Effect of various levels of supplementation with sodium pivalate on tissue carnitine concentrations and urinary excretion of carnitine in the rat

In previous studies, sodium pivalate has been administered to rats in their drinking water (20 mmoles/L; equivalent to 0.3% of the diet) as a way to lower the concentration of carnitine in tissues and to produce a model of secondary carnitine deficiency. Although this level of supplementation results in a marked decrease in carnitine concentration in a variety of tissues, it does not produce the classical signs of carnitine deficiency (i.e., decreased fatty acid oxidation and ketogenesis). The present study was designed (1) to determine if increasing the level of pivalate supplementation (0.6, 1.0% of the diet) would further reduce the concentrations of total and free carnitine in rat tissues without altering growth or food intake, and (2) to examine the effect of length of feeding (4 vs. 8 weeks) on these variables. Male, Sprague-Dawley rats were randomly assigned to either a control (0.2% sodium bicarbonate) or experimental diet (0.3, 0.6, 1.0% sodium pivalate) for either four or eight weeks. Animals (n = 6/group) were housed in metabolic cages; food and water were provided ad libitum throughout the study. Supplementation with sodium pivalate did not alter water intake or urine output. Ingestion of a diet containing 1.0% pivalic acid decreased food intake (g/day; P < 0.05), final body weight (P < 0.007), and growth rate (P < 0.001) after four weeks. The concentration of total carnitine in plasma, heart, liver, muscle, and kidney was reduced in all experimental groups (P < 0.001), regardless of level of supplementation or length of feeding. The concentration of free carnitine in heart, muscle, and kidney was also reduced (P < 0.001) in rats treated with pivalate for either four or eight weeks. The concentration of free carnitine in liver was reduced in animals supplemented with pivalate for eight weeks (P < 0.05), but no effect was observed in livers from rats treated for four weeks. Excretion of total carnitine and short chain acylcarnitine in urine was increased in pivalate supplemented rats throughout the entire feeding period (P < 0.001). Free carnitine excretion was increased during Weeks 1 and 2 (P < 0.01), but began to decline during Week 3 in experimental groups. During Weeks 6 and 8, free carnitine excretion in pivalate supplemented rats was less than that of control animals (P < 0.01). In summary, no further reduction in tissue carnitine concentration was observed when rats were supplemented with sodium pivalate at levels greater than 0.3% of the diet. Food intake (g/day) and growth were decreased in rats fed a diet containing 1.0% sodium pivalate. These data indicate that maximal lowering of tissue carnitine concentrations is achieved by feeding diets containing 0.3% sodium pivalate or less.     

Cefetamet pivoxil treatment causes loss of carnitine reserves that can be prevented by exogenous carnitine administration

Two groups of pediatric patients receiving cefetamet pivoxil treatment (3 x 500 mg daily) for 7 days were studied. In the first group (Group A) the drug was administered alone; in the second group (Group B) the drug was given in combination with a molar excess of carnitine (3 x 1 g). Medication with cefetamet pivoxil alone was associated with a large urinary excretion of pivaloylcarnitine: Approximately 71% of the daily pivalate intake could be eliminated as carnitine ester in the urine. In this group, the plasma level and the urinary output of free carnitine decreased. By contrast, in Group B, the administration of molar excess of carnitine aided stochiometric elimination of pivalate as carnitine ester, and the plasma levels and carnitine-free urinary output were unchanged. The data show that medication of cefetamet pivoxil results in the formation of pivaloylcarnitine in children; the sustained loss of carnitine esters can ultimately lead to carnitine deficiency. Molar excess of exogenous carnitine aids in the elimination of pivalate derived from cefetamet pivoxil therapy and helps to maintain the carnitine reserves.     

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.     

