marker of lipid peroxidation (Fig. 4). Hepatocellular MDA levels in old untreated rats were more than 4-fold higher than the levels seen in young rats, a significant increase (P = 0.0001). Similar to results shown for oxidant production, we observed a small, but significant, increase in steady-state MDA levels in liver tissue from old rats fed ALCAR alone (Fig. 4); on average, a similar increase in young rats was not significant. These results again suggest that high ALCAR alone, although improving metabolism and cognitive function, also increased oxidative stress in the liver. When LA was given along with ALCAR, we observed that there was a significant decline in MDA levels (Fig. 4). Most importantly, hepatic MDA concentrations in old ALCAR+LA fed rats no longer statistically differed from those found in young untreated animals. Discussion We previously demonstrated that feeding old rats ALCAR markedly improves the average mitochondrial membrane potential, a key indicator of mitochondrial function, to a level no longer significantly different from that of young rats (18). This reversal of membrane potential appears to be caused, in part, by replenishment of carnitine, a betaine that shuttles fatty acids into the mitochondrion for beta-oxidation. ALCAR administration also appears to reverse the age-related decline in cardiolipin levels. Cardiolipin is a key phospholipid cofactor for a number of mitochondrial substrate transporters as well as the protein complexes in the electron transport chain. Thus, age-related decline in cardiolipin could profoundly and adversely affect mitochondria. Our results, in combination with studies by Hagen, Paradies, Gadaleta, and others (15, 16), clearly demonstrate that ALCAR improves metabolic function in a number of tissues, most likely by improving substrate and electron flux through mitochondria. ALCAR does not, however, improve one aspect of mitochondrial decay in old rats, namely, the age-related increase in oxidants. Electron transfer through the mitochondrial electron transport chain becomes less efficient with age, which leads to increased oxidant leakage. ALCAR at the 1.5% level used in our initial experiments appears to increase electron flow through the electron transport chain, which further increases the appearance of ROS. In support of this concept, we observed higher oxidant appearance and lower hepatocellular antioxidant status after ALCAR supplementation (18). Feeding 1.5% ALCAR to old rats improved the age-related decline in metabolic rate, but increased oxidant appearance to a small, yet significant, degree. This ALCAR-induced increase in hepatocellular oxidative stress may be unique to the liver or caused by the relatively high levels of ALCAR used in this feeding study. In recent studies where old rats were fed 1.5% ALCAR, we did not observe any ALCAR-induced increased oxidative stress in the heart but saw a significant improvement in mitochondrial function and cellular metabolism (T.M.H., J. Suh, and D. Heath, unpublished results). In other studies using lower ALCAR doses [0.5% (wt/vol)], Liu et al.(22) noted no ALCAR-induced changes in parameters of oxidative stress in rat brain, yet found that this dose significantly improved cognitive function in aged animals (9). Thus, smaller doses of ALCAR may effectively improve metabolic function without higher oxidative stress. The rationale for the present study was to determine whether other mitochondrial metabolites fed along with ALCAR could improve metabolic parameters and lower the age-related increase in oxidative stress. We chose to cosupplement LA with ALCAR because LA is easily taken up into a variety of tissues and can be reduced to a powerful antioxidant, dihydrolipoic acid (23). Even though LA/dihydrolipoic acid is quickly removed from most cells, this compound also induces cystine/cysteine uptake and can thereby increase glutathione synthesis (25). LA supplementation maintains and actually reverses the age-related decline in hepatocellular and myocardial ascorbate and glutathione levels, even when cells were incubated with tert-butylhydroperoxide, a model alkyl peroxide (32, 33). Thus, LA may not only act synergistically with ALCAR to improve mitochondrial-supported bioenergetics but may also improve general antioxidant status, which declines with age. LA elicits other cell responses that may complement the actions of ALCAR on the cell. LA enhances glucose uptake by increasing glucose transporters at the surface of cells (24). It is also a cofactor for alpha-keto acid dehydrogenases found in the mitochondria, and its supplementation in the diet of aging animals may thus correct any age-associated decline in alpha-ketoglutarate dehydrogenase and pyruvate dehydrogenase caused by lost cofactors. Humphries and Szweda (34) showed that pyruvate dehydrogenase and alpha-ketoglutarate dehydrogenase can be modified by adduction with 4-hydroxy-nonenal (R. Moreau and T. M. Hagen, personal communication), rendering it unable to transfer acetyl groups. MDA and 4-hydroxy-nonenal also inactivate carnitine acyltransferase and decrease the binding affinity for substrates (35). Thus, LA may act synergistically with ALCAR to improve both fatty acid and glucose catabolism and energy production. Indeed, we have previously shown that LA alone also increases oxygen consumption and mitochondrial membrane potential, although not as effectively as ALCAR (14). Supplementing the diet of old rats with ALCAR+LA significantly improves many of the most frequently encountered age-related changes in mammals - namely loss of energy metabolism, increased oxidative stress, decreased physical activity, and as shown in ref. 9, impaired cognitive function. This affect on cognitive function has been previously observed for both ALCAR and LA (36), but to our knowledge, has not been observed for the combination of the two supplements. How ALCAR and LA affect short-term memory is not well understood, but may be caused by a number of factors, including increased neurotransmitter production, improved mitochondrial function, and/or calcium handling by the neuron (20, 21, 36). We have also recently found that LA alone significantly reduces the age-related accumulation of iron and copper in the brain (J. H. Suh, personal communication). Thus, the LA component may also increase neuro-cognitive function by potentially lowering iron and copper-induced oxidative stress. Presently, only short-term feeding regimens of ALCAR+LA have been given to aged animals. The present study suggests that long-term feeding experiments are warranted to monitor how effectively ALCAR+LA supplementation ameliorates oxidative stress, loss of metabolic function, and mild cognitive impairment seen in older animals... J Neurochem. 2001 Dec. Transport of L-[3H]carnitine and acetyl-L-[3H]carnitine at the blood-brain barrier (BBB) was examined by using in vivo and in vitro models. In vivo brain uptake of acetyl-L-[3H]carnitine, determined by a rat brain perfusion technique, was decreased in the presence of unlabeled acetyl-L-carnitine and in the absence of sodium ions. Similar transport properties for L-[3H]carnitine and/or acetyl-L-[3H]carnitine were observed in primary cultured brain capillary endothelial cells (BCECs) of rat, mouse, human, porcine and bovine, and immortalized rat BCECs, RBEC1. Uptakes of L-[3H]carnitine and acetyl-L-[3H]carnitine by RBEC1 were sodium ion-dependent, saturable with K(m) values of 33.1 +/- 11.4 microM and 31.3 +/- 11.6 microM, respectively, and inhibited by carnitine analogs. These transport properties are consistent with those of carnitine transport by OCTN2. OCTN2 was confirmed to be expressed in rat and human BCECs by an RT-PCR method. Furthermore, the uptake of acetyl-L-[3H]carnitine by the BCECs of juvenile visceral steatosis (jvs) mouse, in which OCTN2 is functionally defective owing to a genetical missense mutation of one amino acid residue, was reduced. The brain distributions of L-[3H]carnitine and acetyl-L-[3H]carnitine in jvs mice were slightly lower than those of wild-type mice at 4 h after intravenous administration. These results suggest that OCTN2 is involved in transport of L-carnitine and acetyl-L-carnitine from the circulating blood to the brain across the BBB. Pediatr Hematol Oncol. 2001 Dec. Carnitine is not only obtained from animal-derived foods but also synthesized in the body. It plays an important role in the energy metabolism of many tissues, including heart and skeletal muscles. Iron is known to be essential for the biosynthesis of carnitine. Although many conditions are well known to cause secondary carnitine deficiency, iron deficiency, which is a very common condition in children, is not well studied as a cause of secondary carnitine deficiency in humans. This study demonstrates the coexistence of iron deficiency and low carnitine levels in otherwise healthy children. The mean carnitine concentration of 18 otherwise healthy children with iron deficiency anemia was significantly lower compared to the mean carnitine concentration of healthy children without iron deficiency anemia. Based on the evidence about the effect of low iron on carnitine stores in experimental animals, we proposed that low serum carnitine levels in these children may be secondary to iron deficiency. However, further studies need to be done to further clarify this relationship. J Neurosci Res. 2001 Oct 15. The effects of a carnitine derivative, acetyl-L-carnitine (ALCAR), on the cognitive and cholinergic activities of aging rats were examined. Rats were given ALCAR (100 mg/kg) per os for 3 months and were subjected to the Hebb-Williams tasks and a new maze task, AKON-1, to assess their learning capacity. The learning capacity of the ALCAR-treated group was superior to that of the control. Cholinergic activities were determined with synaptosomes isolated from the cortices. The high-affinity choline uptake by synaptosomes, acetylcholine synthesis in synaptosomes, and acetylcholine release from synaptosomes on membrane depolarization were all enhanced in the ALCAR group. This study indicates that chronic administration of ALCAR increases cholinergic synaptic transmission and consequently enhances learning capacity as a cognitive function in aging rats. Rev Neurol. 2001 Oct. Hyperactivity is a significant problem for almost all young males affected by fragile X syndrome (FXS), the most common inherited disease causing mental retardation. Therapeutical approaches are actually based on Central Nervous System (CNS) stimulants lacking a well defined rationale and efficacy while they further decrease the patient's limited attention span. A pilot study on 17 fragile X male treated with L-acetylcarnitine (LAC) over one year, showed a significant reduction of their hyperactivity behaviour tested by the Conners Abbreviated Parent-Teacher Questionnaire. LAC use in FXS patients derives from the hypothesis that the biochemical and physiological properties this substance has may preserve brain activity. LAC is a small, hydrosoluble molecule that easily diffuses in the extracellular space and enters any cell in the nervous system through specific transporters. Different cerebral areas use this molecule differently to metabolize glucose and lipids to provide for ATP and neurotrasmitters synthesis. The acetyl group LAC carriers represents a key metabolic signaling element possibly mediating its effect in the CNS. The exogenous administration of LAC may affect brain activity in FXS by: I) modulation of fuel partitioning for energy production, which at the mitochondrial level is associated with the Kreb's cycle metabolic role in neurotransmitter synthesis; II) remodelling of lipid membrane in terms of LAC actively determining the production of polyunsaturated fatty acids; III) preferential effect on the attention component of the cholinergic system which relies on its peculiar modality of communication in the CNS. Based on the above premises an explorative, double-blind, placebo controlled, multicenter study is ongoing. A total population of 160 children from nine European centers will be enrolled. The objective of this study is to determine the effect of LAC on the hyperactive behaviour of FXS children as evaluated by the administration of the Conners Abbreviated Parent Questionnaire. Med Hypotheses. 2001 May. A new technique for controllable elevation of night time growth hormone (GH) release in adult humans involves a synergy between oral intake of the naturally occurring compounds acetyl-L-carnitine (500 mg) and L-ornithine (25-100 mg) taken at night time sleep after a 3 to 4 hour fast. The set point for normal hypothalamic GH release appears to include a 'whole body' mitochondrial State 3 status 'feed back loop' controlled by systemic acetyl-L-carnitine levels. Mol Psychiatry. 2000 Nov. Acetyl-L-carnitine (ALCAR) contains carnitine and acetyl moieties, both of which have neurobiological properties. Carnitine is important in the beta-oxidation of fatty acids and the acetyl moiety can be used to maintain acetyl-CoA levels. Other reported neurobiological effects of ALCAR include modulation of: (1) brain energy and phospholipid metabolism; (2) cellular macromolecules, including neurotrophic factors and neurohormones; (3) synaptic morphology; and (4) synaptic transmission of multiple neurotransmitters. Potential molecular mechanisms of ALCAR activity include: (1) acetylation of -NH2 and -OH functional groups in amino acids and N terminal amino acids in peptides and proteins resulting in modification of their structure, dynamics, function and turnover; and (2) acting as a molecular chaperone to larger molecules resulting in a change in the structure, molecular dynamics, and function of the larger molecule. ALCAR is reported in double-blind controlled studies to have beneficial effects in major depressive disorders and Alzheimer's disease (AD), both of which are highly prevalent in the geriatric population. Biochim Biophys Acta. 2000 Jun 26. The mitochondrial carnitine system plays an obligatory role in beta-oxidation of long-chain fatty acids by catalyzing their transport into the mitochondrial matrix. This transport system consists of the malonyl-CoA sensitive carnitine palmitoyltransferase I (CPT-I) localized in the mitochondrial outer membrane, the carnitine:acylcarnitine translocase, an integral inner membrane protein, and carnitine palmitoyltransferase II localized on the matrix side of the inner membrane. Carnitine palmitoyltransferase I is subject to regulation at the transcriptional level and to acute control by malonyl-CoA. The N-terminal domain of CPT-I is essential for malonyl-CoA inhibition. In liver CPT-I activity is also regulated by changes in the enzyme's sensitivity to malonyl-CoA. As fluctuations in tissue malonyl-CoA content are parallel with changes in acetyl-CoA carboxylase activity, which in turn is under the control of 5'-AMP-activated protein kinase, the CPT-I/malonyl-CoA system is part of a fuel sensing gauge, turning off and on fatty acid oxidation depending on the tissue's energy demand. Additional mechanism(s) of short-term control of CPT-I activity are emerging. One proposed mechanism involves phosphorylation/dephosphorylation dependent direct interaction of cytoskeletal components with the mitochondrial outer membrane or CPT-I. We have proposed that contact sites between the outer and inner mitochondrial membranes form a microenvironment which facilitates the carnitine transport system. In addition, this system includes the long-chain acyl-CoA synthetase and porin as components. Basic Res Cardiol. 