Carnitine-acylcarnitine translocase deficiency with severe hypoglycemia and auriculo ventricular block. Translocase assay in permeabilized fibroblasts.

Deficiency of the enzymes of mitochondrial fatty acid oxidation and related carnitine dependent steps have been shown to be one of the causes of the fasting-induced hypoketotic hypoglycemia. We describe here carnitine-acylcarnitine translocase deficiency in a neonate who died eight days after birth. The proband showed severe fasting-induced hypoketotic hypoglycemia, high plasma creatine kinase, heartbeat disorder, hypothermia, and hyperammonemia. The plasma-free carnitine on day three was only 3 microM, and 92% of the total carnitine (37 microM) was present as acylcarnitine. Treatments with intravenous glucose, carnitine, and medium-chain triglycerides had been tried without improvements. Measurements in fibroblasts confirmed deficient oxidation of palmitate and showed normal activities of the carnitine palmitoyltransferases I and II and of the three acyl-CoA dehydrogenases. A total deficiency of the carnitine-acyl-carnitine translocase was found in fibroblasts using the carnitine acetylation assay (1986. Biochem. J. 236:143-148). This assay has been further simplified by seeking conditions permitting application to permeabilized fibroblasts and lymphocytes.

Uneven distribution of palmitoyl carnitine in solutions because of migration to air/water interphase.

Standard solutions of palmitoyl carnitine could not be prepared in water because, even at below critical micelle concentrations, palmitoyl carnitine did not distribute uniformly in solutions. Evidence indicates that palmitoyl carnitine prefers to leave the bulk phase to segregate readily at the water/air and water/apolar interphases. Thus, in metabolic and kinetic studies, the actual concentration of long-chain acyl carnitines available for reactions at any instant can be drastically different from that calculated from the amounts added.

Clofibrate enhancement of mitochondrial carnitine transport system of rat liver and augmentation of liver carnitine and gamma-butyrobetaine hydroxylase activity by thyroxine.

The possibilities that the hypotriglyceridemic effect of clofibrate involves activation of carnitine-dependent oxidation of fatty acids in liver and that this may be partially mediated through thyroxine have been examined. 0.25% clofibrate in diet for 10-15 days, was found to increase carnitine 3-fold in livers of male as well as female rats. Liver carnitine was nearly doubled by L-thyroxine, 6 mg/kg of diet fed for 10 days, and so was the activity of gamma-butyrobetaine hydroxylase. Clofibrate decreased carnitine in heart and urine; thyroxine did not affect these parameters but increased serum carnitine by 26%. Clofibrate feeding doubled the concentration of hepatic long-chain acyl(-)carnitine, mitochondrial carnitine, and the rate of mitochondrial carnitine-acylcarnitine translocase reaction, and enhanced acetoacetate production in liver homogenates as well as mitochondrial oxidation of palmitoylcarnitine in the presence of malonate. The ratio of esterified to free carnitine in urine and serum was also increased by clofibrate. These results suggest that clofibrate and thyroxine may exert their hypotriglyceridemic effect, in part, through the activation of carnitine-mediated transport of fatty acids in liver mitochondria.

Differential effects of phosphatidylcholine and cardiolipin on carnitine palmitoyltransferase activity.

Rates of carnitine palmitoyltransferase-catalyzed conversion of palmitoylcarnitine to palmitoyl-CoA are markedly decreased with the progress of this reaction presumably owing to the build up of inhibitory palmitoyl-CoA in the enzyme vicinity. High, above micellar, concentrations of palmitoylcarnitine, phosphatidylcholine liposomes and high KCl concentrations increased the activity, apparently by facilitating the removal of palmitoyl-CoA from the enzyme surface. The presence of cardiolipin was found to be inhibitory. The enzyme activity followed in the direction of palmitoylcarnitine formation with low palmitoyl-CoA concentration as substrate, was inhibited by phosphatidylcholine, but stimulated by cardiolipin. Both of these lipids markedly stimulated the enzyme activity followed by the isotope exchange procedure which requires progression of both the forward and the backward reactions. The results indicate that one of the effects of phospholipids on carnitine palmitoyltransferase activity is exerted from the ability of these substances to bind the amphipathic reactants of this enzyme, particularly long-chain acyl-CoA. The possibility that the activity of the membrane-bound carnitine palmitoyltransferase may at times be affected by changes in the concentrations and composition of the various phospholipids in the enzyme's vicinity is raised by these findings.

Freeze-thawing causes masking of membrane-bound outer carnitine palmitoyltransferase activity: implications for studies on carnitine palmitoyltransferases deficiency.