Free carnitine and acetyl carnitine plasma levels and their relationship with body muscular mass in athletes

Summary The purpose of the present study was to investigate the relationship between plasma carnitine concentration and body composition variation in relation to muscular and fat masses since there is no experimentally proved correlation between plasma carnitine and body masses. We used bioelectric impedance analysis (BIA), to determine body composition and to have a complete physical fitness evaluation. The post-absorptive plasma free carnitine and acetyl carnitine plasma levels, body composition as Fat-Free Mass (FFM) and Fat Mass (FM) in kg, as well as in percent of body mass, were analysed in 33 healthy subjects. A significant negative correlation was found between plasma acetyl carnitine and FFM in weight (kg) as well as in percent of body mass (respectively p < 0.0001; p < 0.01); a significant positive correlation was found only between FM in percent and plasma acetyl carnitine (p < 0.01). The observed negative correlation between plasma acetyl carnitine and muscular mass variation might reflect an oxidative metabolic muscle improvement in relation to muscular fat free mass increment and might be evidence that muscle metabolism change is in relation to plasma acetyl carnitine concentration.     

Effects of acute moderate-intensity exercise on carnitine metabolism in men and women

The purpose of this investigation was to describe the dynamics of carnitine metabolism during an acute episode of exercise. Twenty-eight subjects (14 male; 14 female) exercised for 40 min on a bicycle ergometer at 55% of their maximal aerobic capacities. Blood samples were obtained at rest, 10, 20, 30, and 40 min of exercise, and 15-min postexercise. Muscle biopsies of the vastus lateralis were performed before and after exercise. Results demonstrated that the percent of acylated plasma carnitine increased significantly (P less than 0.05) across all subjects from 17.3% at rest to 22.3% by 40 min of exercise and continued to increase to 22.8% 15-min postexercise. Total muscle carnitine levels fell significantly (P less than 0.001) across all subjects from 4.21 (1.27) (means +/- SD) mumol/g wet weight at rest to 3.29 (1.27) mumol/g wet weight after exercise. Well-trained males and females had almost identical levels of muscle carnitine [4.35(1.86) and 4.34 (0.64) mumol/g wet weight, respectively]. These levels were somewhat higher but not significantly higher than their moderately trained counterparts [3.86(1.34) and 4.28(1.18) males and females, respectively]. Carnitine palmitoyl transferase (E.C. 2.3.1.21) activity also declined significantly (P less than 0.05) across all subjects after exercise. This study is the first to demonstrate a potential loss of acylated carnitine forms from muscle to plasma during acute exercise, possibly reflecting an increase in carnitine turnover. Alterations in carnitine status may represent another metabolic adaptation to chronic exercise training.     

The nutrition of choline, carnitine, and related compounds in the blowfly, Phormia regina Meigen

Fifteen choline analogues were tested as substitutes for choline in the larval diet of Phormia regina. From the results the structural requirements for an adequate choline substitute are further defined. The choline analogue, (CH₃)₃+NCH₂CH₂CH₂OH, although not an inhibitor in the presence of choline, inhibits growth when choline is replaced in the diet by carnitine or γ-butyrobetaine. This compound presumably inhibits one of the reactions which metabolizes carnitine or γ-butyrobetaine to β-methyl choline.     

Plasma carnitine and acetyl-carnitine levels at different times of the day

An interest in both biochemical and clinical carnitine investigation has recently developed. A more complete and extensive study is obtained if acetyl-carnitine as well as carnitine are investigated. This research, using an improved and simplified method for carnitine and acetyl-carnitine determination in the same sample (1 ml) without radioisotopic tracer use, investigates if there are the same differences in their plasma levels at different times of the day. The sample was eluted in a chromatographic column (55 X 15 mm) containing Sephadex G-25M with phosphate buffer (25 mmol/l, pH 7.4). The fraction containing acetyl and free carnitine was divided and employed separately for two assays. The carnitine assay uses an enzymatic reaction catalyzed by carnitine acetyl-transferase (CAT) and measurements are carried out spectrophotometrically. The calibration curve shows r = 0.987 and sensitivity at 5 mumol/l (reference plasma values: 38 +/- 3 mumol/l in 9 subjects). The acetyl-carnitine assay is carried out concentrating the sample by lyophilization and then measuring the enzymatic coupled reactions catalyzed by CAT, malate dehydrogenase and citrate synthase fluorimetrically. The calibration curve gives r = 0.991 and sensitivity at 1.4 mumol/l (reference plasma values: 2.8 +/- 0.3 mumol/l in 9 subjects). Both assay methods are measured at the end point. The carnitine and acetyl-carnitine measured in the plasma of 6 normal subjects at different times of the day vary respectively from 28 to 37 mumol/l and from 1.1 to 5.2 mumol/l in agreement with plasma free fatty acid (FFA) variation from 230 to 779 microEq/l.(ABSTRACT TRUNCATED AT 250 WORDS)     