2000 Apr. This review focuses on the regulation of myocardial fatty acids and glucose metabolism in physiological and pathological conditions, and the role of L-carnitine and of its derivative, propionyl-L-carnitine. Fatty acids are the major oxidation fuel for the heart, while glucose and lactate provide the remaining need. Fatty acids in cytoplasm are transformed to long-chain acyl-CoA and transferred into the mitochondrial matrix by the action of three carnitine dependent enzymes to produce acetyl-CoA through the beta-oxidation pathway. Another source of mitochondrial acetyl-CoA is from the oxidation of carbohydrates. The pyruvate dehydrogenase (PDH) complex, the key irreversible rate limiting step in carbohydrate oxidation, is modulated by the intra-mitochondrial ratio acetyl-CoA/CoA. An increased ratio results in the inhibition of PDH activity. A decreased ratio can relieve the inhibition of PDH as shown by the transfer of acetyl groups from acetyl-CoA to carnitine, forming acetylcarnitine, a reaction catalyzed by carnitine acetyl-transferase. This activity of L-carnitine in the modulation of the intramitochondrial acetyl-CoA/CoA ratio affects glucose oxidation. Myocardial substrate metabolism during ischemia is dependent upon the severity of ischemia. A very severe reduction of blood flow causes a decrease of substrate flux through PDH. When perfusion is only partially reduced there is an increase in the rate of glycolysis and a switch from lactate uptake to lactate production. Tissue levels of acyl-CoA and long-chain acylcarnitine increase with important functional consequences on cell membranes. During reperfusion fatty acid oxidation quickly recovers as the prevailing source of energy, while pyruvate oxidation is inhibited. A considerable body of experimental evidence suggests that L-carnitine exert a protective effect in in vitro and in vivo models of heart ischemia and hypertrophy. Clinical trials confirm these beneficial effects although controversial results are observed. The actions of L-carnitine and propionyl-L-carnitine cannot be explained as exclusively dependent on the stimulation of fatty acid oxidation but rather on a marked increase in glucose oxidation, via a relief of PDH inhibition caused by the elevated acetyl-CoA/CoA ratio. Enhanced pyruvate flux through PDH is beneficial for the cardiac cells since less pyruvate is converted to lactate, a metabolic step resulting in the acidification of the intracellular compartment. In addition, L-carnitine decreases tissue levels of acyl moieties, a mechanism particularly important in the ischemic phase. Biol Neonate. 2000. Carnitine is a key molecule in energy production from various substrates. Although it is generally believed that it plays no role in the metabolism of medium-chain triglycerides, quite a few data exist to the contrary. In the present study we investigated the effect of carnitine on ketogenesis in small-for-date neonates fed formulae of equal caloric value and fat content that was predominantly long-chain triglycerides or medium-chain triglycerides (46% of total fat). According to our results there was a statistically significant interaction between carnitine and the chain length of the administered fat with respect to ketone production. Increased ketogenesis was only shown by the neonates receiving medium-chain triglycerides and carnitine. Our results provide further evidence for the involvement of carnitine in medium-chain triglyceride metabolism. J Child Neurol. 1999 Mar. Rett syndrome is a severe neurodevelopmental disorder of unknown etiology, occurring almost exclusively in female patients. The etiology and functional significance of plasma carnitine deficiency seen in some patients with Rett syndrome is unknown. To investigate whether L-carnitine might be of benefit in Rett syndrome, a randomized, placebo-controlled, double-blind crossover trial of L-carnitine has been completed in 35 subjects. Eight-week treatment phases were completed for both a placebo and L-carnitine. Outcome was measured by parents/caregivers and at medical follow-up using three established tools: the Rett Syndrome Motor Behavioral Assessment, the Hand Apraxia Scale, and the Patient Well-Being Index. Analysis comparing change between baseline and week 8 of treatment for L-carnitine and the placebo showed that both parents/caregivers and medical follow-up detected improvements in the subjects' well-being. In addition, medical review showed an improvement on the Hand Apraxia Scale for a higher proportion of girls on L-carnitine. Identification of predictors of clinical improvement has been limited by the power of the study. These findings suggest that L-carnitine is of benefit in some patients with Rett syndrome. While L-carnitine did not lead to major functional changes in ability, the type of changes reported could still have a substantial impact on the girls and their families. Information is still needed, however, to determine if only subgroups of girls with the disorder are responsive to L-carnitine and the appropriate duration of therapy. Early Hum Dev. 1998 Dec. Carnitine performs a crucial role in the energy supply of tissues during fetal life and in the neonatal period by controlling the influx of fatty acids into mitochondria. Carnitine also facilitates the oxidation of pyruvate and branched chain amino acids, and contributes to the protection of cells from the deleterious actions of acyl CoAs. Carnitine further acts as a secondary antioxidant, favouring fatty acid replacement within previously oxidatively damaged membrane phospholipids. Availability of L-carnitine is essential in the developing fetus for processes underlying fetal maturation. L-carnitine is also essential for development of hepatic ketone synthesis, a central pathway for neonatal energy metabolism. Ketone bodies inhibit the oxidation of both glucose and lactate, sparing these metabolic substrates for biosynthetic functions. Altern Med Rev. 1998 Oct. A trimethylated amino acid roughly similar in structure to choline, carnitine is a cofactor required for transformation of free long-chain fatty acids into acylcarnitines, and for their subsequent transport into the mitochondrial matrix, where they undergo beta-oxidation for cellular energy production. Mitochondrial fatty acid oxidation is the primary fuel source in heart and skeletal muscle, pointing to the relative importance of this nutrient for proper function in these tissues. Although L-carnitine deficiency is an infrequent problem in a healthy, well-nourished population consuming adequate protein, many individuals within the population appear to be somewhere along a continuum, characterized by mild deficiency at one extreme, and tissue pathology at the other. Conditions which seem to benefit from exogenous supplementation of L-carnitine include anorexia, chronic fatigue, coronary vascular disease, diphtheria, hypoglycemia, male infertility, muscular myopathies, and Rett syndrome. In addition, preterm infants, dialysis patients, and HIV+ individuals seem to be prone to a deficiency of L-carnitine, and benefit from supplementation. Although available data on L-carnitine as an ergogenic aid is not compelling, under some experimental conditions pretreatment has favored aerobic processes and resulted in improved endurance performance. Excerpts from the full text article: Introduction Although L-carnitine was originally discovered in 1905, its crucial role in metabolism was not elucidated until 1955, and primary L-carnitine deficiency was not described until 1972. The most significant source of L-carnitine in human nutrition is meat, although humans are also capable of synthesizing L-carnitine from dietary amino acids. It has generally been assumed that a well-balanced diet contains both a significant amount of carnitine, and all of the essential amino acids and micronutrients needed for carnitine biosynthesis; however, increasingly investigators have identified conditions and individuals for which L-carnitine appears to be a conditionally-essential nutrient. Thus, although L-carnitine deficiency is an infrequent problem in a healthy, well-nourished population consuming adequate protein, many individuals within the population appear to be somewhere along a continuum characterized by mild deficiency at one extreme and tissue pathology at the other. Biochemistry A trimethylated amino acid similar in structure to choline, carnitine (see Figure 1) is a cofactor required for transformation of free long-chain fatty acids into acylcarnitines, and for their subsequent transport into the mitochondrial matrix, where they undergo betaoxidation for cellular energy production. Mitochondrial fatty acid oxidation is the primary fuel source in heart and skeletal muscle, pointing to the relative importance of this nutrient for proper function in these tissues. Carnitine synthesis begins with methylation of the amino acid L-lysine by Sadenosylmethionine (SAM). Methionine, magnesium, ascorbic acid, iron, P5P, and niacin, along with the cofactors responsible for regenerating SAM from homocysteine (5-methyltetrahydrofolate, methylcobalamin, and betaine) are all required for endogenous carnitine synthesis (see Figure 2). In order to form L-carnitine from lysine, three consecutive methylation reactions are required, with SAM acting as the methyl donor, resulting in the formation of trimethyllysine. Trimethyllysine is enzymatically transformed into hydroxytrimethyllysine in a reaction requiring alphaketoglutarate, oxygen, ascorbic acid and iron. The next step in the endogenous synthesis of L-carnitine requires pyridoxal 5'-phosphate (vitamin B6) and results in the formation of trimethylaminobutyraldehyde. Trimethylaminobutyrate (or gammabutyrobetaine) is then formed in a reaction requiring NADH (vitamin B3 dependent). The gamma-butyrobetaine is finally hydroxylated into carnitine in a reaction which again requires alpha-ketoglutarate, oxygen, ascorbic acid and iron. A pivotal enzyme in carnitine synthesis, betaine aldehyde dehydrogenase is the same enzyme responsible for synthesis of betaine from choline. Two recent studies suggest this enzyme has a preference for the cholinebetaine conversion, since choline supplementation appeared to decrease carnitine synthesis.1,2 Pharmacokinetics The pharmacokinetic properties of an orally administered dose of L-carnitine have not been unequivocally described in the literature, and might have considerable variability depending upon the relative carnitine stores of an individual. Evidence indicates L-carnitine is absorbed in the intestine by a combination of active transport and passive diffusion.3 There appears to be no significant advantage of supplementing an oral dose of L-carnitine in amounts greater than 2 g, since pharmacokinetic studies suggest mucosal absorption of carnitine is saturated at about a 2 g dose.4 Maximum blood concentrations are reached approximately 3.5 hours following an oral dose, with a half-life of about 15 hours. Elimination of carnitine occurs primarily through the kidneys.5 Although evidence suggests dietary carnitine is not totally absorbed and is in part degraded in the gastrointestinal tract of humans, there is some disagreement on the actual bioavailability of an oral dose. Rebouche and Chenard gave a radio-labeled dose of L-carnitine orally with a meal to subjects who had been adapted to a low-carnitine diet or a high-carnitine diet in order to determine the metabolic fate of dietary carnitine in humans. Their results suggested an oral bioavailability of 54 to 87 percent depending upon the dose of L-carnitine consumed.6 Bach et al also presented evidence suggesting relatively high absorption of L-carnitine. Following a 2 g oral dose of L-carnitine, they reported an increase in total blood carnitine levels of about 57 percent and in the free form of L-carnitine of about 81 percent.5 In contrast to these observations, several researchers have suggested much lower bioavailability. Sahajwalla et al indicated an absolute bioavailability of approximately 18 percent in healthy volunteers.7 Harper et al similarly reported a relatively low oral bioavailability. Following a 2 g dose they estimated bioavailability to be approximately 16 percent.4 Baker et al evaluated the changes in free and acylcarnitine activity in plasma, whole blood, red blood cells, and urine following oral doses of L-carnitine (a single dose of 500 mg or 2500 mg, or by ingestion of a daily dose of 2500 mg for 10 days). Increased levels of free carnitine were observed in the urine following all dosage regimens, while a single or consistent daily dose of 2500 mg of carnitine increased free and acylcarnitine in plasma, whole blood and urine to the greatest degrees. However, irrespective of dosage, these researchers observed no significant change in red blood cell carnitine levels, suggesting either a slow repletion of tissue stores of carnitine following an oral dose, or a low capability to transport carnitine into tissues under normal conditions.8 It is not known whether similar findings would be observed in conditions characterized by functional deficiency; however, based upon consistent clinical observations, it is likely some degree of tissue repletion occurs following an oral dose. Deficiency Although L-carnitine is supplied exogenously as a component of the diet and can also be synthesized endogenously, evidence suggests both primary and secondary deficiencies do occur. Carnitine deficiency can be acquired or a result of inborn errors of metabolism. Carnitine levels of vegetarians are reported to be below normal. Infants fed carnitine-free formulas are also in jeopardy of deficiency, since endogenous synthesis is not adequate to cover systemic needs during the first few days of the postnatal period. Primary carnitine deficiency, although rare, is characterized by low plasma, red blood cell, and tissue levels of carnitine, and generally presents with symptoms such as muscle fatigue, cramps, and myoglobinemia following exercise. Secondary carnitine deficiency is not as rare and is most commonly associated with dialysis, although intestinal resection, severe infections, and liver disease can also induce a secondary deficiency. Other symptoms of a chronic carnitine deficiency can include hypoglycemia, progressive myasthenia, hypotonia, or lethargy. Because of carnitine's role in fatty acid metabolism, elevated triglycerides as a lab finding might, among a variety of possibilities, be indicative of a relative deficiency of carnitine. Pathological manifestations of chronic deficiency include accumulation of neutral lipid within skeletal muscle, heart tissue and liver, a disruption of muscle fibers, and an accumulation of large aggregates of mitochondria within the skeletal and smooth muscle. Because of these changes, a deficiency can result in cardiomyopathy, congestive heart failure, encephalopathy, hepatomegaly, impaired growth and development in