Carnitine-dependent transport of fatty acids into mitochondria is believed to require participation of two carnitine palmitoyltransferase (CPT) activities, one outer, overt (CPTo) and the other inner, latent (CPTi). For exposing the CPTi and monitoring of the total CPT activity, freeze-thawing and sonication have been frequently employed as membrane-disruptive procedures, particularly when examining for CPT-deficiency diseases. Our evaluations have shown, however, that freeze-thawing and sonication yield misleading data for both the CPT activities owing to their previously unrecognized masking and unmasking effects on CPT activities. Formation of vesicular/sheath structures with mixed membrane orientation that prevents the access of medium substrate to enzymes on both aspects of the membrane at the same time appears responsible for these results. That such procedures can yield inexact data when monitoring the latency and sidedness of other membrane-bound biocatalysts as well needs to be recognized. We show that in muscle mitochondria also, a malonyl-CoA-inhibitable CPTo activity resides in the outer membrane, while a malonyl-CoA-insensitive, CPTi, activity is present in the inner membrane. Our results rationalize why Zierz and Engel ((1987) Neurology 37, 1785) were unable to obtain evidences for a latent CPT activity in mitochondria particularly of muscles. Although simple methods to allow an unambiguous quantitation of the two CPT activities in tissue extracts remain unavailable, evaluation of the possibility that two different CPT deficiencies occur appears justified.

Functional studies on in situ-like mitochondria isolated in the presence of polyvinyl pyrrolidone.

Mitochondria isolated and maintained in sucrose mannitol medium show a large intermembrane space and a condensed matrix unlike the appearance of in situ mitochondria. Mitochondria resembling in situ organelles are obtained when the isolation medium is supplemented with certain macromolecules such as polyvinyl pyrrolidone. We found that the in situ appearance was acquired also by the conventionally isolated mitochondria when they were exposed to 2% polyvinyl pyrrolidone supplemented medium. Paradoxically, however, these in situ looking mitochondria proved functionally inferior in that their brief incubation without substrates led to a marked loss of their ability to respire with subsequently added substrates such as pyruvate, acylcarnitines or glutamate. The oxidation of succinate was, however, not so affected. This phenomenon was shared by heart and skeletal muscle mitochondria of different animal species but not by rat liver mitochondria. The inhibition of respiration could not be related to the failure to oxidize NADH, to the tieing up of mitochondrial free CoASH, or to the increased matrix space of mitochondria that was observed in the presence of polyvinyl pyrrolidone. The polyvinyl pyrrolidone-exposed mitochondria regained their respiratory ability on being freed from polyvinyl pyrrolidone. The same phenomenon was seen also when the medium contained 2% albumin or 20% Ficoll.

Derivatives of monoglycerides as apoptotic agents in T-cells.

Recently, lipids have received considerable attention for their potential to induce apoptosis when added exogenously to cells. In this study, we directly demonstrate that murine T-cells undergo rapid apoptosis following treatment with various forms of monoglycerides, which are a family of naturally occurring lipids consisting of a single fatty acid moiety attached to a glycerol backbone. The potency of these lipids varied depending on their chemical structure, whereas glycerol backbone or corresponding fatty acids alone were ineffective. Moreover, monoglyceride-mediated apoptosis was suppressed either by Bcl-2 overexpression, treatment with a broad inhibitor of caspases, or RNA and protein synthesis inhibitors. In addition, treatment of cells with derivatives of monoglycerides induced a calcium flux, which could be inhibited by both extracellular (EGTA) or intracellular (EGTA-AM) calcium chelators. To our knowledge, this is the first report demonstrating a role for derivatives of monoglycerides as inducers of apoptosis in mammalian cells.

Plasma carnitine and lipid-lowering drugs.

Oral carnitine has been reported to have a lipid-lowering effect with concomitant elevation of high density lipoprotein cholesterol (HDL-C) levels in normo- and hyperlipidemic individuals. Unexpectedly, basal carnitine concentrations were found to be abnormally high in subjects receiving a combination of probucol (1 g/day) and clofibrate (2 g/day), and who also had reduced HDL-C levels. Changes in plasma carnitine levels were found to correlate with clofibrate therapy and to be readily reversible with cessation of this drug. These increases of circulating carnitine were not accompanied by a rise in HDL-C.

Effects of high-fat diet and fasting on levels of acyl-coenzyme A binding protein in liver, kidney, and heart of rat.