Metabolism of labeled carnitine in the rat

In two series of rats, the concentration of carnitine in plasma was 39.9 and 37.8 μmol/ liter, in skeletal muscle tissue 2.97 and 3.26 μmol/g dry wt and the urinary excretion 3.2 and 2.4 μmol/24 h. The renal clearance of carnitine was calculated to 88 and 76 ml/24 h. L-[Me-¹⁴C]Carnitine and DL-[Me-¹⁴C]carnitine have been administered to rats. Only labeled l-carnitine has been found on chromatographic analysis of plasma, urine, and muscle tissue. The specific radioactivity of carnitine in plasma, urine, and muscle tissue has been followed for up to 16 days. A two-compartment metabolic model has been used to interpret the result of the experiment with labeled l-carnitine and the rate constants and compartment sizes have been calculated. The total body content of carnitine was 57 μmol (about 35 μmol/100 g body wt) and the daily turnover was about 7% of the body pool. The daily synthesis of carnitine in the rat is estimated to about 2 μmol/100 g body wt.     

Inhibition of carnitine acetyltransferase by bile acids: implications for carnitine analysis

Carnitine acetyltransferase is used in a radioenzymatic assay to measure the concentration of carnitine. While determining the concentration of carnitine in rat bile, we found that the apparent concentration increased as bile was diluted (6.7 +/- 1.0 and 66.6 +/- 9.4 nmol/ml in undiluted and 20-fold diluted bile, respectively). The present study was designed to investigate whether a component of bile inhibited carnitine acetyltransferase. Inhibition was evaluated by measuring carnitine concentration in bile or by determining the recovery of a known amount of carnitine in the presence of bile. Inhibitory activity was extractable in organic solvents, stable to heat and base treatments, resistant to trypsin and lipase digestions, and removable by cholestyramine, a bile acid-binding resin. These results suggested that the inhibitory activity was associated with bile acids. Direct evidence was obtained by showing a reduced detectability of carnitine in the presence of individual bile acids. Chenodeoxycholic acid was the most potent inhibitor. Inhibition was unrelated to the detergent properties of bile acids. Kinetic studies revealed that carnitine acetyltransferase was inhibited competitively by chenodeoxycholic acid with a Ki of 520 microM. Bile acids also interfered in the quantitation of carnitine in cholestatic plasma. Carnitine concentration in such plasma was underestimated (17.5 +/- 2.1 mmol/ml). Reduction of bile acid concentration by a 20-fold dilution of cholestatic plasma resulted in a 3-fold higher carnitine concentration (54.6 +/- 9.0 nmol/ml). Results demonstrate that, because of the inhibition of carnitine acetyltransferase by bile acids, the radioenzymatic assay will underestimate carnitine concentration in bile or in cholestatic plasma. Accurate measurement requires either the removal of bile acids or a marked reduction in their concentration.     

Quantitation of efletirizine in human plasma and urine using automated solid-phase extraction and column-switching high-performance liquid chromatography

A heart-cut column-switching, ion-pair, reversed-phase HPLC system was used for the quantitation of efletirizine (EFZ) in biological fluids. The analyte and an internal standard (I.S.) were extracted from human EDTA plasma by C₁₈ solid-phase extraction (SPE) using a RapidTrace^® workstation. The eluent from the SPE was evaporated, reconstituted and injected onto the HPLC column. Urine samples were diluted and injected directly without the need of extraction. The compounds of interest were separated from most of the extraneous matrix materials by the first C₁₈ column, and switched onto a second C₁₈ column for further separation using a mobile phase of stronger eluting capability. Linearity range was 10–2000 ng ml⁻¹ for plasma and 0.05–10 μg ml⁻¹ for urine. The lower limit of quantitation (LOQ) was 10 ng from 1 ml of plasma, with a signal-to-noise ratio of 15:1. Inter-day precision and bias of quality control samples (QCs) were <5% for plasma and <7% for urine. Selectivity was established against six other antihistamines, three analogs of efletirizine, and on 12 control plasma lots and nine control urine lots. Recovery was 90.0% for EFZ and 89.5% for I.S. from plasma. One hundred samples can be processed in every 2.75 h on a 10-module RapidTrace^® workstation with minimal human attention. Method ruggedness were tested on three brands of SPE and six different lots of one SPE brand. Performance ruggedness was demonstrated by different analysts on multiple HPLC systems. Analyte stability through sample storage, extraction process (benchtop, freeze–thaw, refrigeration after extraction) and chromatography (on-system, reinjection) was established.     