infants, and neuromuscular disorders. Diet and Nutritional Interactions It is assumed a diet adequate in protein will supply enough exogenous, and promote any additional endogenous, synthesis needed to supply an individual's requirements for L-carnitine. However, since L-carnitine is found primarily in animal proteins, with red meat regarded as the richest source, it is theoretically possible to consume a high protein diet consisting of beans, legumes, or egg whites and still promote a relative deficiency. Since a vegetarian diet is very low in exogenous carnitine, and is potentially low in some of the substrates required for carnitine synthesis, there is some concern that following a strict vegetarian diet might produce a carnitine deficiency in some individuals. A case report in the literature seems to lend credence to this possibility. In this report, a 12-year-old consuming a vegetarian diet had recurrent episodes of vomiting, lethargy, and hypoglycemia dating back 11 years. Investigations revealed a systemic carnitine deficiency that, when corrected, promptly improved his condition.9 Results indicate the macronutrient composition of a diet, other than protein content, can influence carnitine metabolism. In seven healthy men receiving the same amount of dietary carnitine, plasma free carnitine rose significantly in individuals following a highfat, low-carbohydrate diet, while no change in carnitine level was observed in individuals on a high-carbohydrate, low-fat diet. Renal excretion of carnitine increased only on the higher fat diet as well. This evidence suggests a high-fat, low-carbohydrate diet might be capable of boosting endogenous synthesis of carnitine and its metabolites.10 Coupled with this observation is the clinical finding that some individuals consuming a high carbohydrate diet for prolonged periods of time present with vague symptoms of fatigue and hypoglycemia, symptoms which can be indicative of a relative carnitine deficiency. In these individuals it might be prudent to assess carnitine status. Although medium-chain triglycerides (MCTs) have generally been assumed to be utilized as an energy substrate independently of carnitine, Rossle et al have shown the administration of MCTs does impact plasma concentrations of free carnitine and acylcarnitines, suggesting MCTs might not be metabolized independently of carnitine.11 Since carnitine deficiency can also impair ketogenesis, individuals consuming a ketogenic diet high in MCTs should be monitored for signs or symptoms suggestive of carnitine deficiency. Davis et al have shown that very low calorie diets (between 420-600 kcal/day) can have a negative effect on both plasma and urinary carnitine levels. Although plasma shortchain acylcarnitine esters increased and free carnitine declined significantly, protein consumption appeared to exert a sparing effect on carnitine since individuals consuming a preponderance of calories as meat/fish/poultry maintained significantly higher levels of plasma total carnitine.12 Ascorbic acid is required for the biosynthesis of L-carnitine, so it is not surprising to find a deficiency of ascorbic acid will decrease endogenous biosynthesis of carnitine.13,14 Experimental evidence suggests some forms of vitamin B12 affect carnitine synthesis. In rats, administration of methylcobalamin, cyanocobalamin, and hydroxycobalamin have been shown to stimulate carnitine synthesis. This is probably due to the use of vitamin B12 in the re-methylation of homocysteine to methionine, since biochemically, SAM must donate methyl groups to complete the endogenous generation of carnitine from lysine.15 Although evidence is currently unavailable, it is possible deficiencies in other nutrients required for optimal levels of SAM, such as methionine, magnesium, folic acid, and betaine, might impair endogenous synthesis of L-carnitine. Although the precise role of riboflavin (B2) in carnitine metabolism has not been elucidated, a case report suggests this vitamin might be an important component in optimizing carnitine levels in some individuals. In the report, Triggs et al describe a 29-year-old female with severe nausea and vomiting of pregnancy, migraines, psychiatric illness, non-epileptic seizures, and valproate-induced coma. Treatment with riboflavin normalized her plasma free carnitine level, and had a beneficial impact on her headaches and behavior.16 Since adequate dietary lysine is required as a substrate for carnitine synthesis, a deficiency of this amino acid or the other cofactors - iron, vitamin C, B6, niacin - might also compromise carnitine status. Due to the choline-betaine pathway sharing an enzyme with the lysine to carnitine pathway, which was mentioned earlier,1,2 choline supplementation might decrease carnitine synthesis; therefore, it might be of greater benefit to supplement with betaine rather than its precursors, choline or phosphatidylcholine. In individuals consuming high amounts of choline, phosphatidylcholine, or lecithin (a rich source of phosphatidylcholine), additional supplementation of L-carnitine or an assessment of L-carnitine status might be warranted. Drug Interactions Anticonvulsant therapy, including phenobarbital, valproic acid, phenytoin, and carbamazepine, has a significant lowering effect on carnitine levels.17 Pivampicillin treatment is also known to negatively impact carnitine metabolism.18 L-carnitine should be used cautiously if at all with pentylenetetrazol, a respiratory stimulant drug, since evidence suggests the combination might enhance the side-effects of the drug.19 Evidence suggests L-carnitine might prevent the cardiac complications secondary to interleukin-2 immunotherapy in cancer patients.20 Experimental evidence also suggests L-carnitine might be able to prevent cardiac toxicity secondary to the administration of adriamycin.21 L-Carnitine, when used concurrently with zidovudine (AZT), appears to prevent the AZT-induced destruction of myotubules, preserve the structure and volume of mitochondria, and prevent the accumulation of lipids.22 Anorexia In patients with anorexia nervosa, carnitine and adenosylcobalamin accelerated body weight gain and normalization of gastrointestinal function. Latent fatigue was reported to disappear and mental performance increase under this treatment regimen.23 Korkina et al reported the combined use of carnitine and adenosylcobalamin eliminated fluctuations in the work rate and improved the scope and productivity of intellectual work in patients with anorexia nervosa in the stage of cachexia, although latent fatigue in the population studied was not fully removed.24 Children with infantile anorexia also appear to respond well to a combination of carnitine and adenosylcobalamin. One group of children was given 2000 mcg adenosylcobalamin and 1000 mg carnitine, while the other group was given cyproheptadine, an anti-histamine used to stimulate appetite. Results of adenosylcobalamin and carnitine treatment were judged good by the authors, were comparable to the effects of the pharmaceutical agent, and were produced with no side-effects.25 Athletic Performance Carnitine is promoted as a supplement needed to improve the body's ability to use stored fat as fuel. Supplementation purportedly enhances lipid oxidation, increases VO2 max, and decreases plasma lactate accumulation during exercise. [...] Siliprandi et al, in a small double-blind cross-over study of ten moderately-trained male subjects, gave either 2 grams of L-carnitine or placebo orally one hour prior to exercise. Supplementation with L-carnitine induced a significant post-exercise decrease of plasma lactate and pyruvate and a concurrent increase of acetylcarnitine.27 Vecchiet et al randomly gave 2 grams of L-carnitine or a placebo to subjects one hour before they began exercise. At the maximal exercise intensity, treatment with L-carnitine increased both maximal oxygen uptake and power output. The authors also reported, at similar, non-maximal, exercise intensities, participants receiving L-carnitine had reduced oxygen uptake, carbon dioxide production, pulmonary ventilation, and plasma lactate.28 While some of the results with L-carnitine supplementation have been promising, not all research is in agreement. Heinonen, in his review of carnitine supplementation and physical exercise, concluded that its impact on performance in athletes is equivocal: it does not enhance fatty acid oxidation, spare glycogen or postpone fatigue during exercise; it does not stimulate pyruvate dehydrogenase activity; and it does not reduce body fat or help with weight loss.29 [...] Colambani et al investigated the effects of L-carnitine supplementation on metabolism and performance of endurance-trained athletes during and after a marathon run. In a doubleblind cross-over field study, seven male subjects received two grams of L-carnitine two hours before the start of a marathon run and again after 20 km of running. Although the administration of L-carnitine was associated with a significant increase in the plasma concentration of all analyzed carnitine fractions, significant changes in running time, plasma concentrations of carbohydrate metabolites (glucose, lactate, and pyruvate), as well as fat metabolites (free fatty acids, glycerol, and beta-hydroxybutyrate), hormones (insulin, glucagon, and cortisol), and enzyme activities (creatine kinase and lactate dehydrogenase) were not observed.33 [...] Chronic Fatigue Syndrome and Mitochondrial Myopathy Researchers investigating the oral administration of L-carnitine as a potential treatment for chronic fatigue syndrome observed clinical improvement in 12 of 18 patients. They also reported the trend for the greatest improvement occurred between weeks four and eight of treatment. One patient was unable to complete the trial due to the development of diarrhea.34 Campos et al found plasma carnitine "insufficiency," (defined as plasma esterified carnitine to free carnitine ratio above 0.25) in 21 of 48 (43.8%) patients with mitochondrial myopathy. They proceeded to treat the patients classified as "insufficient" with L-carnitine (50-200 mg/kg four times daily) and observed improvements in muscle weakness in 19 of 20 patients, failure to thrive in 4 of 8, encephalopathy in 1 of 9, and cardiomyopathy in 8 of 8 patients.35 [...] Hypoglycemia Since one of the manifestations of carnitine deficiency is hypoglycemia, it is not surprising that several investigators have reported a beneficial impact of L-carnitine administration on plasma glucose and insulin levels following intravenous infusion of glucose. Negro et al observed that the addition of both 2 g and 4 g of L-carnitine to 500 ml solutions of 5 percent and 10 percent glucose reduced the increase in plasma glucose levels.56 Grandi et al reported a similar improvement in glucose metabolism following the addition of 2 g of L-carnitine to a 5-percent glucose solution.57 Whether these observations would translate to a beneficial clinical effect in individuals with a tendency to reactive blood sugar is not currently known; however, due to the safety of L-carnitine and its tendency to improve fatigue (a common concomitant symptom of individuals with reactive blood sugar), a clinical trial with L-carnitine seems warranted. Pregnancy and Pediatric Applications Among the physiological changes characteristic of intrauterine development is an increase in body stores of carnitine, thought to occur due to the increased demand of the fetus on maternal supplies. Following delivery, interruption of the materno-fetal supply, along with the inability of the newborn to meet body requirements by endogenous synthesis, leaves the infant dependent upon exogenous intake. In circumstances of the absence of exogenous intake due to carnitine-free nutrition, tissue carnitine reserves decline in infants. Preterm infants are predictably in even greater jeopardy of having a relative carnitine deficiency.61 In contrast, a gradual increase of carnitine stores is a normal response of infants to breast feeding or the use of carnitine-containing formulas.62 Genger et al have reported an increased need for carnitine during pregnancy. In a small trial of women with diagnosed placental insufficiency, they observed a tendency toward beneficial outcomes following carnitine administration (1 g twice daily orally for one week followed by 1 g daily for the second week). In spite of the small numbers in this trial, the relative short duration of supplementation combined with the trend toward improved outcomes certainly merits further investigation of L-carnitine for this condition.63 Since physiologically, L-carnitine activates surfactant synthesis, it is not surprising that supplementation to women with imminent premature delivery provides a substantial benefit to the infant in the postnatal period. Results indicate a combination of L-carnitine (4 g/day for five days) and betamethasone given to women in the prenatal period can reduce both the incidence of respiratory distress syndrome and the mortality of premature newborns. In this trial, the incidence of respiratory distress syndrome of infants was approximately one-half (7.3% vs 14.5%) and the mortality rate was 1.8 percent compared with 7.3 percent in the group receiving the combined intervention as compared to betamethasone alone.64 In a case of three siblings presenting with apnea and periodic breathing, along with biochemical defects consistent with a non-specific abnormality of beta oxidation, one of the infants died of sudden infant death syndrome; however, the two surviving infants had a rapid resolution of both respiratory and metabolic abnormalities subsequent to treatment with Lcarnitine.65 Winter et al have suggested, based upon their clinical experience, that "secondary plasma carnitine deficiency is a common pediatric finding. The presence of failure to thrive, recurrent infections, hypotonia, encephalopathy, cardiomyopathy, or nonketotic hypoglycemia requires investigation of carnitine status."66 These authors reported normalization of cardiomyopathy in eight infants subsequent to the correction of a secondary L-carnitine deficiency. Kothari and Sharma have also reported a modest improvement in left ventricular function subsequent to administration of L-carnitine (50 mg/kg/day) in 13 children with idiopathic dilated cardiomyopathy.67 Progressive cardiomyopathy, with or without chronic muscle weakness, is the most common presentation (median age of onset, three years) in children with a defect in intracellular uptake of carnitine resulting in carnitine deficiency. Episodes of fasting hypoglycemia during the first two years of life are also a common presentation in affected infants. These children have low levels of plasma carnitine and a decreased rate of carnitine uptake. Typically, parents of affected children have intermediate values lying between those of normal control subjects and the affected children, suggestive of recessive inheritance. Stanley et al have suggested that early recognition of this disorder and the subsequent treatment with high doses of oral carnitine might be a lifesaving intervention for these children.68 Carnitine deficiency might be a complicating factor in cystic fibrosis. Wos et al reported five cases of infants