Carnitine and deoxycarnitine concentration in rat tissues and urine after their administration

Administration of L-carnitine to rats was followed by an increase of deoxycarnitine in urine. Conversely, administration of deoxycarnitine caused an increase of carnitine. The latter treatment also produced a transient but significant diminution of L-carnitine in heart, skeletal muscle and kidney, but not in liver and plasma. Administration of D-carnitine to rats previously loaded with deoxycarnitine significantly depleted the elevated deoxycarnitine concentration in skeletal muscle and kidney while increasing it in plasma. These results suggest that the tissue exchange between L-carnitine and deoxycarnitine, already demonstrated in vitro, occurs also in vivo.     

Inhibition studies on acetyl group incorporation into chloroplast fatty acids

Abstract. Whilst L-acetylcarnitine acted as a substrate for fatty acid synthesis by isolated pea leaf chloroplasts, D-acetylcarnitine did not. This result, together with those obtained using the inhibitors D-carnitine and deoxycarnitine, indicated that L-acetylcarnitine was not being hydrolysed to free acetate prior to incorporation into chloroplast fatty acids. Seventy-five per cent and 66% inhibitions of L-acetylcarnitine incorporation into fatty acids, brought about by adding equimolar quantities of D-carnitine and deoxycarnitine, respectively, were suggestive of competitive inhibition at two points: an integral membrane translocator in the chloroplast envelope: and the carnitine acetyltransferase enzyme of the chloroplast stroma, which converts L-acetylcarnitine to acetyl CoA. Isotope competition experiments between acetate and L-acetylcarnitine confirmed that L-acetylcarnitine was the preferred substrate for pea chloroplast fatty acid synthesis.     

Novel action of carnitine: inhibition of aggregation of dispersed cells elicited by clusterin in vitro

A novel effect of carnitine and O-acylcarnitine derivatives has been described. The presence of these compounds has been shown to inhibit the aggregation of erythrocytes otherwise elicited by the addition of clusterin or fetuin. The specificity of carnitine action has been investigated by comparing influences of chemically related compounds. The concentrations required for inhibition by approximately 50% of aggregation of erythrocytes by clusterin under in vitro conditions defined were determined to be 1.5 mM for L(-) or D(+) enantiomers of carnitine; 0.5 mM for decanoyl(-)- or (+)-carnitine; 0.13 mM for lauroyl(-)- or (+)-carnitine, and 0.05 mM for myristoyl(-)- or (+)-carnitine. In contrast, concentrations up to 12.5 mM of dimethylcarnitine, deoxycarnitine, acetylcholine, acetyl-beta-methylcholine, or inositol had no detectable inhibitory effect on aggregation elicited by clusterin. Clusterin addition also resulted in the aggregation of three other cell types examined (guinea pig spermatozoa, a cell line derived from testes of neonatal mice called TM4 cells, and Sertoli cells from testes of 20 day-old rats). As in the case with erythrocytes, the presence of carnitine inhibited aggregation of spermatozoa, TM4 cells, and Sertoli cells in suspension. We consider possible mechanisms by which carnitine inhibits aggregation of erythrocytes and other populations of dispersed cells incubated in the presence of clusterin.     

Carnitine transport in rat heart slices: I. The action of thiol reagents on the acetylcarnitine/carnitine exchange

Rat heart slices show a permeability barrier that can be crossed by carnitine but not by sucrose and inulin. The integrity of thiol groups of heart cell membrane is essential for the uptake of carnitine. N-ethylmaleimide inhibits the transport into heart slices which is insensitive to Mersalyl. On the contrary both N-ethylmaleimide and Mersalyl inhibit acetyl carnitine/carnitine exchange. The amount of thiol groups titrated by the above reagents are related to the extent of exchange inhibition.     