Carnitine, acetylcarnitine and the activity of carnitine acyltransferases in seminal plasma and spermatozoa of men, rams and rats.

The concentration of total carnitine (i.e. carnitine plus acetylcarnitine) was measured in seminal plasma and spermatozoa of men and rams. In ram semen, there was a close correlation between the concentration of spermatozoa and that of total carnitine in the seminal plasma, indicating that the epididymal secretion was the sole source of seminal carnitine. The percentage of total carnitine present as acetylcarnitine was 40% in seminal plasma and 70-80% in spermatozoa. The acetylation state of carnitine in seminal plasma was apparently not influenced by the metabolic activity of spermatozoa in ejaculated ram semen as no change was found in the plasma concentration of carnitine or acetylcarnitine up to 45 min after ejaculation. In spermatozoa, the activity of carnitine acetyltransferase (EC 2.3.1.7) was approximately equivalent to that of carnitine palmitoyltransferase (EC 2.3.1.21); and the activity of these enzymes was similar in ram and human spermatozoa but greater in rat spermatozoa. It is concluded that there is no correlation between the content of either total carnitine or the carnitine acyltransferases and the respiratory capacity of spermatozoa.     

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.     

The influence of diet and carnitine supplementation on plasma carnitine, cholesterol and triglyceride in WHHL (Watanabe-heritable hyperlipidemic), Netherland dwarf and New Zealand rabbits (Oryctolagus cuniculus)

1. Plasma carnitine levels in the spontaneously (endogenously) hyperlipidemic Watanabe (WHHL) rabbit are approximately 2-fold higher (P less than 0.001) than in normal rabbits of the New Zealand (NZ) or Netherland Dwarf (NDw) breeds. 2. Plasma carnitine levels in WHHL (44 +/- 3 nmol/ml) can be approximated in NZ and NDw which are rendered exogenously hyperlipidemic by supplementation of the stock chow diet with cholesterol and peanut oil. 3. The induction of endogenous hyperlipidemia in NZ by feeding a sucrose casein rich diet results in a biphasic response of plasma carnitine (elevation followed by normalization). 4. Plasma carnitine in WHHL is readily elevated by supplemental L-carnitine and the elevation is associated with a reduction in plasma triglyceride which shows differences in individual response time; plasma cholesterol is unaffected by supplemental L-carnitine.     

Effects of ciprofibrate and 2-[5-(4-chlorophenyl)pentyl]oxirane-2-carboxylate (POCA) on the distribution of carnitine and CoA and their acyl-esters and on enzyme activities in rats. Relation between hepatic carnitine concentration and carnitine acetyltransferase activity.

The effects of feeding the peroxisome proliferators ciprofibrate (a hypolipidaemic analogue of clofibrate) or POCA (2-[5-(4-chlorophenyl)pentyl]oxirane-2-carboxylate) (an inhibitor of CPT I) to rats for 5 days on the distribution of carnitine and acylcarnitine esters between liver, plasma and muscle and on hepatic CoA concentrations (free and acylated) and activities of carnitine acetyltransferase and acyl-CoA hydrolases were determined. Ciprofibrate and POCA increased hepatic [total CoA] by 2 and 2.5 times respectively, and [total carnitine] by 4.4 and 1.9 times respectively, but decreased plasma [carnitine] by 36-46%. POCA had no effect on either urinary excretion of acylcarnitine esters or [acylcarnitine] in skeletal muscle. By contrast, ciprofibrate decreased [acylcarnitine] and [total carnitine] in muscle. In liver, ciprofibrate increased the [carnitine]/[CoA] ratio and caused a larger increase in [acylcarnitine] (7-fold) than in [carnitine] (4-fold), thereby increasing the [short-chain acylcarnitine]/[carnitine] ratio. POCA did not affect the [carnitine]/[CoA] and the [short-chain acylcarnitine]/[carnitine] ratios, but it decreased the [long-chain acylcarnitine]/[carnitine] ratio. Ciprofibrate and POCA increased the activities of acyl-CoA hydrolases, and carnitine acetyltransferase activity was increased 28-fold and 6-fold by ciprofibrate and POCA respectively. In cultures of hepatocytes, ciprofibrate caused similar changes in enzyme activity to those observed in vivo, although [carnitine] decreased with time. The results suggest that: (1) the reactions catalysed by the short-chain carnitine acyltransferases, but not by the carnitine palmitoyltransferases, are near equilibrium in liver both before and after modification of metabolism by administration of ciprofibrate or POCA; (2) the increase in hepatic [carnitine] after ciprofibrate or POCA feeding can be explained by redistribution of carnitine between tissues; (3) the activity of carnitine acetyltransferase and [total carnitine] in liver are closely related.     