with cystic fibrosis, impaired liver function, and neurological symptoms who, subsequent to a high carnitine diet and enteral administration, experienced a concomitant improvement in clinical condition with the progressive normalization of serum carnitine levels.69 Rett Syndrome Two case reports in the medical literature indicate administration of L-carnitine might provide some benefits to individuals with Rett syndrome. Plioplys and Kasnicka treated a 17-year-old female with L-carnitine (50 mg/kg/day). Over the two-month course of treatment an improvement in alertness, eye contact, and interaction with her environment were observed. These improvements were all lost following discontinuation of L-carnitine, and were regained after L-carnitine was reintroduced.70 Researchers administered L-carnitine (beginning at 75 mg/kg/day and increasing to 150 mg/kg/day) to a 3 1/2-year-old girl diagnosed with Rett syndrome. They observed improvements in physical activity, muscle hypotonia, communication, and sleep time. Similar to the findings of Plioplys and Kasnicka, a wash-out period followed by reintroduction of L-carnitine supplementation confirmed the efficacy of this regime.71 Precautions L-carnitine is listed as pregnancy category B, indicating animal studies have revealed no harm to the fetus, but that no adequate studies in pregnant women have been conducted. However, since L-carnitine has been given to pregnant women late in pregnancy with resulting positive outcomes and since L-carnitine is a normally occurring component of the diet, it is unlikely this supplement has any negative impact on pregnancy in normally supplemented amounts. In general, the recommendation for its use follows that of other nutritional substances which have not been overtly studied during human pregnancy; that being, use the supplement cautiously and only if clearly indicated by either laboratory or clinical status. The racemic mixture (D,L-carnitine) should be avoided. D-carnitine is not biologically active and might interfere with the proper utilization of the L isomer. In uremic patients, use of the racemic mixture has been correlated with myasthenia-like symptoms in some individuals. Adverse Reactions A variety of mild gastrointestinal symptoms have been reported, including transient nausea and vomiting, abdominal cramps, and diarrhea. A change in body odor has also been observed in a few individuals. Typically, reducing the dose will result in improvements in these adverse reactions. No reports of L-carnitine toxicity from overdosage exist. In mice, the LD50 is 19.2 g/ kg. Studies indicate no mutagenicity; however, experiments to determine the long-term carcinogenicity have not been conducted. Dosage As a general guideline, the average therapeutic dose is 1000 mg given two to three times daily for a total of 2000-3000 mg. No advantage appears to exist in giving an oral dose greater than 2000 mg at one time, since absorption studies indicate saturation at this dose. Conclusions L-carnitine has been used as a nutritional supplement for more than two decades. Although it has a well-deserved reputation as a safe and effective addition to nutritional protocols for a range of clinical conditions, its therapeutic role in coronary disease is perhaps its primary claim to fame. Addition of L-carnitine to a protocol for angina and ischemia results in improved exercise tolerance, reduced frequency of angina episodes, and beneficial changes to the ECG. L-carnitine seems to predictably improve risk factor markers of coronary disease; however, the single most impressive aspect of L-carnitine supplementation in coronary conditions has been the consistent bottom-line impact in reducing the clinical end point of congestive heart failure mortality. With additional research now indicating a place for L-carnitine in assisting with clinically challenging conditions such as chronic fatigue syndrome, HIV, and hypoglycemia, and with the ever expanding role of L-carnitine in pediatric health, it appears the use of this dietary supplement will continue to expand. Acta Paediatr Jpn. 1997 Aug. The serum free carnitine levels of 33 children with recurrent pulmonary infection and 30 healthy children were measured and found to be 26.12 +/- 0.98 nmol/mL and that of the control group 38.98 +/- 0.79 nmol/mL on the average. The mean free carnitine level was statistically determined to be significantly lower when compared with that of the control group (P < 0.01). The results indicate that oral L-carnitine therapy is recommended for pediatric patients with recurrent pulmonary infection. Neurosci Lett. 1997 Feb 28. The effect of acetyl-L-carnitine (ALC) on behavioral deficits following neonatal anoxia (N2 100% for 25 min at 30 h after birth) was studied in the rat. Transient hyperactivity at P20-P45 postnatal days and permanent spatial memory deficits were shown by anoxic rats. A chronic ALC treatment (50 mg/kg per die injected intraperitoneally from P2, after anoxia, to P60) significantly reduced the transient increase in sniffing, rearing and locomotory activity of anoxic rats, but, mostly, ameliorated the spatial memory performances in a maze at P30-P40 and in a water maze at P50-P60. No behavioral changes were seen in ALC-treated animals that received sham-exposure at birth. On the basis of these results, the use of ALC for the treatment of perinatal asphyctic insults in children is suggested. Biochem Biophys Res Commun. 1997 Feb 13. The brain uptake of acetylcarnitine was investigated in rhesus monkeys using different position labeled acetyl-L-carnitine and related molecules with 11C by positron emission tomography. The uptake values of radio-labeled acetylcarnitine into the brain were quite different depending on the labeling positions of 11C. That is, the uptake values of L-[methyl-11C]carnitine and acetyl-L-[methyl-11C]carnitine were almost the same and extremely low, while the uptake of [1-11C]-acetyl-L-carnitine was slightly higher. The uptake value of [2-11C]acetyl-L-carnitine was by far the highest among the 11C-labeled acetyl-L-carnitine and L-carnitine. The uptake of [2-11C]acetyl-L-carnitine into the brain was suppressed by the intravenous administration of glucose. These results suggest that endogenous serum acetyl-L-carnitine has some roles on conveying an acetyl moiety into the brain especially under an energy crisis, and that an unknown metabolic pathway of [2-11C]acetyl moiety might be rather active in the brain. Vasc Med. 1997. In order to help to clarify the mode of action of carnitine derivatives, plasma levels of adenosine, ATP and inosine were evaluated following the infusion of 0.75, 0.50 and 0.25 mg/kg/min propionyl-L-carnitine (PLC) for 30 min in patients affected with peripheral arterial disease. Moreover, the effects of 0.75 mg/kg/min acetyl-L-carnitine (ALC) and L-carnitine (LC) were studied in the same conditions. Finally, the activity of 7.5 mg/kg/min PLC administered for 3 min was also evaluated. PLC and ALC produced a significant increase in plasma levels of adenosine and ATP, whereas LC induced less relevant changes. The administration of the compounds did not affect the adenosine/inosine ratio. Peak plasma levels of adenosine preceded in any case those of ATP. The possibility can be suggested that the pharmacological activity of PLC, ALC, and LC may be mediated, at least in part, by an interference with the endogenous purine system. Since these effects may be related to physiological mechanisms of tissue protection, new pharmacological perspectives for the compounds may arise. Tohoku J Exp Med. 1996 Jun. In isovaleric acidemia (IVA), accumulated isovaleryl-CoA in the mitochondrion induces variable metabolic disturbances. To remove intramitochondrial isovaleryl groups, glycine therapy has been advocated primarily. On the other hand, secondary carnitine deficiency has been documented in this disorder and carnitine supplementation alone has been reported to be effective. In the present study, we administered carnitine and glycine to patients with IVA, and investigated serum carnitine and urinary excretion of total and free carnitine, acylcarnitine profile (i.e., isovalerylcarnitine and acetylcarnitine), and isovalerylglycine. By adding carnitine to glycine supplementation, more isovalerylglycine, not only isovalerylcarnitine, was excreted in the urine. Acetylcarnitine was detected in the urine only when sufficient carnitine was supplemented. We concluded that combined therapy of glycine and carnitine is more effective and safer to eliminate isovaleryl-CoA in IVA than conventional therapy using either glycine or carnitine. Urinary acetylcarnitine concentration might be a good marker indicating the optimal dose of L-carnitine supplementation. Klin Padiatr. 1996 May-Jun. Background: Rett syndrome can be diagnosed only clinically. Several biochemical abnormalities are known, but none of them is characteristic. To our knowledge only one study on carnitine deficiency and one case of successful carnitine therapy have been reported. Patient: A five years old girl with normal milestones in the first months of life became retarded in the second year with muscle hypotonia of unknown cause and loss of known abilities. Later on recurrent washing movements of the hands, hyperventilation and microcephaly were observed and the diagnosis of Rett syndrome was established. Method: A muscle biopsy was performed for the determination of enzymes of the respiratory chain and polarographic respirometry in permeabilized muscle fibres at the age of 3 1/2 years. Carnitine in plasma and urine was determined before and during a therapy with carnitine. Results: The activities of some enzymes of the respiratory chain were slightly decreased as was oxygen consumption in the permeabilized muscle fibres. However muscle morphology and histochemistry were normal. With normal carnitine in the muscle, plasma carnitine was clearly decreased but showed a normal ratio of acylcarnitine to free carnitine. Carnitine substitution [supplementation?] was started at the age of 3 1/2 years with 75 mg/kg/day and was later increased to 150 mg/kg/day. The treatment showed not only a normalisation of plasma carnitine but also an improvement of physical activity, muscle hypotonia, communication and sleep time. A wash out for one month and resumption of therapy confirmed the efficacy of this regime. Conclusions: The reason for the carnitine deficiency in the patient with Rett syndrome is not known. A primary carnitine deficiency is excluded by normal muscle carnitine. An explanation for the efficacy of the carnitine therapy is not known, although one could speculate that carnitine provides a transport system for acetyl groups, stimulates acetylcholine formation in the brain and in this way improves the disturbance of the cholinergic system. Pharmacol Res. 1996 Jan. L-Carnitine (L-C) is involved in the transport of acyl groups into mitochondria for beta-oxidation, although its role in the adult brain is still uncertain. We have shown before that the uptake of L-carnitine into cultured rat cortical neurones was dependent on temperature as well as the Na gradient and is inhibited by compounds resembling its structure, like gamma-aminobutyric acid (GABA), but most potently by specific GABA uptake blockers. In this study we have characterised this uptake process further. We have shown that the uptake of L-carnitine may be dependent on Cl ions, in addition to Na ions, but non on Ca ions. The L-C uptake was inhibited by substituent anions in the order gluconate (83%) > isethionate (32%), with propionate being ineffective, whereas GABA uptake was inhibited most potently by propionate substitution (79%) and equally by isethionate and gluconate (67%). This L-C uptake process was not affected by the amino acids, glutamine or lysine, up to 1 mM concentration, although beta-alanine at 500 microM caused a 38% inhibition. The uptake of L-C was also significantly inhibited by structurally-related compounds, with a carbon chain length of three to six atoms, possessing an amine group and/or a carboxyl group. At a concentration of 500 microM, 3-aminopropane sulphonic acid (53%), gamma-butyrobetaine (31%), gamma-hydroxybutyric acid (34%) and 4 methylaminobutyric acid (33%). Other compounds were effective only at the lower concentration of 10 microM, such as butyric acid (25%), nicotinic acid (26%), isonicotinic acid (26%), hexanoic acid (23%) and at 100 microM, like 6-aminocapric acid (22%). Drugs suggested to affect membrane properties, such as chlorpromazine, was without effect at 1 or 10 microM, whereas flunarizine (FLU) at 1 microM inhibited both L-C (24%) and GABA uptake (17%). Other drugs like the cholinesterase inhibitors, tacrine and eserine, also had a small inhibitory effect on L-C uptake, reducing it at 1 microM by 22 and 21% respectively, although higher concentrations were toxic (> 100 microM). Pretreatment of the cells with neuraminidase (50 U ml-1, 10 min) reduced the subsequent uptake of both L-C (18%) and GABA (42%). Hypoxia (3 h) also significantly attenuated L-C uptake (42%), however part of these effects were related to the loss of cell viability. In summary, L-C uptake occurs by a complex mechanism which at least in part may occur by a Na/Cl cotransport mechanism, which could be similar, to that of GABA or may even in part occur via the GABA transporter. J Child Neurol. 