Characterization of the unidirectional transport of carnitine catalyzed by the reconstituted carnitine carrier from rat liver mitochondria

The carnitine carrier from rat liver mitochondria was purified by chromatography on hydroxyapatite and celite and reconstituted in egg yolk phospholipid vesicles by adsorbing the detergent on polystyrene beads. In the reconstituted system, in addition to the carnitine/carnitine exchange, the purified protein catalyzed a uni-directional transport (uniport) of carnitine measured as uptake into unloaded proteoliposomes as well as efflux from prelabelled proteoliposomes. In both cases the reaction followed a first-order kinetics with a rate constant of 0.023-0.026 min-1. Besides carnitine, also acylcarnitines were transported in the uniport mode. N-Ethylmaleimide inhibited the uni-directional transport of carnitine completely. The uniport of carnitine is not influenced by the delta pH and the electric gradient across the membrane. The activation energy for uniport was 115 kJ/mol and the half-saturation constant on the external side of the proteoliposomes was 0.53 mM. The maximal rate of the uniport at 25 degrees C was 0.2 mumol/min per mg protein, i.e. about 10 times lower than that of the reconstituted carnitine transport in exchange mode.     

Inhibition of cell-cell adhesion and morphogenesis of Dictyostelium by carnitine.

Carnitine (gamma-trimethylammonium beta-hydroxy-butyric acid) possesses the novel property of preventing cell aggregation elicited by clusterin or by fibrinogen (I.B. Fritz and K. Burdzy, J. Cell. Physiol., 140:18-28 [1989]). In investigations reported here, we show that carnitine also affects cell-cell adhesion in Dictyostelium discoideum, a cellular slime mold whose cells interact in specific and complex manners during discrete stages of development. Two types of cell adhesion systems sequentially appear on the surface of developing Dictyostelium cells, involving the surface glycoprotein gp24 which mediates EDTA-sensitive binding sites, and the surface glycoprotein gp80 which mediates the EDTA-resistant binding sites. Addition of increasing concentrations of D(+)-carnitine and L(-)-carnitine resulted in a progressive inhibition of both the EDTA-sensitive binding sites and the EDTA-resistant binding sites of Dictyostelium cells at different stages of development. In contrast, comparable or higher concentrations of choline, acetyl-beta-methylcholine, or deoxycarnitine had no detectable effects on cell aggregation. Concentrations of carnitine required for 50% inhibition of EDTA-resistant adhesion sites were found to be dependent upon levels of gp80 expressed by Dictyostelium, with greatest inhibition by carnitine of reassociation of cells containing the lowest levels of gp80. Removal of carnitine from cells by washing resulted in the rapid restoration of the ability of Dictyostelium to form aggregates and to resume normal development. We discuss possible mechanisms by which carnitine inhibits the aggregation of cells.     

Kinetic characterization of the reconstituted carnitine carrier from rat liver mitochondria

The carnitine carrier was purified from rat liver mitochondria and reconstituted into liposomes by removing the detergent from mixed micelles by Amberlite. Optimal transport activity was obtained with 1 microgram/ml and 12.5 mg/ml of protein and phospholipid concentration, respectively, with a Triton X-100/phospholipid ratio of 1.8 and with 16 passages through the same Amberlite column. The activity of the carrier was influenced by the phospholipid composition of the liposomes, being increased in the presence of cardiolipin and decreased in the presence of phosphatidylinositol. In the reconstituted system the incorporated carnitine carrier catalyzed a carnitine/carnitine exchange which followed a first-order reaction. The maximum transport rate of external [3H]carnitine was 1.7 mmol/min per g protein at 25 degrees C and was independent of the type of countersubstrate. The half-saturation constant (Km) for carnitine was 0.51 mM. The affinity of the carrier for acylcarnitines was in the microM range and depended on the carbon chain length. The activation energy of the carnitine/carnitine exchange was 133 kJ/mol. The carrier function was independent of the pH in the range between 6 and 8 and was inhibited at pH below 6.     

