Addition Of L-carnitine Research Articles

L-Carnitine delays the killing of cultured hepatocytes by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine

The role of fatty acid metabolism in chemical-dependent cell injury is poorly understood. Addition of l-carnitine to the incubation medium of cultured hepatocytes delayed cell killing initiated by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Protection by l-carnitine was stereospecific and observed as late as 1 h following addition of MPTP. d-Carnitine, but not iodoacetate, reversed the l-carnitine effect. Monoamine oxidase A and B activities, MPTP/ N-methyl-4-phenylpyridinium levels, and MPTP-dependent loss of mitochondrial membrane potential measured by release of [ 3H]triphenylmethylphosphonium were not altered by addition of l-carnitine. Significant changes in MPTP-induced depletion of total cellular ATP did not occur with excess l-carnitine. Although the mechanism of cytoprotection exerted by l-carnitine remains unresolved, the data suggest that l-carnitine does not significantly alter: (i) mitochondrial-dependent bioactivation of MPTP; (ii) MPTP-dependent loss of mitochondrial membrane potential; or (iii) MPTP-mediated depletion of total cellular ATP content. We conclude that alterations of fatty acid metabolism may contribute to the toxic consequences of exposure to MPTP. Moreover, the lack of l-carnitine-mediated cytoprotection of monolayers incubated with 4-phenylpyridine or potassium cyanide suggests: (i) a link between fatty acid metabolism and mitochondrial membrane-mediated, bioactivation-dependent cell killing; and (ii) that inhibition of NADH dehydrogenase may not totally explain the mechanism of MPTP cytotoxicity.

Morphometric Evidence of the Trophic Effect of <i>L</i>-Carnitine on Human Skeletal Muscle

We investigated the effect of long-term i.v. administration of L-carnitine on human muscle fibers using morphometric parameters. We administered 2g/day L-carnitine to patients undergoing hemodialysis for at least 12 months. At the end of this period a marked increase in serum and muscle carnitine levels was observed in all patients, together with hypertrophy and predominance of type 1 fibers. L-carnitine was withheld for 4 months, during which time serum and muscle levels gradually decreased and no changes were observed in muscle fibers. Subsequent addition of L-carnitine to dialysis fluid for another 4 months stabilized lower levels. At the end of this period reduction of diameter of type 1 fibers was observed. Type 2 fibers remained unchanged. Moreover, type 1 fibers remained predominant in all cases. Hence, we suggest that carnitine has a specific trophic effect on type 1 fibers which are characterized by an oxidative metabolism.

Cultured Cells as a Screen for Novel Treatments of Alzheimer's Disease

Two easily measured abnormal properties of fibroblasts from patients with Alzheimer's disease have been utilized to develop an in vitro test system for screening novel therapeutic agents for Alzheimer's disease. The abnormal properties selected for study were increased isoproterenol-stimulated cyclic adenosine monophosphate production and decreased pH (measured by the weak-acid distribution method). L-Carnitine was tested as a potential therapeutic agent, since it has been used to treat a variety of experimental metabolic encephalopathies. The addition of L-carnitine normalized both of these properties in the Alzheimer cells. Tissue culture may aid as a preliminary screen for identifying novel approaches to the treatment of Alzheimer's disease.

Ca 2+-mediated action of long-chain acyl-CoA on liver mitochondria energy-linked processes

The decrease of steady-state transmembrane potential (delta psi) and loss of accumulated Ca2+ are magnified if palmitoyl-CoA is added to rat liver mitochondria exposed to Ca2+ and phosphate. The extent of this damage increases with increasing concentration of long-chain acyl-CoA. Addition of L-carnitine with or without the addition of palmitoyl-CoA considerably delays the deenergization. In the latter case, there is a substantial decrease in the assayed endogenous long-chain acyl-CoA content. This protective action of L-carnitine is abolished by L-aminocarnitine, a powerful inhibitor of carnitine palmitoyl transferase (palmitoyl-CoA: L-carnitine O-palmitoyltransferase, EC 2.3.1.21.). The removal of Ca2+ by EGTA, or the inhibition of its uptake by Ruthenium red or Mg2+ further enhances the degree of protection.

Involvement of long-chain acyl CoA in the antagonistic effects of halothane and L-carnitine on mitochondrial energy-linked processes

Incubation of rat liver mitochondria in the presence of halothane induced a consistent impairment of mitochondrial oxidative phosphorylation without significantly affecting the steady-state of transmembrane electrical potential. These alterations of mitochondrial energy-linked processes were associated with a consistent accumulation of long-chain acyl CoA. Addition of L-carnitine partially prevented the effects of halothane on oxidative phosphorylation and completely abolished the halothane-induced long-chain acyl CoA accumulation. The possibility is discussed that the damaging action of halothane on mitochondrial functions might be partially ascribed to the noxious action of the excess of long-chain acyl CoA induced the anesthetic.