1995 Nov. The objective of this article is to review primary and secondary causes of carnitine deficiency, emphasizing recent advances in our knowledge of fatty acid oxidation. It is now understood that the cellular metabolism of fatty acids requires the cytosolic carnitine cycle and the mitochondrial beta-oxidation cycle. Carnitine is central to the translocation of the long chain acyl-CoAs across the inner mitochondrial membrane. The mitochondrial beta-oxidation cycle is composed of a newly described membrane-bound system and the classic matrix compartment system. Very long chain acyl-CoA dehydrogenase and the trifunctional enzyme complex are embedded in the inner mitochondrial membrane, and metabolize the long chain acyl-CoAs. The chain shortened acyl-CoAs are further degraded by the well-known system in the mitochondrial matrix. Numerous metabolic errors have been described in the two cycles of fatty acid oxidation; all are transmitted as autosomal recessive traits. Primary or secondary carnitine deficiency is present in all these clinical conditions except carnitine palmitoyltransferase type I and the classic adult form of carnitine palmitoyltransferase type II deficiency. The sole example of primary carnitine deficiency is the genetic defect involving the active transport across the plasmalemmal membrane. This condition responds dramatically to oral carnitine therapy. The secondary carnitine deficiencies respond less obviously to carnitine replacement. These conditions are managed by high carbohydrate, low fat frequent feedings, and vitamin/cofactor supplementation (eg, carnitine, glycine, and riboflavin). Medium chain triglycerides may be useful in the dietary management of patients with inborn errors of the cytosolic carnitine cycle or the mitochondrial membrane-bound long chain specific beta-oxidation system. Pharmacol Res. 1995 Mar-Apr. L-carnitine plays a central role in mitochondrial function and is found to be differentially distributed in the brain. We have shown before that the uptake of L-carnitine into cultured rat cortical neurones was temperature-dependent, as well as potently inhibited by factors affecting the sodium gradient as well as by molecules resembling its structure, e.g. D-carnitine, acetyl-L-carnitine and gamma-aminobutyric acid (GABA). GABA was the most potent inhibitor of L-carnitine uptake. In the present study we have found that specific GABA uptake blockers, nipecotic acid, cis-4-hydroxynipecotic (HNA), guvacine, 2,4-diaminobutyric acid (DABA) and NO 711 inhibit L-carnitine uptake even more potently than GABA. However, apart from NO 711, they caused about the same maximal inhibition, 67.4% at 50 microM for guvacine, compared to 60.5% by GABA. NO 711 was extremely potent and blocked 80.5% of the L-carnitine uptake. In contrast, the GABAA receptor agonists, isonipecotic acid and isoguvacine, or the antagonist bicuculline, at similar concentrations (50 microM), did not significantly inhibit the uptake of the L-carnitine. However, bicuculline at relatively high concentration (500 microM) was inhibitory (38%). The GABAB receptor agonist, baclofen, or antagonist, phaclofen, were ineffective, although 5-aminovaleric acid did significantly inhibit uptake at both 50 and 500 microM, causing 22 and 48% inhibition respectively. Like bicuculline, it was not as effective as GABA or the specific GABA uptake blockers. The results indicate that the uptake of L-carnitine by rat cortical neurones occurs in part by a process that can be potently inhibited by GABA and GABA uptake blockers. Brain Res Mol Brain Res. 1994 Aug. The ability of the primary rat cortical cells to take up L-carnitine increased with the age of the cultures and plateaued at around day 11 up to 25 days in vitro (DIV) when a slight decline was evident and by 32 DIV there was a major decrease in L-carnitine uptake. The uptake of L-carnitine displayed complex components. Elimination of mitochondrial energy supply by NaCN (1 mM), rotenone (1.25 microM) and DNP (50 microM), caused a small but significant decrease in the uptake (21, 11 and 16%, respectively). The uptake was highly dependent on the Na gradient, since ouabain (0.5 mM) and Na free buffer (replaced by 250 mM sucrose), reduced uptake by 54 and 63%, respectively. There was competition of L-carnitine uptake by molecules resembling its structure, e.g. gamma-aminobutyric acid (GABA), acetyl-L-carnitine (ALC), D-carnitine, L-aminocarnitine and L-choline, with GABA being the most potent inhibitor (57% at 50 microM) and L-choline not being significantly active. The Na-dependent uptake of L-carnitine was saturable with a high Km (692 microM) and Vmax (839 pmol/min/mg). This Na-dependent component was not further additive with the GABA (500 microM) or the DNP (50 microM) inhibitable component, suggesting that it represented the same phenomenon, probably the Na gradient dependent transport of L-carnitine. The results indicate that the uptake of L-carnitine occurs by Na-dependent saturable process as well as non-saturable, Na-independent processes. At least the former uptake mechanism is potently inhibited by GABA. Brain Dev. 1994 Mar-Apr. We present five cases of children with severe neurologic handicaps whose management was complicated by excessive lethargy. Treatment with L-carnitine in a dosage range of 35-50 mg/kg/day resulted in a marked improvement in alertness and arousability. In four cases, when L-carnitine was discontinued for a month, they all promptly became lethargic. When L-carnitine was re-started, the lethargy resolved and the improvement has been maintained for up to 14 months. In three children who were tested, serum carnitine levels (total and free) were normal before starting L-carnitine treatment. Clin Ter. 1994 Feb. Neuropsychologic tests were performed in subjects with Down syndrome in order to assess the effect of a 90-day treatment with L-acetyl-carnitine (LAC). Findings were evaluated statistically (Wilcoxon test) and compared to three further groups of subjects: untreated Down syndrome, mental deficiency due to other causes treated and not treated with LAC (Mann-Whitney U-test). Treated Down syndrome patients showed statistically significant improvements of visual memory and attention both in absolute terms and in comparison with the other groups. No improvement was found in mentally deficient non-Down subjects, so that the favourable effect of LAC appears to be specific for Down patients. In view of the analogies of the pathology and neurochemistry between Down syndrome and Alzheimer degenerative deficiency (deficit of cholinergic transmission) it is suggested that the action of LAC in these pathologies is related to its direct and indirect cholinomimetic effect. Neurosci Lett. 1994 Jan 3. Pulsatile gonadotropin-releasing hormone (GnRH) secretion from perifused hypothalamic cells and GT1-1 neuronal cells was significantly increased after culture in medium containing 100 microM acetyl-L-carnitine (ALC). This action of ALC was largely due to an increase in the spike amplitude of GnRH release. In addition, the receptor-mediated release of GnRH by N-methyl-D-aspartic acid and endothelin was significantly increased in perifused cells cultured in ALC-enriched medium. Stimulatory effects of ALC on basal, high K(+)- and agonist-induced GnRH release were also observed during long-term culture of primary hypothalamic neurons. Similar effects of ALC were evident in cultured GT1-1 cells and were accompanied by a significant increase in cell number. These observations in normal and transformed GnRH neurons demonstrate that ALC promotes the growth and secretory activity of neuropeptide-producing cells of the hypothalamus. Exp Gerontol. 1994 Jan-Feb. The hypothesis that some neurodegenerative events associated with ageing of the central nervous system (CNS) may be due to a lack of neurotrophic support to neurons is suggestive of a possible reparative pharmacological strategy intended to enhance the activity of endogenous neurotrophic agents. Here we report that treatment with acetyl-l-carnitine (ALCAR), a substance which has been shown to prevent some impairments of the aged CNS in experimental animals as well as in patients, is able to increase the levels and utilization of nerve growth factor (NGF) in the CNS of old rats. The stimulation of NGF levels in the CNS can be attained when ALCAR is given either for long or short periods to senescent animals of various ages, thus indicating a direct effect of the substance on the NGF system which is independent of the actual degenerative stage of the neurons. Furthermore, long-term treatment with ALCAR completely prevents the loss of choline acetyltransferase (ChAT) activity in the CNS of aged rats, suggesting that ALCAR may rescue cholinergic pathways from age-associated degeneration due to lack of retrogradely transported NGF. Pediatr Res. 1994 Jan. The purpose of this study was to determine whether treatment with L-carnitine or acetyl-L-carnitine enhances the turnover of lipid or branched-chain amino acid oxidation in patients with inborn errors of metabolism. Increasing i.v. doses of L-carnitine and acetyl-L-carnitine were given to one patient with medium-chain acyl-CoA dehydrogenase deficiency and to another with isovaleric acidemia. Both patients were in stable condition and receiving oral L-carnitine supplements. The excretion of carnitine and disease-specific metabolites was measured. The incorporation of L-carnitine in the intracellular pool was demonstrated using stable isotopes and mass spectrometry. Increasing doses of either i.v. L-carnitine or acetyl-L-carnitine did not stimulate the excretion of octanoylcarnitine in the patient with medium-chain acyl-CoA dehydrogenase deficiency, nor did it raise the plasma levels of either cis-4-decenoate or octanoylcarnitine. Similarly, increasing doses of either i.v. L-carnitine or acetyl-L-carnitine did not enhance the excretion of isovalerylcarnitine in a patient with isovaleric acidemia. The excretion of isovalerylglycine actually decreased. We conclude that there was no evidence of enhanced fatty acid beta-oxidation or enhanced branched-chain amino acid oxidation in vivo by the administration of high doses of L-carnitine or acetyl-L-carnitine in these two patients. Because only one individual with each disorder was studied, the data are only indicative and may not necessarily be representative of all individuals with these disorders. Definite settlement of this issue will require further studies in additional subjects. Adv Exp Med Biol. 1994. In summary, we propose that acute ammonia intoxication leads to increased extracellular concentration of glutamate in brain and results in activation of the NMDA receptor. Activation of this receptor mediates ATP depletion and ammonia toxicity since blocking the NMDA receptor with MK-801 prevents both phenomena. Ammonia-induced metabolic alterations (in glycogen, glucose, pyruvate, lactate, glutamine, glutamate, etc) are not prevented by MK-801 and, therefore, it seems that they do not play a direct role in ammonia-induced ATP depletion nor in the molecular mechanism of acute ammonia toxicity. The above results suggest that ammonia-induced ATP depletion is due to activation of Na+/K(+)-ATPase, which, in turn, is a consequence of decreased phosphorylation by protein kinase C. This can be due to decreased activity of PKC or to increased activity of a protein phosphatase. We also show that L-carnitine prevents glutamate toxicity in primary neuronal cultures. The results shown indicate that carnitine increases the affinity of glutamate for the quisqualate type (including metabotropic) of glutamate receptors. Also, blocking the metabotropic receptor with AP-3 prevents the protective effect of L-carnitine, indicating that activation of this receptor mediates the protective effect of carnitine. We suggest that the protective effect of carnitine against acute ammonia toxicity in animals is due to the protection against glutamate neurotoxicity according to the above mechanisms. South Med J. 1993 Dec. A 17-year-old girl with Rett syndrome, who was taking no other medications, was treated with L-carnitine (50 mg/kg/day). Within 2 months of initiation of treatment, she became much more alert, developed good eye contact, started reaching for objects with both hands, and answered simple questions with one or two words. L-carnitine was discontinued and within 1 week she lapsed into her pretreatment condition of lethargy with no interest in her environment, not reaching for objects, poor eye contact, and not speaking. One week after L-carnitine was resumed, she again became alert, started reaching for objects, and saying one or two words. Her serum carnitine levels (free and total) were within normal limits before and after L-carnitine treatment, but were higher while she was taking L-carnitine. Her serum ammonia was within normal limits prior to starting L-carnitine. L-carnitine appears to be an effective treatment for this girl with advanced Rett syndrome. Pediatr Res. 1993 Jul. Reduced plasma and tissue concentrations of carnitine, a cofactor required for fatty acid oxidation, are present in patients with inherited disorders of mitochondrial acyl-CoA oxidation that are associated with accumulations of acylcarnitines. To determine whether the secondary carnitine deficiency in these patients is due to excessive urinary loss of acylcarnitines, the development of carnitine deficiency was examined in patients with four different acyl-CoA oxidation disorders, including medium-chain and long-chain fatty acyl-CoA dehydrogenase deficiencies, isovaleric acidemia, and propionic acidemia. After a 3-mo period of treatment with oral carnitine to raise plasma total carnitine concentrations to or above normal, patients were started on a carnitine-free diet and the changes in plasma total and free carnitine levels and urinary total and free carnitine excretion were followed for 5 d. Patients with all four disorders showed a return of plasma carnitine levels and urinary carnitine excretion to baseline within 2 to 4 d. The rapidity of these changes could not be explained solely by excessive acylcarnitine wasting. Continued excretion of free carnitine in all patients indicated the additional presence of an impairment in renal transport of free carnitine. Consistent with this interpretation, estimates of renal thresholds for free carnitine gave values that were less than that for a control child in all four disorders and ranged as low as one half those reported in normal individuals. These results suggest that secondary carnitine deficiency in the acyl-CoA oxidation disorders is due to indirect as well as direct effects of accumulated acylcarnitines. Muscle Nerve. 1993 Jul. Abnormal carnitine distribution in muscle was found in 22 of 77 patients (29%), with mitochondrial myopathy. Furthermore, total (TC) and free (FC) carnitine levels in muscle were lower in patients than in controls (P < 0.01). Muscle long-chain acylcarnitines (LCAC) were significantly increased in these patients (P < 0.01). Muscle carnitine deficiency was found in 31.5% of patients with lipid storage myopathy (LSM) and in 25.6% of patients with ragged-red fibers (RRF). Therefore, carnitine deficiency can be found in patients with mitochondrial myopathy even in the absence of LSM. Muscle levels of TC and FC were lower in patients with respiratory chain defects than in those with normal respiratory chain (P < 0.01). In contrast, LCAC levels were significantly increased (P < 0.05). Carnitine levels did not differ significantly, among patients with different respiratory-chain defects. Consequently, these patients, owing to their biochemical block, reduce progressively the muscle carnitine pool and subsequent LCAC rise, due to long-chain fatty acid (LCFA) accumulation. Nutrition. 1993 May-Jun. Carnitine in the human body is derived from the intake of preformed dietary carnitine and biosynthesized carnitine, stemming from the metabolism of lysine and methionine. Carnitine is synthesized in liver and kidney, stored in skeletal muscle, and excreted mainly in urine. Carnitine has two main functions, i.e., transporting long-chain fatty acids into the mitochondrial matrix for beta-oxidation to provide cellular energy and modulating the rise in intramitochondrial acyl-CoA/CoA ratio, which relieves the inhibition of many intramitochondrial enzymes involving glucose and amino acid catabolism. Thus, the main consequence of carnitine deficiency is impaired energy metabolism. Human carnitine deficiency can be either hereditary or acquired. Hereditary carnitine deficiency can be grouped into three clinical entities: myopathic carnitine deficiency, systemic carnitine deficiency, and organic acidurias. Acquired carnitine deficiency is due to inadequate intake, increased requirement, and increased loss of carnitine. The definite diagnosis of carnitine deficiency is based on the determination of free- and acylcarnitine levels in serum, urine, and/or tissues. The estimated safe and adequate daily carnitine intake for adults is 150-500 mumol/day whereas pharmacological doses of carnitine are required for the treatment of hereditary carnitine deficiency. Muscle Nerve. 