Influence of carnitine supplementation on muscle substrate and carnitine metabolism during exercise

We examined 1) the effect of L-carnitine supplementation on free fatty acid (FFA) utilization during exercise and 2) exercise-induced alterations in plasma levels and skeletal muscle exchange of carnitine. Seven moderately trained human male subjects serving as their own controls participated in two bicycle exercise sessions (120 min, 50% of VO2max). The second exercise was preceded by 5 days of oral carnitine supplementation (CS; 5 g daily). Despite a doubling of plasma carnitine levels, with CS, there were no effects on exercise-induced changes in arterial levels and turnover of FFA, the relation between leg FFA inflow and FFA uptake, or the leg exchange of other substrates. Heart rate during exercise after CS decreased 7-8%, but O2 uptake was unchanged. Exercise before CS induced a fall from 33.4 +/- 1.6 to 30.8 +/- 1.0 (SE) mumol/l in free plasma carnitine despite a release (2.5 +/- 0.9 mumol/min) from the leg. Simultaneously, acylated plasma carnitine rose from 5.0 +/- 1.0 to 14.2 +/- 1.4 mumol/l, with no evidence of leg release. Consequently, total plasma carnitine increased. We concluded that in healthy subjects CS does not influence muscle substrate utilization either at rest or during prolonged exercise and that free carnitine released from muscle during exercise is presumably acylated in the liver and released to plasma.     

Effect of exogenous carnitine on carnitine homeostasis in the rat

The interaction of exogenous carnitine with whole body carnitine homeostasis was characterized in the rat. Carnitine was administered in pharmacologic doses (0-33.3 mumols/100 g body weight) by bolus, intravenous injection, and plasma, urine, liver, skeletal muscle and heart content of carnitine and acylcarnitines quantitated over a 48 h period. Pre-injection urinary carnitine excretion was circadian as excretion rates were increased 2-fold during the lights-off cycle as compared with the lights-on cycle. Following carnitine administration, there was an increase in urinary total carnitine excretion which accounted for approx. 60% of the administered carnitine at doses above 8.3 mumols/100 g body weight. Urinary acylcarnitine excretion was increased following carnitine administration in a dose-dependent fashion. During the 24 h following administration of 16.7 mumols [14C]carnitine/100 g body weight, urinary carnitine specific activity averaged only 72 +/- 4% of the injection solution specific activity. This dilution of the [14C]carnitine specific activity suggests that endogenous carnitine contributed to the increased net urinary carnitine excretion following carnitine administration. 5 min after administration of 16.7 mumol carnitine/100 g body weight approx. 80% of the injected carnitine was in the extracellular fluid compartment and 5% in the liver. Plasma, liver and soleus total carnitine contents were increased 6 h after administration of 16.7 mumols carnitine/100 g body weight. 6 h post-administration, 37% of the dose was recovered in the urine, 12% remained in the extracellular compartment, 9% was in the liver and 22% was distributed in the skeletal muscle. In liver and plasma, short chain acylcarnitine content was increased 5 min and 6 h post injection as compared with controls. Plasma, liver, skeletal muscle and heart carnitine contents were not different from control levels 48 h after carnitine administration. The results demonstrate that single, bolus administration of carnitine is effective in increasing urinary acylcarnitine elimination. While liver carnitine content is doubled for at least 6 h following carnitine administration, skeletal muscle and heart carnitine pools are only modestly perturbed following a single intravenous carnitine dose. The dilution of [14C]carnitine specific activity in the urine of treated animals suggests that tissue-blood carnitine or acylcarnitine exchange systems contribute to overall carnitine homeostasis following carnitine administration.     

Carnitine transport in submitochondrial particles and reconstituted proteoliposomes.