Effect of L-Carnitine on Cultured Murine Melanoma Cells Exposed to Azelaic Acid

The cytotoxic effect of azelaic acid on murine melanoma cells in culture is due, at least in part, to an antimitochondrial action. We investigated the possibility that the addition of carnitine to the medium may increase the transport of azelaic acid into the mitochondria and thereby increase its cytotoxic effect. Using mitochondrial cross-sectional area measured from electron micrographs as a criterion for mitochondrial damage, we found that the addition of L-carnitine to the culture medium had no effect either alone or with a low (10(-3) M) concentration of azelaic acid. At a high concentration (5 X 10(-2) M) azelaic acid caused swelling and disruption of the mitochondria to such an extent that this was not increased by carnitine. At 10(-2) M azelaic acid, however, some swelling of the mitochondria occurred which was significantly increased by the addition of carnitine. This indicates that carnitine-mediated transport of the diacid into the mitochondria had occurred. We conclude that carnitine may reduce the time or concentration needed for azelaic acid to have a toxic effect on the malignant melanocyte.

Stabilising action of carnitine on energy linked processes in rat liver mitochondria

Rat liver mitochondria exposed to stressing conditions — Ageing at room temperature, incubation in the presence of t-butyl hydroperoxide or damaging concentrations of Ca2+ and phosphate- undergo a rapid fall in their membrane potential (Δψ) with a concomitant release of endogenous Mg2+ and accumulated Ca2+. Addition of L-carnitine to the incubation medium considerably delays mitochondrial deenergization. A similar, though lower, protection has also been observed in L-carnitine pretreated and subsequently washed rat liver mitochondria. Furthermore mitochondria isolated from livers of starved rats, treated with L-carnitine 30 minutes before death and exposed to the same stressing conditions show similar delay in the decrease of Δψ and concurrent energy linked processes as compared with untreated animals. Both the in vitro and in vivo results strongly indicate that the stabilising action of L-carnitine on liver mitochondria is due to the removal of membrane bound long chain acyl CoA.

The metabolic fate of branched-chain amino acids and 2-oxo acids in rat muscle homogenates and diaphragms

1. 1. After incubation of muscle preparations with [U- 14C]branched-chain amino acids or 2-oxo acids, radioactive metabolites were separated, identified and quantified. 2. 2. Homogenates of rat heart and skeletal muscle incubated with 4-methyl-2-oxopentanoate accumulated isovalerate, 3-hydroxyisovalerate and the corresponding carnitine esters. 3. 3. Incubation with 3-methyl-2-oxobutanoate resulted in the production of isobutyrate, 3-hydroxyisobutyrate and their carnitine esters. Addition of L-carnitine increased the production of the esters. 4. 4. The enzymes 3-methylcrotonyl-CoA carboxylase and 3-hydroxyisobutyric acid dehydrogenase apparently are inactive during incubation of muscle homogenates. 5. 5. With liver homogenates the degradation of both 2-oxo acids was more complete. 6. 6. Rat hemidiaphragms incubated with leucine, valine and isoleucine accumulated the corresponding branched-chain 2-oxo acids, fatty acids and hydroxylated fatty acids. 7. 7. The degradation of valine was markedly limited by the release of these metabolites. 8. 8.Considerable amounts (relatively smaller for valine) of radioactivity were also recovered in CO 2 and glutamine and glutamate. 9. 9. Incubations with branched-chain 2-oxo acids gave the same radioactive products, except for glutamine and glutamate. 10. 10. Radioactivity was never found in lactate, pyruvate or alanine. 11. 11. These data indicate that the carbon-chains of amino acids entering the citric acid cycle in muscle, are not used for oxidation or for alanine synthesis, but are converted exclusively to glutamine.

Stimulation of the glycine cleavage system by short-chain fatty acids in isolated rat liver mitochondria.

The effect of short-chain fatty acids on glycine catabolism by the glycine cleavage system was investigated in isolated rat liver mitochondria. Metabolic flux through the glycine cleavage system was monitored by measuring the production of 14CO2 from [1-14C]glycine. Propionate, butyrate, pentanoate, and octanoate, and to a lesser extent acetate, all significantly stimulated 14CO2 production from [1-14C]glycine by isolated rat liver mitochondria maintained in state 4. Concomitant with the stimulation of 14CO2 production was a decrease in the measured intramitochondrial content of NADPH which we have previously demonstrated correlates with the metabolic flux through the glycine cleavage system [Hampson, R.K., Barron, L.L., & Olson, M.S. (1983) J. Biol. Chem. 258, 2993-2999]. The propionate-mediated stimulation of mitochondrial 14CO2 production from [1-14C]glycine was not diminished by the addition of L-carnitine but was abrogated nearly completely by the addition of oligomycin. Incubation of the mitochondria with short-chain fatty acids evoked a large decrease in the measured intramitochondrial ATP content and a large increase in the AMP content. However, manipulation of the intramitochondrial adenine nucleotide profile demonstrated that no direct correlation existed between ATP, ADP, or AMP and 14CO2 production from [1-14C]glycine. These experimental results indicate that short-chain fatty acid oxidation causes the oxidation of the NADP(H) redox couple in isolated rat liver mitochondria, resulting in the stimulation of the metabolic flux through the glycine cleavage system.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of carnitine on the ischemic arrested heart.