1993 Feb. Plasma carnitine "insufficiency," (plasma esterified carnitine to free carnitine ratio above 0.25) was found in 21 of 48 (43.8%) patients with mitochondrial myopathy, of whom 4 also showed both total and free carnitine deficiencies in plasma. In addition, plasma levels of SCAC and LCAC were higher in patients with mitochondrial myopathy than in controls (P < 0.001 and P < 0.01, respectively). Patients diagnosed as having plasma carnitine insufficiency or deficiency were treated with L-carnitine (50-200 mg/kg per day in four daily doses). Muscle weakness improved in 19 of 20 patients, failure to thrive in 4 of 8, encephalopathy in 1 of 9, and cardiomyopathy in 8 of 8 patients. Plasma carnitine "insufficiency" provides an additional clue to the diagnosis of mitochondrial myopathy and an indication for L-carnitine therapy. J Steroid Biochem Mol Biol. 1992 Oct. Acetyl-L-carnitine (ALC) is known to affect several aspects of neuronal activity. To evaluate the neuroendocrine actions of this compound, several endocrinological parameters were followed in ALC-treated and control animals during recovery from dark-induced anestrus. In treated animals, serum luteinizing hormone (LH) and prolactin levels were higher than those of controls during the proestrous and estrous phases of the cycle, and serum estradiol levels were higher during estrus. No significant changes were observed in serum levels of follicle-stimulating hormone and progesterone. Uterine weight was increased in ALC-treated rats during proestrus and estrus, but not in diestrus. The basal release of gonadotropin-releasing hormone (GnRH) from perifused hypothalamic slices of ALC-treated animals was elevated at proestrus and diestrus, and GnRH release elicited by high K+ was higher during all three phases of the cycle. The basal release of LH from perifused pituitaries of treated animals was elevated in diestrus, and the LH response to GnRH was higher in estrus and diestrus I. Depolarization with K+ caused increased LH secretion during proestrus and estrus in treated animals. In contrast to these effects of ALC treatment in vivo, no direct effects of ALC were observed during short- or long-term treatment of cultured pituitary cells. These results indicate that ALC treatment influences hypothalamo-pituitary function in a cycle stage-dependent manner, and increases the secretory activity of gonadotrophs and lactotrophs. Since no effects of ALC on basal and agonist-induced secretory responses of gonadotrophs were observed in vitro, it is probable that its effects on gonadotropin release are related to enhancement of GnRH neuronal function in the hypothalamus. Eur J Pharmacol. 1992 Jul 21. The effect of acetyl-L-carnitine, a compound reported to be beneficial for senile patients, on the release of dopamine (DA) from the striatum was studied by using in vivo brain dialysis in anesthetized rats coupled with HPLC-electrochemical detection. Striatal infusion of acetyl-L-carnitine increased the efflux of DA with no apparent changes in efflux of DA metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and 4-hydroxy-3-methoxyphenylacetic acid (HVA). The DA-releasing effect of acetyl-L-carnitine was concentration- and Ca(2+)-dependent, and was abolished by omega-conotoxin fraction GVIA and tetrodotoxin, inhibitors of the voltage-dependent Ca2+ and Na+ channels, respectively. Nomifensine, an inhibitor of DA reuptake did not alter the DA-releasing property of acetyl-L-carnitine. DA released from the striatum by acetyl-L-carnitine was decreased by reserpine pretreatment whereas the d-amphetamine-evoked DA outflow was not affected. In contrast to acetyl-L-carnitine, d-amphetamine reduced the extracellular concentrations of DOPAC and HVA. We conclude from the present data that acetyl-L-carnitine evokes DA release from the vesicular pools of the nigrostriatal dopaminergic neurons by a Ca(2+)-dependent, exocytotic process. Acta Neurol (Napoli). 1992 Feb. Daily addition of acetyl-L-carnitine (100 microM) to cultured cerebellar granule cells since the first day of maturation led to an increased rate of expression of D-[3H]aspartate uptake (an established marker of maturation of glutamatergic neurons) and of N-methyl-D-aspartate (NMDA) receptors linked to large conductance ion channels permeable to Ca2+. Acetyl-L-carnitine treatment also increased neuronal survival, as reflected by a greater percentage of cultures retaining functional NMDA receptors after 15 days of maturation. These results support the view that acetyl-L-carnitine exerts neuronotrophic activity and prevents age-dependent neuronal degeneration. J Int Med Res. 1991 Mar-Apr. Carnitine has a fundamental biological role as a long-chain fatty acid carrier across the mitochondrial membrane and in ketone body formation. Although the body normally synthesizes carnitine, in certain circumstances such as total parenteral nutrition and haemodialysis a dietary supplement may be needed to maintain adequate levels. Several considerations suggest that carnitine is a truly essential nutrient in infancy and in other situations where the energy requirement is particularly high, e.g. pregnancy and breast feeding. There are, for example, congenital deficit syndromes due to enzymatic inadequacies. There is also the possible role of carnitine in serious metabolic disorders such as organic acidaemias and, above all, it has multiple physiological functions in major metabolic pathways which are essential for development and growth. Acta Paediatr Jpn. 1990 Aug. A large quantity of propionylcarnitine in the urine of patients with propionic acidemia and methylmalonic aciduria was demonstrated. The amount excreted depended on the administered L-carnitine dose from 25 to 75 mg/kg/day. A high level of propionylcarnitine was also detected in the amniotic fluid of fetuses at risk of methylmalonic aciduria. Glutaric aciduria type 1 was characterized by excessive urinary excretion of glutarylcarnitine. In a neonate with glutaric aciduria type 2, several specific acylcarnitines were detected in the urine. These included isovaleryl-, acetyl-, isobutyryl-, and butyrylcarnitine as major carnitine esters and glutaryl-, and octanoylcarnitine as minor components. However, the pattern of acylcarnitines excreted changed from isovalerylcarnitine (via leucine) to isobutyrylcarnitine (via valine) during early life. In patients diagnosed as Reye syndrome, tissue carnitine deficiency was not always recognized and no decrease in the free/total carnitine ratio was found in the liver or muscle. The clinical and pathophysiological manifestations seen in these disorders are considered to relate to mitochondrial activity. Therefore, it is necessary to measure acylcarnitine fractions in the urine in order to obtain more precise information about mitochondrial function because carnitine and acylcarnitine compounds may express the metabolic state of mitochondria. Neurochem Res. 1990 Jun. Synthesis of [3H]acetylcholine from [3H]acetyl-L-carnitine was demonstrated in vitro by coupling the enzyme systems choline acetyltransferase and carnitine acetyltransferase. Likewise, both [3H] and [14C] labeled acetylcholine were produced when [3H]acetyl-L-carnitine and D-[U-14C] glucose were incubated with synaptosomal membrane preparations from rat brain. Transfer of the acetyl moiety from acetyl-L-carnitine to acetylcholine was dependent on concentration of acetyl-L-carnitine and required the presence of coenzyme A, which is normally produced as an inhibitory product of choline acetyltransferase. These results provide further evidence for a role of mitochondrial carnitine acetyltransferase in facilitating transfer of acetyl groups across mitochondrial membranes, thus regulating the availability in the cytoplasm of acetyl-CoA, a substrate of choline acetyltransferase. They are also consistent with a possible utility of acetyl-L-carnitine in the treatment of age-related cholinergic deficits. J Clin Chem Clin Biochem. 1990 May. In animal cells long chain fatty acids are transferred into the mitochondria for oxidation as acylcarnitines. Carnitine palmitoyltransferase I in the outer membrane, and carnitine translocase plus carnitine palmitoyltransferase II in the inner membrane catalyse the transfer. Carnitine palmitoyltransferase I is inhibited by malonyl-CoA, an intermediate in fatty acid synthesis. In the liver of fasted, diabetic, or thyreotoxic animals this enzyme shows increased activity and less inhibition by malonyl-CoA. Peroxisomes also contain carnitine acyltransferases and a beta-oxidation enzyme system. This system is particularly active in the shortening of very long chain fatty acids. The carnitine acyltransferases of the peroxisomes presumably are active in the transfer of the shortened acyl-CoAs and the acetyl-CoA to the mitochondria for complete oxidation. The carnitine acyltransferases of the mitochondria can catalyse the formation of propionylcarnitine and branched chain acylcarnitines from branched chain amino acids, and methylthiopropionylcarnitine from methionine. Their formation may represent a "security valve" preventing acyl-CoA accumulation in the mitochondria. The liver, which normally releases carnitine for other tissues, releases the branched chain acylcarnitines even more easily. This may be important for the development of secondary carnitine deficiency in some inborn errors of metabolism which are accompanied by the accumulation of acyl-CoAs in the tissue. Neurosci Lett. 1989 Dec 15. The effect of acetyl-L-carnitine (ALC) on the spontaneous release of acetylcholine (ACh) in the striatum and hippocampus of freely moving rats was investigated using brain microdialysis coupled with HPLC-electrochemical detection. Systemic administration of ALC, in a dose-dependent manner, stimulated ACh release in both areas, while the D-enantiomer was substantially ineffective. The effect of ALC was strongly Ca2+ dependent and tetrodotoxin (TTX) sensitive. These features of an exocytotic and impulse flow-dependent mechanism suggest that the increase in ACh release is the result of ALC activation of a physiological mechanism in cholinergic neurons. J Nutr. 1989 Aug. Acyl-CoA thioesters are generated during the oxidation of organic acids in mammalian systems. Vitamin B-12 deficiency is associated with decreased L-methylmalonyl-CoA mutase activity, and consequent accumulation of propionyl-CoA and methylmalonyl-CoA. The formation of propionylcarnitine from propionyl-CoA and carnitine provides an alternative pathway to remove propionyl-CoA from cells. Hepatocytes isolated from vitamin B-12-deficient rats metabolized propionate (1 mM) to CO2 and glucose at only 23% and 12%, respectively, of the rates observed in hepatocytes from control animals. In contrast, no difference was seen in rates of pyruvate metabolism by hepatocytes from control and vitamin B-12-mdeficient rats. Addition of carnitine (10 mM) to hepatocyte incubations increased the rate of propionylcarnitine formation 10- to 20-fold without altering conversion of propionate to CO2 or glucose. The rate of propionylcarnitine formation was not affected by vitamin B-12 deficiency. When carnitine (10 mM) was added, propionylcarnitine generation represented 65-71% of total propionate utilization in hepatocytes isolated from vitamin B-12-deficient rats. Gluconeogenesis from [1-14C]pyruvate was inhibited by 1 mM propionate in hepatocytes from vitamin B-12-deficient rats. No effect of 1 mM propionate on glucose formation from pyruvate was seen using hepatocytes from control rats. Intraperitoneal administration of L-carnitine resulted in a significant increase in urinary propionylcarnitine excretion from vitamin B-12-deficient rats, but not from control animals. The results demonstrate that exogenous carnitine can significantly enhance propionyl-group utilization via the formation of acylcarnitines under the conditions of impaired acyl-CoA metabolism associated with vitamin B-12 deficiency. Introduction Carnitine, in addition to its well-established role as an obligate for the mitochondrial oxidation of long-chain fatty acids, interacts with other metabolic pathways by generating acylcarnitines from the corresponding acyl-CoAs. Under normal physiological conditions, the generation of short-chain acylcarnitines (acyl-group chain length less than 10 carbon atoms) from short-chain acyl-CoAs operates near steady state, with the distribution of total carnitine between free (unesterified) carnitine and the short-chain acylcarnitines reflecting the metabolic state of the tissue. Under pathophysiological conditions of acyl-CoA accumulation secondary to a metabolic defect, the generation of acylcarnitines provides a mechanism to remove the acyl group and liberate free CoA. This reaction may be critical for maintaining normal cellular function under conditions where organic acids (and the corresponding acyl-CoAs) accumulate. This mechanism is thought to be in part responsible for the efficacy of carnitine in the treatment of some hereditary organic acidurias characterized by acyl-CoA accumulation secondary to an enzyme deficiency, such as in propionic acidaemia and the methylmalonic acidurias. The resulting increase in urinary acylcarnitine excretion may lead to insufficient endogenous carnitine to meet metabolic requirements. Understanding of the inter-relationship between the changes in endogenous carnitine metabolism, the consequences of the acyl-CoA buildup and overall cellular function is limited by a lack of experimental data. Evidence obtained in vitro demonstrates that high concentrations of unusual organic acids such as propionate can disrupt a number of metabolic pathways, including gluconeogenesis, fatty acid oxidation and pyruvate oxidation. For propionate, many of the cellular effects can be related to the accumulation of propionyl-CoA and methylmalonyl-CoA. Under conditions where propionate interferes with cellular metabolism, addition of carnitine results in a partial normalization of metabolism associated with a large increase in propionylcarnitine production. The use of carnitine in the treatment of patients with organic acidurias has resulted in clinical improvement of the patients and increased urinary excretion of the specific acylcarnitine corresponding to the accumulating acyl-CoA. The current studies were initiated to establish an animal model in vivo of impaired acyl-CoA utilization to permit controlled studies of the inter-relationships between carnitine metabolism, the underlying metabolic defect and overall cellular function. In the vitamin B-12 (cobalamin)-deficient rat, the activity of L-methylmalonyl-CoA mutase is decreased. This results in the secondary accumulation of propionyl-CoA and methylmalonyl-CoA generated during the normal metabolism of several amino acids and odd-chain-length fatty acids. This situation is directly analogous to the human methylmalonic acidurias. The current studies demonstrate that in the vitamin B-12-deficient rat there is a marked increase in tissue short-chain acylcarnitine contents and in the urinary excretion of acylcarnitines, including significant amounts of propionylcarnitine, associated with the metabolic abnormalities of vitamin B-12 deficiency. [...] Results