Influx and efflux measurements of carnitine with submitochondrial particles lead to the conclusion that carnitine can cross the inner mitochondrial membrane by either facilitated diffusion or more rapidly by a carnitine-carnitine exchange. Both, the facilitated diffusion and the exchange are inhibited by N-ethylmaleimide or mersalyl at low concentrations. Reconstituted particles prepared from liposomes and either submitochondrial particles or an octyl β-glucoside-solubilized preparation were active in catalyzing carnitine-carnitine exchange.     

Identification and purification of the carnitine carrier from rat liver mitochondria

The carnitine carrier from rat liver mitochondria, solubilized in Triton X-100 and partially purified on hydroxyapatite, was identified and completely purified by specific elution from celite in the presence of cardiolipin. On SDS-gel electrophoresis, the purified celite fraction consisted of a single band with an apparent Mr of 32,500. When reconstituted into liposomes the carnitine transport protein catalyzed an N-ethylmaleimide-sensitive carnitine/carnitine exchange. It was purified 970-fold with a recovery of 43% and a protein yield of 0.04% with respect to the mitochondrial extract. The properties of the reconstituted carrier, i.e., requirement for a countersubstrate, substrate specificity and inhibitor sensitivity, were similar to those of the carnitine transport system as characterized in intact mitochondria.     

The mitochondrial carnitine/acylcarnitine carrier: function, structure and physiopathology

The carnitine/acylcarnitine carrier (CAC) is a transport protein of the inner mitochondrial membrane that belongs to the mitochondrial carrier protein family. In its cytosolic conformation the carrier consists of a bundle of six transmembrane α-helices, which delimit a water filled cavity opened towards the cytosol and closed towards the matrix by a network of interacting charged residues. Most of the functional data on this transporter come from studies performed with the protein purified from rat liver mitochondria or recombinant proteins from different sources incorporated into phospholipid vesicles (liposomes). The carnitine/acylcarnitine carrier transports carnitine and acylcarnitines with acyl chains of various lengths from 2 to 18 carbon atoms. The mammalian transporter exhibits higher affinity for acylcarnitines with longer carbon chains. The functional data indicate that CAC plays the important function of catalyzing transport of acylcarnitines into the mitochondria in exchange for intramitochondrial free carnitine. This results in net transport of fatty acyl units into the mitochondrial matrix where they are oxidized by the β-oxidation enzymes. The essential role of the transporter in cell metabolism is demonstrated by the fact that alterations of the human gene SLC25A20 coding for CAC are associated with a severe disease known as carnitine carrier deficiency. This autosomal recessive disorder is characterized by life-threatening episodes of coma induced by fasting, cardiomyopathy, liver dysfunction, muscle weakness, respiratory distress and seizures. Until now 35 different mutations of CAC gene have been identified in carnitine carrier deficient patients. Some missense mutations concern residues of the signature motif present in all mitochondrial carriers. Diagnosis of carnitine carrier deficiency requires biochemical and genetic tests; treatment is essentially limited to important dietetic measures. Recently, a pharmacological approach based on the use of statins and/or fibrates for the treatment of CAC-deficient patients with mild phenotype has been proposed.     

Membrane transport of fatty acylcarnitine and free L-carnitine by rat liver microsomes.

Recent studies have suggested that parts of the hepatic activities of diacylglycerol acyltransferase and acyl cholesterol acyltransferase are expressed in the lumen of the endoplasmic reticulum (ER). However the ER membrane is impermeable to the long-chain fatty acyl-CoA substrates of these enzymes. Liver microsomal vesicles that were shown to be at least 95% impermeable to palmitoyl-CoA were used to demonstrate the membrane transport of palmitoylcarnitine and free L-carnitine - processes that are necessary for an indirect route of provision of ER luminal fatty acyl-CoA through a luminal carnitine acyltransferase (CAT). Experimental conditions and precautions were established to permit measurement of the transport of [14C]palmitoylcarnitine into microsomes through the use of the luminal CAT and acyl-CoA:ethanol acyltransferase as a reporter system to detect formation of luminal [14C]palmitoyl-CoA. Rapid, unidirectional transport of free L-[3H]carnitine by microsomes was measured directly. This process, mediated either by a channel or a carrier, was inhibited by mersalyl but not by N-ethylmaleimide or sulfobetaine - properties that differentiate it from the mitochondrial inner membrane carnitine/acylcarnitine exchange carrier. These findings are relevant to the understanding of processes for the reassembly of triacylglycerols that lipidate very low density lipoprotein particles as part of a hepatic triacylglycerol lipolysis/re-esterification cycle.     