Involvement of protein kinase C in prostaglandin D2 synthesis by cultured astrocytes

The role of protein kinase C (PKC) and calcium in the stimulation of prostaglandin D₂ (PGD₂) synthesis was investigated in primary rat astroglial cultures using the phorbol esters phorbol 12-myristate, 13-acetate (PMA), phorbol 12,13-dibutyrate (PDB) and the calcium ionophore A₂₃₁₈₇. Both phorbol esters and the ionophore were able to stimulate PGD₂ synthesis in a concentration dependent manner. The inactive stereoisomers of PMA and PDB had no significant effect. Combinations of subthreshold concentrations of phorbol esters (10 nM PMA or 10 nM PBD) potentiated PG formation induced by 100 nM A₂₃₁₈₇. An even more pronounced effect was observed when phorbol ester concentrations were increased to 100nM. The contribution of extra- and intracellular calcium in phorbol ester or A₂₃₁₈₇ stimulated PGD₂ synthesis was evaluated by carrying out experiments with calcium-free media plus EGTA or with the intracellular calcium-chelating agent TMB-8. Ionophore stimulated PGD₂ release was shut down to basal values upon removal of extracellular calcium, whereas phorbol ester stimulated PGD₂ formation persisted at a reduced level. It was unabated also upon further addition of EGTA. In the presence of TMB-8, however, phorbol ester stimulated PGD₂ synthesis was completely suppressed. These data strongly suggest that PKC has an additional effect on the activation of phospholipase A₂ and subsequent prostanoid synthesis, which is independent from extracellular calcium and, thus, support the concept of more than one metabolic pathway in astrocytes that synergistically regulate phospholipase A₂ activity.     

Sensitive high-performance liquid chromatographic method for the determination of the lactone form and the lactone plus hydroxy-acid forms of the new camptothecin derivative DX-8951 in human plasma using fluorescence detection

A sensitive quantitation of the lactone form and the lactone plus hydroxy-acid forms of DX-8951, a camptothecin derivative, in human plasma has been investigated by high-performance liquid chromatography (HPLC). This assay method consisted of two analytical procedures. In Procedure I, the lactone form was collected by the stepwise separation on a C₁₈ cartridge. In Procedure II, the lactone plus hydroxy-acid forms were collected using another batch of the plasma sample by co-elution of the two forms from a C₁₈ cartridge with acidic solution. The hydroxy-acid form of DX-8951 was quantitated from the difference of the lactone plus hydroxy-acid forms and the lactone form. Thereafter, these pre-treated samples were assayed by HPLC under the same HPLC conditions with a spectrofluorometer and a reverse-phase ODS column. The mobile phase was acetonitrile/0.05 M potassium dihydrogen phosphate (pH 3) (18:82, v/v) at a flow-rate of 1.0 ml/min. For the assay of the lactone form and the lactone plus hydroxy-acid forms of DX-8951 in plasma, analytical method were validated over the range 0.2–50 ng/ml.     

