Free Carnitine Concentrations Research Articles

Screening for medium chain acyl-CoA dehydrogenase deficiency using electrospray ionisation tandem mass spectrometry

OBJECTIVETo establish criteria for the diagnosis of medium chain acyl-CoA dehydrogenase (MCAD) deficiency in the UK population using a method in which carnitine species eluted from blood spots are butylated...

Randomised controlled trial of L-carnitine as a nutritional supplement in preterm infants

AIMSTo evaluate the effect of L-carnitine supplementation (25 mg/kg/d) on the growth and incidence of hypoglycaemia in preterm infants.METHODSA double blind, placebo controlled randomised trial, stratified for gestational age, was...

Improvement in exercise tolerance in isovaleric acidaemia with L-carnitine therapy.

The effect of 4 weeks' treatment with oral-L-carnitine (100 mg/kg per day) on carnitine status and metabolic parameters during an incremental ramp exercise test in a 12-year-old girl with isovaleric acidaemia was examined to determine its possible therapeutic role. The maximum work rate achieved increased from 110 to 120 watts; oxygen consumption at anaerobic threshold from 600 to 800 L/min; peak oxygen consumption from 1270 to 1450 L/min; and oxygen pulse, a measure of cardiac output, from 7.0 to 8.1 L/beat. These changes were associated with increases in plasma and urinary free and acyl carnitine concentrations but no change in physical activity. This observed effect of L-carnitine on exercise performance may be on cardiac or skeletal muscle function or both. We conclude that, in this single patient with isovaleric acid-aemia, L-carnitine supplementation had objective benefits and further studies on more patients are warranted.

Markers of cellular dysoxia during orthotopic liver transplantation in pigs.

To characterize global, regional, and end-organ markers of cellular dysoxia during orthotopic liver transplantation and early reperfusion in pigs. Descriptive study. University hospital research laboratory. 7 fasted, anesthetized, and mechanically ventilated Yorkshire pigs underwent orthotopic liver transplantation. Oxygen consumption (VO2) and oxygen delivery (DO2) were both calculated using standard formulae. Gastric interstitial pH and the gastroarterial partial pressure of carbon dioxide (PCO2) gradient were measured with a gastric tonometer. The following were determined from arterial blood samples: serum lactate to pyruvate ratio, serum 3-hydroxybutyrate to acetoacetate ratio, plasma free fatty acids, and plasma free and total carnitine levels. Data were collected 1 h after induction of anesthesia (I), at the end of the anhepatic phase (A), and 1 h after reperfusion (R), Median (range) VO2 values obtained at the specified time points were: I 318 (206-1860), A 210 (152-408), R 330 (214-424) ml/kg per min, respectively (NS); DO2 values were: I 1828 (1382-3259), A 1219 (452-2492), R 1741 (1345-12,071) ml/kg per min, respectively (NS). The lactate to pyruvate ratio, reflecting the redox potential of the cytosol, progressively increased: I 22 (9-46), A 29 (16-68), R 43 (23-55), (p < 0.05). Gastric interstitial pH, as well as the gastroarterial PCO2 gradient values at the specified time points did not reach statistical significance. Levels of ketone bodies (3-hydroxybutyrate+acetoacetate) remained lower than 0.120 mmol/l. The ketone body ratio did not significantly vary over time (NS). Plasma esterified and free carnitine concentrations and free fatty acid values remained within normal limits (NS). Among these markers, the ketone body ratio presented the largest area under the receiver operating characteristic curve as a marker of postoperative mortality, with an inflexion point at 0.9. In this study, orthotopic liver transplantation was associated with significant variations over time in the redox potential of the cytosol. Postoperative mortality was, however, related to the redox state of the liver mitochondria. Our data suggest the occurrence of abnormal tissue oxygenation during liver transplantation.

Urinary, plasma, and erythrocyte carnitine concentrations during transition to a lactoovovegetarian diet with vitamin B-6 depletion and repletion in young adult women