On the presence of carnitine acetyl transferase in human platelets

Summary The presence of carnitine acetyl transferase (E.C.2.3.1.7) activity has been found for the first time in human platelets. The enzymic activity was measured by a radiometric method based on the separation of labelled acetylcarnitine and carnitine on a cation exchange column. Carnitine acetyl transferase activity closely paralleled the activity distribution of the mitochondrial marker carnitine palmitoyl-transferase. Contrary to the marker enzyme, human platelet carnitine acetyl-transferase is rather thermosensitive: 60% of its activity is lost after 10 min when kept at 37°C.     

Reexamination of the 1H NMR assignments and solution conformations of L-carnitine and O-acetyl-L-carnitine using C-2 stereospecifically labeled monodeuterated isomers

The two C-2 monodeuterated isomers of L-carnitine were synthesized by enzymatic hydration of crotonobetaine in D2O and by enzymatic proton exchange of L-[2-2H2]carnitine in H2O. These reactions, catalyzed by an induced Escherichia coli carnitine hydrolyase proceed stereospecifically. The two isomers of L-[2-2H]carnitine were examined by 1H NMR at 500 MHz, which allowed us to independently monitor the pD dependence and coupling constants of the H-2 protons. The results obtained indicate that there is little effect of the carboxyl charge on the conformational state(s) of L-carnitine about the C-2/C-3 bond. The NMR data obtained in this study do not support previous solution studies of the pH-dependent conformational changes for DL-carnitine nor the proposed conformation of O-acetyl-DL-carnitine in the crystalline state.     

Metabolic interactions between peroxisomes and mitochondria with a special focus on acylcarnitine metabolism

Carnitine plays an essential role in mitochondrial fatty acid β-oxidation as a part of a cycle that transfers long-chain fatty acids across the mitochondrial membrane and involves two carnitine palmitoyltransferases (CPT1 and CPT2). Two distinct carnitine acyltransferases, carnitine octanoyltransferase (COT) and carnitine acetyltransferase (CAT), are peroxisomal enzymes, which indicates that carnitine is not only important for mitochondrial, but also for peroxisomal metabolism. It has been demonstrated that after peroxisomal metabolism, specific intermediates can be exported as acylcarnitines for subsequent and final mitochondrial metabolism. There is also evidence that peroxisomes are able to degrade fatty acids that are typically handled by mitochondria possibly after transport as acylcarnitines. Here we review the biochemistry and physiological functions of metabolite exchange between peroxisomes and mitochondria with a special focus on acylcarnitines.     

Chymotrypsin activates cardiac mitochondrial carnitine-acylcarnitine translocase.

The carnitine-acylcarnitine translocase facilitates carnitine and acylcarnitine transport into the mitochondrial matrix during beta-oxidation. Our results demonstrate that chymotrypsin can activate the maximal velocity of N-ethylmaleimide (NEM)-sensitive carnitine or palmitoylcarnitine exchange 7-fold, while doubling the affinity of the translocase for carnitine. Chymotrypsin activation is strictly dependent on the presence of free or short-chain acylcarnitine in the proteolysis medium, the extent of activation decreasing as the acylcarnitine chain length in the proteolysis medium increases. Chymotrypsin treatment decreases the apparent I50 value (inhibitor concentration required to give half-maximal inhibition) of the translocase for inhibition by NEM only under conditions which produce translocase activation. Modification of submitochondrial particle membranes by chymotrypsin does not result in gross ultrastructural changes or in an increase in the passive permeability of these membranes to carnitine. The data suggest that carnitine binding produces a change in translocase conformation which allows chymotrypsin modification to occur. This modification alters the kinetic and inhibitor-binding properties of the translocase.     