Capillary electrophoresis with contactless conductivity detection for the determination of carnitine and acylcarnitines in clinical samples

A capillary electrophoresis method with contactless conductivity detection was developed for the quantification of carnitine and six acylcarnitines in plasma and urine samples. The running buffer employed consisted of 500 mmol/L acetic acid, 1.0 mmol/L hydroxypropyl-β-cyclodextrin and 0.05% Tween at a pH of 2.6. Under these conditions, the isomeric valproyl- and octanoyl-carnitines could be distinguished. The linearity was in the range from 5.0 to 200.0 μmol/L with correlation coefficients between 0.9992 and 0.9997. The limits of detection were between 1.0 and 3.2 μmol/L. Intra- and inter-day precisions as %RSD were better than 10%. The method allows for direct determination without derivatisation or extraction processes. The method was applied for the quantification of carnitine and acetylcarnitine in plasma pre- and post-exercise, and to measure valproylcarnitine in plasma and urine of patients undergoing valproate therapy.     

An inverse relationship between plasma carnitine and triglycerides in selected Macaca arctoides and Macaca nemistrina fed a low-fat chow diet

Plasma carnitine and triglycerides were measured in five male Macaca arctoides and one female Macaca nemistrina during the course of feeding a low-fat (5.2% w/w), high carbohydrate diet and a high-fat (15.9% w/w), low carbohydrate diet. For each individual monkey, an inverse relationship was observed between plasma carnitine and triglyceride levels when the low-fat diet was fed but not when the high-fat diet was fed. The mechanism of the different responses to diet was not investigated but may be related to the primary source of the plasma triglycerides (i.e. endogenous origin or exogenous origin) or to differing hormonal effects. A close coupling between carnitine and triglyceride metabolism may be part of a sensitive homeostatic control mechanism that responds to endogenously-synthesized triglyceride.     

Carnitine and carnitine esters in rat bile and human duodenal fluid

The recent discovery of carnitine and its esters in rat bile has led to much speculation about its role. The objectives of these studies were to investigate the origin of carnitine esters in rat bile and to study the presence of carnitine in human bile-rich duodenal fluid. Bile was collected from chow-fed (n = 11), fasted (72 h, n = 6), and fasted plus 2-tetradecylglycidic acid administered (72 h, n = 5) male adult rats under sodium pentobarbital anaesthesia. Carnitine and carnitine ester content was measured in the bile and compared with serum and liver carnitine. Bile from fed rats was found to contain 80% acylcarnitine, one-third of this as long chain carnitine esters. Fasting caused no change in the secretion rate of acylcarnitine into the bile, although long chain carnitine ester secretion almost doubled. Conversely, 2-tetradecylglycidic acid treatment caused a decrease in long chain carnitine ester secretion into bile. Duodenal fluid was collected from patients with suspected cholelithiasis (n = 10) before and after pancreozymin-cholecystokinin injection. Although carnitine concentration was variable, it was consistently 80% esterified. These data associate bile carnitine with hepatic carnitine metabolism and establish the presence of carnitine and carnitine esters in the human intestinal lumen.     

GFP-Human high-affinity carnitine transporter OCTN2 protein: subcellular localization and functional restoration of carnitine uptake in mutant cell lines with the carnitine transporter defect.

Individuals with the plasmalemmal high-affinity carnitine transporter defect present with progressive infantile-onset carnitine-responsive cardiomyopathy, lipid storage myopathy, recurrent hypoglycemic hypoketotic encephalopathy, and failure to thrive. The carnitine uptake defect (CUD) has been documented in their cultured skin fibroblasts, lymphoblasts, and/or myoblasts. The cDNA encoding the high-affinity sodium-dependent human carnitine transporter OCTN2 has recently been cloned. We used the green fluorescent protein (GFP) as a living marker for positively transfected cells in our expression studies of the high-affinity carnitine transporter OCTN2 cDNA in cell lines with the CUD. Transfection of cell lines from 12 unrelated patients (nine fibroblast and three lymphoblastoid) with a GFP construct harboring the wild-type full-length OCTN2 cDNA was done using LipoTAXI. Transient and stable expression of the recombinant GFP-human carnitine transporter OCTN2 cDNA was surveyed, and transient transfection of the fibroblast and stable transfection of the lymphoblastoid cell lines were achieved. There was functional restoration of carnitine uptake in the transfected mutant cell lines, thereby confirming the identity of the transfected cDNA. In addition, we report the first demonstration of the subcellular localization of an in-frame fusion GFP-human high-affinity carnitine transporter OCTN2 protein in the plasma membrane by confocal laser-scanning fluorescence microscopy.