Acyl-coenzyme A (CoA) binding protein (ACBP) is a 10-kd protein that binds acyl-CoA moieties and stimulates medium-chain fatty acid synthesis by goat mammary gland fatty acid synthetase. Its exact role in intermediary lipid metabolism has not been fully elucidated. It is hypothesized that ACBP is directly involved in the metabolism of lipid. In the present study, purified rat liver ACBP was used to generate a polyclonal antisera for radioimmunoassay of ACBP in tissue specimens isolated from fasted rats and rats fed normal rat chow and a high-fat diet. In addition, purified ACBP was used to examine its effect on the activity of mitochondrial outer membrane (OM) carnitine palmitoyltransferase (CPT0). Fasting for 24 hours significantly decreased tissue levels of ACBP in the liver (69.0 +/- 7.2 v 46.7 +/- 5.0 pg/ng DNA), whereas feeding of a high-fat diet for 48 hours caused ACBP levels to increase (69.0 +/- 7.2 v 103.9 +/- 18.0). Hepatic levels of this protein continued to increase and remained elevated with prolonged exposure to the high-fat diet (28 days). A similar pattern of change was observed in the kidney, but the magnitude of change was less. Heart ACBP did not respond acutely to the high-fat diet, but did increase after prolonged exposure (28 days). Fasting had no effect on ACBP levels in kidney and heart. Addition of ACBP to an in vitro assay system significantly increased the activity of CPT0 (from 5.2 +/- 0.8 to 72.1 +/- 5.3 nmol palmitoylcarnitine formed.min-1.mg-1 protein) when measured under inhibiting concentrations of palmitoyl-CoA (40 mumol/L).(ABSTRACT TRUNCATED AT 250 WORDS)

Radioisotopic assay of femtomole quantities of total adenine nucleotides, ATP plus ADP, and AMP.

AMP is converted to ATP by incubating overnight with pyruvate kinase, phosphoenolpyruvate and adenylate kinase in the presence of endogenous ATP (ADP) as primer. In a subsequent incubation in the presence of pyruvate kinase, phosphoenolpyruvate, radioactive glucose and hexokinase, ATP and ADP are estimated together by coupling their recycling to the formation of glucose 6-phosphate. The latter is separated by precipitation using 76% (v/v) acetone for radioactivity measurement in the same Eppendorf tube. The sensitivity of these simple procedures matches or exceeds those of luciferase methods of nucleotide determination.

Carnitine-acylcarnitine translocase deficiency.

Carnitine-acylcarnitine translocase deficiency, like other defects of mitochondrial fatty acid oxidation, is an autosomal, recessively inherited disorder. When the deficiency is near total, it is usually fatal, affects life soon after birth, and constitutes one of the causes of skeletal muscle myopathy, cardiac and liver abnormalities, and childhood sudden death. The presenting features have included neonatal distress, convulsions, hypoglycemia, hyperammonemia, hypoketonemia, intermittent dicarboxyluria, hypothermia, apnea, neurological deterioration, and hypocarnitinemia with grossly elevated acylcarnitines. Two cases of partial translocase deficiency (4-6% residual activity) with milder symptoms and without cardiac involvement have also been identified. Evidence so far indicates that the translocase protein is the product of a single gene. In two cases of translocase deficiency, the accompanying mutations have been identified. The benefits of prenatal diagnosis have been provided to the affected families by assays of the translocase and/or fatty acid oxidation in cultured amniotic/villous cells. In one such case genetic counseling was made possible even when the only specimen available from a deceased sibling was the Guthrie card.

One-step synthesis of radioactive acyl-CoA and acylcarnitines using rat liver mitochondrial outer membrane as enzyme source.

Rat liver mitochondrial outer membrane enriched preparations have proven to be a convenient enzyme source for synthesizing coenzyme A (CoA) and carnitine esters of radioactive fatty acids. These membranes are simple to isolate and they retain acyl-CoA ligase and carnitine palmitoyltransferase activities well upon storage. Enzyme purification is not required. A novel aspect of the present procedure is that the same enzymatic incubation step allows both the acyl-CoA and the acylcarnitine esters to be obtained simultaneously when carnitine is present, but produces acyl-CoA ester only when carnitine is not included. Under the conditions described, the conversion of [1-14C]octanoic acid to the respective esters was about 95%; the corresponding figure for [1-14C]palmitic acids was over 70%. The procedure seems suitable for synthesizing the labeled CoA and carnitine esters from a variety of radioactive fatty acids.

Erucic acid metabolism by rat heart preparations.

Rat heart preparations metabolized erucic acid at much slower rates than palmitic acid. This applied for activation reaction, for the conversion of acyl-CoA to acylcarnitine, and for the utilization of acyl group for oxidation. As compared to palmityl-CoA, erucyl-CoA exhibited a lower affinity for carnitine palmityltransferase (EC 2.3.1.23), the respective apparent Michaelis constants were 43 and 83 muM. Presence of erucyl-CoA or erucyl-carnitine slowed the mitochondrial oxidation of palmityl groups apparently because of the slower oxidation of erucyl groups. However, presence of erucate did not inhibit the activation of palmitate. Heart mitochondria obtained from rats fed rapeseed oil (50 cal %) or corn oil diet for 3 days showed similar abilities for the coupled oxidation of various substrates and similar carnitine palmityltransferase activities. Thus, a suggestion of gross mitochondrial malfunction following rapeseed oil consumption was not confirmed.