Lipid oxidation by heart mitochondria from young adult and senescent rats

1. State-3 (i.e. ADP-stimulated) rates of O(2) uptake with palmitoylcarnitine, palmitoyl-CoA plus carnitine, pyruvate plus malonate plus carnitine and octanoate as respiratory substrate were all diminished in heart mitochondria isolated from senescent (24-month-old) rats compared with mitochondria from young adults (6 months old). By contrast, State-3 rates of O(2) uptake with pyruvate plus malate or glutamate plus malate were the same for mitochondria from each age group. 2. Measurements of enzyme activities in disrupted mitochondria showed a decline with senescence in the activity of acyl-CoA synthetase (EC 6.2.1.2 and 6.2.1.3), carnitine acetyltransferase (EC 2.3.1.7) and 3-hydroxy-acyl-CoA dehydrogenase (EC 1.1.1.35), but no change in the activity of carnitine palmitoyltransferase (EC 2.3.1.21) or acyl-CoA dehydrogenase (EC 1.3.99.3). 3. Measurement of dl-[(3)H]carnitine (in)/acetyl-l-carnitine (out) exchange in intact mitochondria showed decreased rates when the animals used were senescent. However, this followed from a decreased intramitochondrial pool of exchangeable carnitine, such that calculated first-order rate constants for exchange were identical in mitochondria from the two age groups. 4. The decline in acyl-CoA synthetase activity is thought to be the reason for the diminished rate of O(2) uptake with octanoate in senescence. The decline in carnitine acetyltransferase activity is considered to be the cause of the diminished rate of O(2) uptake with acetylcarnitine or with pyruvate plus malonate plus carnitine as substrate. The mechanism of the diminished rate of O(2) uptake with palmitoylcarnitine in senescence is discussed.     

Tissue specific depletion of L-carnitine in rat heart and skeletal muscle by D-carnitine

L-carnitine deficiency in heart and skeletal muscle was induced by intraperitoneal injection of D-carnitine into starved or fed rats. Carnitine levels in kidney were slightly lowered, but liver, brain and plasma were unaffected. L-carnitine deficient hearts were unable to maintain normal cardiac function when perfused in an isolated working heart apparatus with palmitate as the only perfused substrate. These findings indicate that tissue levels of carnitine in heart and skeletal muscle are maintained $\text{in vivo}$ by an exchange transport mechanism. It is postulated that the depletion of L-carnitine from these tissues occurs by an exchange of the D- and L-isomer across the cell membrane. The technique may be useful for estimating the levels of carnitine required for fatty acid oxidation and normal cardiac and skeletal muscle function; however, interpretation of such tests may be complicated by the inhibitory effects of the D-isomer upon carnitine transferase enzymes.     

Aggregation of submitochondrial particles by heparin and its application to the study of carnitine transport.

A novel technique for the separation of submitochondrial particles from the external medium, an essential procedure in transport studies, was devised. Very low concentrations of heparin (5-10 micrograms/ml) aggregate the particles and permit their rapid sedimentation in a micro-centrifuge. The transfer of activated fatty acids into mitochondria for oxidation depends on the exchange of matrix carnitine for external fatty-acylcarnitine. To study the matrix face of the carnitine/acylcarnitine translocase, inverted submitochondrial particles were prepared and loaded with L-[14C]carnitine. As found in intact mitochondria, the Km value for L-carnitine was 8 mM, that for palmitoyl-L-carnitine was two orders of magnitude lower, and 11-trimethylaminoundecanoyl-DL-carnitine was a competitive inhibitor. The properties of the carrier exposed to the outer and to the matrix sides of the mitochondrial inner membrane are thus similar.