Characterization of vitamin B-12-deficient rats Rats fed on control or vitamin B- 12-deficient diets maintained on the pair-feeding schedule gained weight at similar rates. Urinary excretion of methylmalonic acid was monitored as an index of the metabolic defect associated with vitamin B- 12 deficiency. After 11 weeks, methylmalonic acid excretion averaged 0.24 + 0.03 and 10.3 + 5.6 ,mol/day in the control (n = II) and vitamin B-12-deficient (n = 11) animals respectively. After 21 weeks on the vitamin B-12-deficient diet, the animals demonstrated a profound vitamin B-12 deficiency, as evidenced by a 230-fold increase in urinary methylmalonic acid excretion, a 740 decrease in hepatic vitamin B-12 content and a 21% decrease in hepatic holo-L-methylmalonyl-CoA mutase activity. Urinary carnitine excretion Over the course of 21 weeks on the special diet, the vitamin B-12-deficient group developed an increase in urinary excretion of short-chain acylcarnitines as compared with the control animals. This difference was demonstrated by both a higher daily elimination of acylcarnitines and an increase in the percentage of total urinary carnitine present as acylcarnitine. The increase in urinary acylcarnitines associated with vitamin B-12 deficiency was evident after 11 weeks on the deficient diet, and after 21 weeks acylcarnitines accounted for 300 of total urinary carnitine in the deficient animals, compared with 150 in the control group. In contrast, urinary excretion of total carnitine was not different in the two groups. The increased urinary excretion of acylcarnitines in the vitamin B- 12-deficient animals was correlated with the urinary methylmalonic acid excretion. Acylcarnitines are generated from the corresponding acyl-CoA, and therefore the specific acylcarnitines excreted in the vitamin B-12-deficient rats should correspond to the acyl-CoAs which accumulate secondarily to the metabolic defect in vitamin B-12 deficiency. Acetylcarnitine and propionylcarnitine were therefore specifically quantified in the urine of control and vitamin B- 12-deficient rats after 21 weeks on the special diets. A h.p.l.c. method was used to resolve acetylcarnitine and propionylcarnitine for postchromatographic quantification [24]. In control rats, acetylcarnitine accounted for most (58 of the total urinary acylcarnitine in the vitamin B-12-deficient rats. The increased content of propionylcarnitine in the urine of the vitamin B-12-deficient rats was verified by using fast-atom-bombardment mass spectrometry, in both absolute (comparison with external standard) and relative (comparison with acetylcarnitine) terms. Plasma and tissue carnitine pools Groups of vitamin B-12-deficient and control animals were killed after 24 weeks on the diet, and their plasma, liver and skeletal-muscle carnitine pools were measured. The vitamin B-12-deficient animals demonstrated decreased plasma concentrations of carnitine and total carnitine as compared with the control animals. No differences in the concentrations of plasma short- or long-chain acylcarnitines were seen. Thus the percentage of total acid-soluble carnitine (the sum of carnitine and short-chain acylcarnitine) present as acylcarnitines was increased in the vitamin B-12-deficient rats (22%) as compared with the control rats (18 %). Comparison of the liver carnitine pools in the two groups of rats showed decreases in carnitine and total carnitine content in the vitamin B-12-deficient rats. The percentage of hepatic total acid-soluble carnitine present as acylcarnitine was increased in the vitamin B-12-deficient rats (59 ). The skeletal-muscle contents of carnitine, short- and long-chain acylcarnitine and total carnitine were equivalent in the two groups. Propionylcarnitine contents in skeletal muscle and liver of control rats were low (12+3 and 16 +4 nmol/g respectively; n = 6). Skeletal-muscle propionylcarnitine content was 51 + 29 nmol/g (n = 7) in the vitamin B-12-deficient rats, accounting for 32 of the muscle short-chain acylcarnitines. Hepatic propionylcarnitine content in the vitamin B-12-deficient rats was 12+ 3 nmol/g (n = 7), and not different from control values. Effect of fasting on carnitine metabolism in the vitamin B-12-deficient rat During fasting, significant changes occur in carnitine metabolism in the rat, including a redistribution of the total carnitine pool into acylcarnitines. After 24 weeks of pair-feeding, a group of control and vitamin B-12-deficient rats were fasted for 48 h. Over the 48 h fast, the plasma/3-hydroxybutyrate concentration rose from 0.12+0.01 mm in the fed state (n = 6) to 0.80+ 0.07 mm (n = 7) in the control rats, and from 0.11+0.01 mM (n=7) to 1.13+0.34 mM (n=5) in the vitamin B-12-deficient rats. With fasting, the urinary excretion of carnitine, acylcarnitines and total carnitine decreased as compared with the fed state, with an increase in the percentage of total carnitine as acylcarnitine. In the vitamin B-12-deficient rats the larger excretion of acylcarnitines as compared with controls seen in the fed state was maintained, and resulted in an increased total carnitine excretion during fasting. In plasma, the free carnitine concentration fell by 180% in control animals during fasting, but it rose by 170% in the vitamin B-12-deficient rats. Plasma short-chain acylcarnitines increased by 86% in control rats and by 136% in the vitamin B-12-deficient group. Increases in short-chain acylcarnitines were also seen in liver during fasting. The percentage of hepatic total acid-soluble carnitine present as short-chain acylcarnitine was higher in the vitamin B-12-deficient rats (53%) as compared with control animals (43%). In skeletal muscle from control rats, the short-chain acylcarnitine content fell with fasting by 22%. In contrast, an increase of 68% was seen in the short-chain acylcarnitine content of skeletal muscle in vitamin B-12-deficient rats during the 48 h fast. Thus fasting accentuated the redistribution of total carnitine towards short-chain acylcarnitines in the vitamin B-12-deficient rats. Discussion Methylmalonic aciduria in the vitamin B-12-deficient rat was associated with a redistribution of urine and tissue total carnitine content towards short-chain acylcarnitines, and away from free carnitine. Propionylcarnitine became a significant constituent of the carnitine pool in vitamin B-12 deficiency. The increase in short-chain acylcarnitines seen in vitamin B-12 deficiency was maintained or accentuated under the metabolic stress of starvation ketosis. In the rat, vitamin B-12 is a required cofactor for two enzymes. The first, methionine synthase, is responsible for methionine generation from homocysteine. The second, L-methylmalonyl-CoA mutase, converts L-methylmalonyl-CoA into succinyl-CoA for further metabolism. In the vitamin B-12-deficient rat L-methylmalonyl-CoA and propionyl-CoA accumulate secondarily to the decreased L-methylmalonyl-CoA mutase activity. This accumulation in turn leads to a large increase in urinary methylmalonic acid excretion. This is qualitatively identical with the defects in the methylmalonic acidurias. In the current studies urinary excretion of methylmalonic acid was used as a marker of the metabolic defect with the development of vitamin B-12 deficiency. After 21 weeks on the experimental diet, the methylmalonic acid excretion in the vitamin B-12-deficient rats was 230 times the control value. Hepatic vitamin B-12 contents and holo-L-methylmalonyl-CoA mutase activity also confirmed the vitamin B-12 deficiency. Increased generation of acylcarnitines from acyl-CoAs may be reflected as either an increase in the absolute amount of acylcarnitine or as a redistribution of the carnitine pool (regardless of total carnitine content) towards acylcarnitines. The changes observed in the carnitine pool with the development of vitamin B-12 deficiency were consistent with both of these concepts. Urinary excretion of acylcarnitines was doubled in the vitamin B-12-deficient rats as compared with controls. The percentage of total acid-soluble carnitine present as acylcarnitine was also higher in urine, plasma and liver from the vitamin B-12-deficient animals as compared with the controls. Overall, the increased urinary excretion of acylcarnitines correlated well with the degree of metabolic impairment resulting from the vitamin B-12 deficiency as assessed by methylmalonic acid excretion. The use of the pair-feeding schedule eliminated energy intake or age, both of which alter carnitine metabolism, as possible etiologies for the differences observed in the two groups of animals. The nature of the acyl-CoA accumulation is also reflected in the specific acyl moieties present in the acylcarnitine pool. Propionylcarnitine excretion was increased 11-fold in vitamin B-12-deficient rats as compared with control animals. Propionylcarnitine has also been identified as a major acylcarnitine in the urine of patients with methylmalonic aciduria. The increase in urinary acylcarnitine excretion with vitamin B-12 deficiency was approximately 300 nmol/day, or which 100 nmol/day could be accounted for by propionylcarnitine. This is consistent with the production of other, as yet unidentified, acylcarnitines in vitamin B-12 deficiency. Acyl-CoAs other than propionyl-CoA or methylmalonyl-CoA might also accumulate and form acylcarnitines secondarily to disruption of normal intermediary metabolism by the propionyl-CoA build-up. Burton & Frenkel identified propionylcarnitine in the liver of vitamin B-12-deficient rats. In the most severely vitamin B-12-deficient animals in the current study, propionylcarnitine was estimated to represent 45% and 30% of total short-chain acylcarnitine in skeletal muscle and liver respectively. The large variability in the degree of methylmalonic aciduria attained in the current studies minimized the changes in mean tissue propionylcarnitine content. Despite the large degree of methylmalonic aciduria in our animals, and the dramatic changes in the CoA pool previously described in vitamin B-12 deficiency, the magnitude of the redistribution of tissue carnitines towards acylcarnitines was relatively small. Although the reasons for this are unclear, the poor conversion of branched-chain acyl-CoAs (such as methylmalonyl-CoA, a major acyl-CoA in vitamin B-12 deficiency) into the corresponding acylcarnitine, may have minimized the changes in the carnitine pool. Starvation is associated with an increase in short-chain acylcarnitines. This phenomenon was maintained or enhanced in the vitamin B-12-deficient rats. For example, although skeletal-muscle short-chain acylcarnitine content fell in the control animals with fasting, consistent with previous observations, short-chain acylcarnitine content of skeletal muscle increased by 68% with fasting in the vitamin B-12-deficient group. This increase in short-chain acylcarnitines may be associated with the increased utilization of branched-chain amino acids by muscle during starvation. The biosynthesis of carnitine requires the methylation of lysine contained in protein to form trimethyl-lysine. The impairment of methionine formation from homocysteine in vitamin B-12 deficiency might therefore interfere with carnitine biosynthesis. As most of the rat's carnitine is in skeletal muscle, the normal total carnitine content in the skeletal muscle of the vitamin B-12-deficient rats after 24 weeks on the diets, along with the normal total urinary carnitine elimination, suggests that there is no major impairment in carnitine biosynthesis in these animals. Thus the increase in short-chain acylcarnitines seen in vitamin B-12 deficiency reflect changes in the CoA pool rather than a broad disturbance in carnitine homoeostasis. The vitamin B-12-deficient rat demonstrates changes in carnitine metabolism in both the fed and fasted states as compared with control animals. These changes correspond both qualitatively and quantitatively to predictions based on the accumulation of specific acyl-CoAs in these animals. The vitamin B-12-deficient rat provides a potential model system for studying the effects of changes in acylcarnitine generation on intermediary metabolism in the methylmalonic acidurias, with possible extrapolation to other organic acidaemias and diseases associated with acyl-CoA accretion. Neurochem Res. 1989 May. Analysis in mouse brain slices of the uptake of acetyl-L-[N-methyl-14C]carnitine with time showed it to be concentrative, and kinetic analysis gave a Km of 1.92 mM and a Vmax of 1.96 mumol/min per ml, indicating the presence of a low-affinity carrier system. The uptake was energy-requiring and sodium-dependent, being inhibited in the presence of nitrogen (absence of O2), sodium cyanide, low temperature (4 degrees C), and ouabain, and in the absence of Na+. The uptake of acetyl-L-carnitine was not strictly substrate-specific; gamma-butyrobetaine, L-carnitine, L-DABA, and GABA were potent inhibitors, hypotaurine and L-glutamate were moderate inhibitors, and glycine and beta-alanine were only weakly inhibitory. In vivo, acetyl-L-carnitine transport across the blood-brain barrier had a brain uptake index of 2.4 +/- 0.2, which was similar to that of GABA. These results indicate an affinity of acetyl-L-carnitine to the GABA transport system. J Inherit Metab Dis. 1989. Medium-chain acyl-CoA dehydrogenase deficiency is a recently described inborn error of metabolism characterized by episodes of coma and hypoketotic hypoglycaemia in response to prolonged fasting. Secondary carnitine deficiency has been documented in these patients as well as the excretion in the urine of medium-chain-length acyl carnitine esters, such as octanoylcarnitine. Based on the potential toxicity of medium-chain fatty acid metabolites and the beneficial responses of patients with other inborn errors of metabolism and secondary carnitine deficiency, oral carnitine has been proposed as treatment for children with medium-chain acyl-CoA dehydrogenase deficiency. We report the results of carefully monitored fasting challenges of an infant with this deficiency both before and after 3 months of oral carnitine therapy. Carnitine supplementation failed to prevent lethargy, vomiting, hypoglycaemia and accumulation of free fatty acids in response to fasting despite normalization of plasma carnitine levels and a marked increase in urinary excretion of acyl-carnitine esters. Potentially toxic medium-chain fatty acids accumulated in the plasma in spite of therapy. Based on this study of one patient, we stress that avoidance of fasting and prompt institution of glucose supplementation in situations when oral intake is interrupted remain the mainstays of therapy for medium-chain acyl-CoA dehydrogenase deficient patients. Biochem J. 1988 Oct 1. In vitamin B-12 (cobalamin) deficiency the metabolism of propionyl-CoA and methylmalonyl-CoA are inhibited secondarily to decreased L-methylmalonyl-CoA mutase activity. Production of acylcarnitines provides a mechanism for removing acyl groups and liberating CoA under conditions of impaired acyl-CoA utilization. Carnitine metabolism was studied in the vitamin B-12-deficient rat to define the relationship between alterations in acylcarnitine generation and the development of methylmalonic aciduria. Urinary excretion of