We investigated the effect of L-carnitine on the recovery of cardiac function after ischemic arrest in the perfused rat heart. L-carnitine was added to a cardioplegic solution, both as a free base and hydrochloride. The addition of L-carnitine as a free base to the solution had no effect on recovery of cardiac function. When L-carnitine HCl was added to the cardioplegic solution, it was necessary to adjust the pH of the solution to 7.4. The hearts arrested with this solution showed a greater incidence of reperfusion dysrhythmias than those in the control or the free base solution, but the overall recovery of cardiac function was the same as control. The hydrochloride of L-carnitine is strongly acidic, and these findings indicate that either the free base or a properly buffered solution must be used to study effects of carnitine upon cardiac function.

Acetyl-coenzyme A hydrolase, an artifact? The conversion of acetyl-coenzyme A into acetate by the combined action of carnitine acetyltransferase and acetylcarnitine hydrolase.

1. The nature of the acetyl-CoA hydrolase (EC 3.1.2.1) reaction in rat and sheep liver homogenates was investigated. 2. The activity determined in an incubated system was 5.10 and 3.28nmol/min per mg of protein for rat and sheep liver homogenate respectively. This activity was not affected by the addition of l-carnitine, but was decreased by the addition of d-carnitine. 3. No acetyl-CoA hydrolase activity could be detected in rat or sheep liver homogenates first treated with Sephadex G-25. This treatment decreased the carnitine concentrations of the homogenates to about one-twentieth. Subsequent addition of l-carnitine, but not d-carnitine, restored the apparent acetyl-CoA hydrolase activity. 4. Sephadex treatment did not affect acetyl-carnitine hydrolase activity of the homogenates, which was 5.8 and 8.1nmol/min per mg of protein respectively for rat and sheep liver. 5. Direct spectrophotometric assay of acetyl-CoA hydrolase, based on the reaction of CoA released with 5,5'-dithiobis-(2-nitrobenzoic acid), clearly demonstrated that after Sephadex treatment no activity could be measured. 6. Carnitine acetyltransferase (EC 2.3.1.7) activity measured in the same assay system in response to added l-carnitine was very low in normal rat liver homogenates, owing to the apparent high acetyl-CoA hydrolase activity, but was increased markedly after Sephadex treatment. The V(max.) for this enzyme in rat liver homogenates was increased from 3.4 to 14.8nmol/min per mg of protein whereas the K(m) for l-carnitine was decreased from 936 to 32mum after Sephadex treatment. 7. Acetyl-CoA hydrolase activity could be demonstrated in disrupted rat liver mitochondria but not in separated outer or inner mitochondrial membrane fractions. Activity could be demonstrated after recombination of outer and inner mitochondrial membrane fractions. The outer mitochondrial membrane fraction showed acetylcarnitine hydrolase activity and the inner mitochondrial membrane fraction showed carnitine acetyltransferase activity. 8. The results presented here demonstrate that acetyl-CoA hydrolase activity in rat and sheep liver is an artifact and the activity is due to the combined activity of carnitine acetyltransferase and acetylcarnitine hydrolase.

The oxidation of fatty-acyl derivatives by mitochondria from bovine fetal and calf hearts.

Functional activity of the "external" carnitine palmitoyltransferase in intact mitochondria, prepared from hearts from various sources, was estimated by measuring respiration by mitochondria in the presence of palmitoyl-CoA plus or minus l-carnitine. When palmitoyl-CoA alone was substrate, respiration was not increased above the basal rate under all conditions examined. Addition of l-carnitine increased respiration, provided the ionic strength of the incubation medium was sufficiently high. In the presence of palmitoyl-CoA plus l-carnitine, the rate of respiration increased as the ionic strength was increased to physiological levels. In contrast, the increase in rate of oxygen consumption by heart mitochondria which followed the addition of palmitoyl-l-carnitine was relatively independent of the ionic strength of the medium.Mitochondrial fractions prepared from fetal bovine hearts were shown to possess "external" carnitine palmitoyltransferase activity, as judged by the ability of l-carnitine to stimulate respiration by mitochondria incubated with palmitoyl-CoA under various conditions. These data were discussed in relation to information available concerning the functions of different carnitine acyltransferases in mitochondria.