methylmalonic acid was increased 200-fold in vitamin B-12-deficient rats as compared with controls. Urinary acylcarnitine excretion was increased in the vitamin B-12-deficient animals by 70%. This increase in urinary acylcarnitine excretion correlated with the degree of metabolic impairment as measured by the urinary methylmalonic acid elimination. Urinary propionylcarnitine excretion averaged 11 nmol/day in control rats and 120 nmol/day in the vitamin B-12-deficient group. The fraction of total carnitine present as short-chain acylcarnitines in the plasma and liver of vitamin B-12-deficient rats was increased as compared with controls. When the rats were fasted for 48 h, relative or absolute increases were seen in the urine, plasma, liver and skeletal-muscle acylcarnitine content of the vitamin B-12-deficient rats as compared with controls. Thus vitamin B-12 deficiency was associated with a redistribution of carnitine towards acylcarnitines. Propionylcarnitine was a significant constituent of the acylcarnitine pool in the vitamin B-12-deficient animals. The changes in carnitine metabolism were consistent with the changes in CoA metabolism known to occur with vitamin B-12 deficiency. The vitamin B-12-deficient rat provides a model system for studying carnitine metabolism in the methylmalonic acidurias. Pediatr Res. 1988 May. At the time of acute presentation, children with carnitine deficiency may have increased free fatty acid concentrations and hypoglycemia. However, whether carnitine replacement affects the plasma concentration of these substrates remains to be determined. Therefore, to evaluate the effect of carnitine replacement on plasma substrate and hormone concentrations, five children with carnitine deficiency (two idiopathic, two secondary to long-chain acyl coenzyme A dehydrogenase deficiency, one secondary to isovaleric acidemia) were fasted overnight before and after treatment with oral carnitine (80 +/- 7 mg.kg-1.day-1). During carnitine supplementation, plasma total carnitine (19 +/- 4 versus 45 +/- 6 nmol/ml, pretreatment versus treatment, respectively) and free carnitine (11 +/- 3 versus 31 +/- 6 nmol/ml), as well as red blood cell total carnitine (0.057 +/- 0.019 versus 0.130 +/- 0.019 nmol/mg of hemoglobin) increased (p less than 0.05). Fasting plasma glucose (83 +/- 4 versus 85 +/- 3 mg/dl) and ketone body (0.54 +/- 0.18 and 0.56 +/- 0.20 mM) concentrations did not change with carnitine supplementation, but plasma free fatty acids (1.28 +/- 0.32 versus 0.77 +/- 0.07 mM) decreased (p less than 0.05). No differences in fasting insulin, growth hormone, or cortisol concentrations were observed. Urinary excretion of free carnitine (0.1 +/- 0.0 versus 2.4 +/- 0.7 mumol/mg creatinine), total carnitine (0.3 +/- 0.1 versus 3.4 +/- 0.9 mumol/mg creatinine) and acyl carnitine (0.2 +/- 0.1 versus 0.9 +/- 0.3 mumol/mg creatinine) increased (p less than 0.05) with carnitine supplementation. The decreased plasma free fatty acid concentrations with carnitine supplementation may be due to more efficient fatty acid oxidation and/or increased urinary excretion of fatty acids as acylcarnitines. Neurochem Res. 1988 Apr. The uptake of L-carnitine was characterized in mouse brain synaptosomal preparations, with an emphasis on mutual interactions with GABA uptake systems. The uptake consisted of nonsaturable diffusion and one saturable energy- and sodium-dependent component. GABA, L-DABA and nipecotate were strong and hypotaurine and homotaurine moderate inhibitors of the uptake. The inhibition by GABA was shown to be competitive. GABA uptake contained two saturable transport components, high- and low-affinity. It was most strongly inhibited by nipecotate and L-DABA, but also by carnitine and hypotaurine. The high-affinity uptake of GABA was competitively inhibited by carnitine, but the inhibition of the low-affinity uptake of GABA was of the mixed type. The results suggest that GABA and carnitine share the same carrier system at synaptosomal membranes. However, GABA is the preferred substrate and the carnitine concentrations which significantly inhibited GABA uptake exceed the physiological carnitine levels in vivo. J Inherit Metab Dis. 1988. Secondary carnitine deficiency in a patient with glutaric acidaemia type II, due to deficient ETF-dehydrogenase activity, is described. The patient responded clinically to a pharmacological dose of riboflavin and a restricted protein diet. In the second year of her life she developed more frequent and severe exacerbations during intercurrent infections from which she did not fully recover. Hypotonia and marked ataxia persisted. Plasma carnitine was entirely complexed as acylcarnitine with no free carnitine detected. Retrospective evaluation of several frozen urine specimens obtained since the age of 10 months revealed undetectable free carnitine with elevated acylcarnitine levels. Marked clinical improvement was observed following L-carnitine supplementation. The hypotonia and ataxia disappeared. The frequency and the severity of the exacerbations were noticeably decreased. The role of L-carnitine in preventing the accumulation of acyl-CoA compounds in inborn errors of organic acid metabolism is further emphasized by this patient. The necessity to evaluate free carnitine, acylcarnitine and acyl/free ratio in the assessment, follow-up and management of patients with inborn errors of organic acid metabolism is discussed. Am J Dis Child. 1987 Jun. Drugs Exp Clin Res. 1987. An open, cross-over study was performed on a population of 24 geriatric patients hospitalized because of depressive syndrome. They were subdivided, according to Hamilton's Scale as modified for the aged, into low- and high-score subgroups. The study period covered 2 months. Half the patients received acetylcarnitine for 1 month and placebo thereafter (Group A); the other half received placebo and acetyl-carnitine thereafter (Group B). Statistical evaluation was twofold: parametrical analysis of variance was carried out on 4 subgroups (A1, A2, B1 and B2) and analysis of the score percentage modifications before and after treatment was performed on Groups A and B. The experimental results showed that acetylcarnitine treatment was highly effective and statistically significant in subgroups A1/B1, A2/B2, A1, B1 and B2. In particular, it should be noted that depressive tendencies were significantly modified in most groups, whereas general somatic symptoms as well as anxiety, asthenia and sleep disturbances proved to be little affected. Clinical evaluation, carried out by calculation of modifications in pre- and post-treatment score percentages, provided clear evidence that acetylcarnitine was particularly effective in patients showing more serious clinical symptoms. The drug caused no side-effects at the doses and regimens used. Neurology. 1986 Jul. A child with myopathy and systemic carnitine deficiency died at age 8 years in an acute metabolic attack. He had glutaric aciduria type II, and his cultured fibroblasts contained normal activity of four different acyl CoA dehydrogenases, but there was deficiency of electron transfer flavoprotein:ubiquinone oxidoreductase (ETF-QO). This enzyme is thought to reduce coenzyme Q in the respiratory chain, funneling reducing equivalents from seven flavoproteins in the beta-oxidation of acyl CoAs. There was massive urinary excretion of the short-chain acylcarnitines that accumulated in mitochondria as a result of the ETF-QO defect. Carnitine therefore acts as a buffer for excessive accumulation of intramitochondrial acyl CoAs, and defective beta-oxidation can cause carnitine insufficiency. Pediatr Neurol. 1986 Mar-Apr. Fourteen children with the following Reye and Reye-like syndromes were studied to determine each patient's carnitine status: valproate-induced Reye-like attack, ornithine transcarbamylase deficiency, systemic carnitine deficiency, methylmalonic acidemia, and propionic acidemia. Reduced free carnitine and increased serum and urine acylcarnitine levels were found in all patients except for 2 with Reye syndrome, in whom serum creatinine levels were mildly elevated and serum free carnitine levels were not reduced. The renal free carnitine reabsorption rate was reduced in all cases. The free carnitine content of autopsied liver samples were reduced in 2 Reye syndrome patients, 2 OTC deficiency patients, and in a single systemic carnitine deficiency patient. The observed secondary free carnitine deficiency may be a factor in the pathogenesis of Reye and Reye-like syndromes. Eur J Pediatr. 1986 Feb. The profound metabolic disturbances which occur in isovaleric acidaemia are due to the intramitochondrial accumulation of isovaleryl coenzyme A (CoA) with a consequent reduction in the availability of free CoA. Secondary carnitine insufficiency is also a feature of this and other disorders of organic acid metabolism. A patient who presented at 2.5 years of age was diagnosed using capillary GC-MS as having isovaleric acidaemia. She showed the full spectrum of abnormal organic acids previously associated with the 'neonatal' form of the disease despite her late presentation, indicating that it is inappropriate to refer to acute early and late onset forms of isovaleric acidaemia. Instead, a spectrum of disease exists, determined by environmental factors, residual enzyme activities and modifying effects of different phenotypes in different individuals. She also showed evidence of carnitine insufficiency. An oral challenge with L-carnitine resulted in the excretion of large amounts of urinary acylcarnitines which were shown by use of fast atom bombardment mass spectrometry to be primarily isovalerylcarnitine. Regular glycine supplementation caused no significant increase in urinary isovalerylglycine and had to be stopped because of side-effects after 5 days. An oral L-carnitine challenge during glycine supplementation resulted in a marked increase in isovalerylglycine excretion, again associated with the excretion of large amounts of isovalerylcarnitine. Carnitine acts by removing (detoxifying) intramitochondrial isovaleryl groups and, in the presence of glycine, it promotes the formation of isovalerylglycine. We believe L-carnitine supplementation is of value in the treatment of isovaleric acidaemia and that, in the present case, L-carnitine together with a moderate dietary restriction has proved to be the optimum form of therapy. Pediatr Res. 1985 May. The medium-chain acyl-coA dehydrogenase deficiency is one of several metabolic disorders presenting clinically as Reye syndrome. Evidence is presented for a characteristic organic aciduria that distinguishes this disorder from Reye syndrome and other masqueraders characterized by dicarboxylic aciduria. The key metabolites, suberylglycine and hexanoylglycine, are excreted in high concentration only when the patients are acutely ill. More significantly, using novel techniques in mass spectrometry, the medium-chain defect is shown to be characterized by excretion of specific medium-chain acylcarnitines, mostly octanoylcarnitine, without significant excretion of a normal metabolite, acetylcarnitine, in four patients with documented enzyme deficiency. Similar studies on the urine of two patients reported with Reye-like syndromes of unidentified etiology have suggested the retrospective diagnosis of medium-chain acyl-coA dehydrogenase deficiency. Administration of L-carnitine to medium-chain acyl-coA dehydrogenase deficiency patients resulted in the enhanced excretion of medium-chain acylcarnitines. Octanoylcarnitine is prominent in the urine both prior to and following L-carnitine supplementation. The detection of this metabolite as liberated octanoic acid, following ion-exchange chromatographic purification and mild alkaline hydrolysis, provides a straightforward diagnostic procedure for recognition of this disorder without subjecting patients to the significant risk of fasting. In view of the carnitine deficiency and the demonstrated ability to excrete the toxic medium-chain acyl-coA compounds as acylcarnitines, a combined therapy of reduced dietary fat and L-carnitine supplementation (25 mg/kg/6 h) has been devised and applied with positive outcome in two new cases. Presse Med. 1984 Apr 28. No abstract available. J Biol Chem. 1981 Jun 10. The kinetic behavior of the carnitine:acylcarnitine translocase was studied in isolated rat heart mitochondria. The kinetic parameters, Km(apparent) and Vmax, for carnitine were determined by measuring the rates of influx of [14C]carnitine using two different methods to quench the exchange reaction. The range of the Km(app) was 0.38-1.50 mM and the Vmax was 0.20-0.34 nmol/mg . min by both methods. Carnitine esters of acetyl isobutyryl, and octanoyl groups were competitive with carnitine for uptake and Ki values for these esters were 1.1, 2.6, and 0.10 mM, respectively. The Km(app) for carnitine was increased in the presence of these carnitine esters, while the Vmax for carnitine was unchanged. Distribution of radiolabel from free [14C]carnitine into acetylcarnitine, isobutyrylcarnitine, and octanoylcarnitine during the incubations was examined by thin layer chromatography and was negligible. The Km values for carnitine and the Ki value for acetylcarnitine are within the concentration ranges of these compounds in the intact heart (Idell-Wenger, J. A., Grotyohann, L. W., and Neely, J. R. (1978) J. Biol. Chem. 253, 4310-4318). the Ki values for isobutyrylcarnitine and octanoylcarnitine may also be within their concentration ranges in vivo, but exact concentrations in heart muscle are not known. These data support the concept that carnitine esters of short (acetylcarnitine), branched (isobutyrylcarnitine), and medium (octanoylcarnitine) chain acyl groups compete with free carnitine for transport into the mitochondria under physiological conditions. Lancet. 1981 May 30. No abstract available. J Neurochem. 1981 Feb. The properties of carnitine transport were studied in rat brain slices. A rapid uptake system for carnitine was observed, with tissue-medium gradients of 38 +/- 3 for L-[14CH3]carnitine and 27 +/- 3 for D-[14CH3]carnitine after 180 min incubation at 37 degrees C in 0.64 mM substrate. Uptake of L- and D-carnitine showed saturability. The estimated values of Km for L- and D-carnitine were 2.85 mM and 10.0 mM, respectively; but values of Vmax (1 mumol/min/ml intracellular fluid) were the same for the two isomers. The transport system showed stereospecificity for L-carnitine. Carnitine uptake was inhibited by structurally related compounds with a four-carbon backbone containing a terminal carboxyl group. L-Carnitine uptake was competitively inhibited by gamma-butyrobetaine (Ki = 3.22 mM), acetylcarnitine (Ki = 6.36 mM), and gamma-aminobutyric acid (Ki = 0.63 mM). The data suggest that carnitine and gamma-aminobutyric acid interact at a common carrier site. Transport was not significantly reduced by choline or lysine. Carnitine uptake was inhibited by an N2 atmosphere, 2,4-dinitrophenol, carbonylcyanide-N-chlorophenylhydrazone, potassium cyanide, n-ethylmaleimide, and ouabain. Transport was abolished by low temperature (4 degrees C) and absence of glucose from the medium. Carnitine uptake was Na+-dependent, but did not require K4+ or Ca2+. |