The purpose of the present study was to determine whether vitamin B-6 depletion and repletion influence carnitine concentrations in the plasma, erythrocytes, and urine of young adult women consuming a low-carnitine (30 micromol/d), lactoovovegetarian diet. Doses of vitamin B-6 were administered sequentially as follows: 1.60 mg/d during adjustment for 9 d, 0.46 mg/d during depletion for 27 d, 1.26 mg/d during the first repletion for 21 d, 1.66 mg/d during the second repletion for 21 d, and 2.06 mg/d during the third repletion for 14 d. Urinary carnitine tended to decline but was not significantly different throughout the 92-d study period. Plasma short-chain acylcarnitine and total carnitine decreased during vitamin B-6 depletion; however, the ratio of plasma acylcarnitine to total carnitine was not significantly different during changes in vitamin B-6 intake. Erythrocyte long-chain acylcarnitine increased during vitamin B-6 depletion, but the ratio of erythrocyte acylcarnitine to total carnitine did not respond to changes in vitamin B-6 intake. Plasma free and total carnitine concentrations were only weakly correlated with plasma pyridoxal-P concentration (r = 0.28 and r = 0.29, respectively; P < 0.01). No significant correlations were observed between urinary carnitine excretion or erythrocyte carnitine concentrations and plasma or erythrocyte pyridoxal-P concentrations. Thus, a vitamin B-6 intake of 0.5 mg/d does not affect carnitine concentrations in biological fluids and therefore is unlikely to affect endogenous carnitine synthesis over 27 d. The changes in carnitine indexes that we observed were probably due to adaptation to a vegetarian pattern of exogenous carnitine consumption.

Carnitine contents in remnant liver, kidney, and skeletal muscle after partial hepatectomy in rats: randomized trial.

Carnitine, an important carrier of free fatty acid that is transported into mitochondria for beta-oxidation, was thought to be one of the key factors in the regulation of liver regeneration. If the carnitine content is insufficient in the hepatocyte, it might impair the energy substrate's transport and the energy charge required for cell regeneration. The purpose of this study is to evaluate the changes of carnitine content in remnant liver, kidney, and skeletal muscle simultaneously after partial hepatectomy in rats. Partial hepatectomy with resection of the median and left lateral lobes was performed on male Wistar rats. Rats with a sham operation comprised a control group. This study was an experimental randomized trial. Ten rats from each group were sacrificed before the operation and at 6, 24, 48, and 72 hours after the operation. The carnitine content, as total and free forms, in remnant liver, kidney, and skeletal muscle were quantified by high-performance liquid chromatography. The carnitine contents in the remnant liver increased significantly at 6, 24, and 48 hours after partial hepatectomy (p < 0.01). The increase of total carnitine content was more obvious than that of the free form. In contrast, the decreasing concentrations of total carnitine and free carnitine in the kidney were significant (p < 0.01). In skeletal muscle the total carnitine content decreased to a small extent, and it was observed only at 6 hours after partial hepatectomy (p < 0.05). It is suggested that remnant liver promoted the generation of carnitine, whereas kidney and skeletal muscle released their stored carnitine at an early stage after partial hepatectomy. As a result, the influx of the carnitine into hepatocytes increased at the regenerative stage. The carnitine content of remnant liver is sufficient during the early posthepatectomy stage.

The effect of starvation on brain carnitine concentration in neonatal rats.

We examined carnitine concentrations in fasted neonatal rat brain to evaluate the effect of starvation on fatty acid metabolism. The free- and acylcarnitine concentrations in neonatal rat brain and heart were determined after a 72-hour starvation period from the 3rd to 6th postnatal day. They were also determined in rats at 3 and 6 days of age fed normally by the mother rats as controls. In the brain, the mean free carnitine concentration in the fasted group showed an increase similar to that in normal rats and there was no difference between the fasted and 6-day-old control rats. However, the mean acylcarnitine concentration was significantly higher in the fasted group than in the control group at both 3 and 6 days of age. Almost all of the increased acylcarnitine in the fasted group was short-chain acylcarnitine. In the heart, there was no difference in the mean free carnitine concentration between the fasted group and control group at 6 days of age. The 6-day-old rats in both the fasted and control groups showed higher levels compared to 3-day-old rats in the control group. The mean acylcarnitine concentration in the fasted group was not different from that in control group at 6 days of age, while the amount of short-chain acylcarnitine was less than that in the control group at 6 days of age. These findings suggest that in the brain, carnitine is accumulated as a result of redistribution during starvation, and is utilized for energy supply by fatty acid oxidation.

The distribution of total, free, short-chain acyl and long-chain acyl carnitine in ocular tissues of the camel (Camelus dromedarius).

Carnitine (CA) plays an important role in the metabolism of fatty acids. Its presence is considerable in tissues that utilize fatty acids as an important source of energy, such as the heart and the skeletal muscle. The presence of free, short-chain acyl and long-chain acyl carnitine was shown for the first time in various tissues of the camel eye. The ratio of acyl carnitine (AC) to free carnitine (FC) was high in aqueous humor, indicating a predominance of AC, while it was low in vitreous humor, indicating a higher concentration of FC. The highest concentration of total carnitine (TC) was observed in the retina, followed by the cornea, while the lowest was found in lens nucleus. Among various forms, FC concentration was the highest in the cornea while it was the lowest in the lens cortex. In the cornea and retina, the proportion of short-chain acylcarnitines (SC) was two-fold when compared to long-chain acylcarnitines (LC), while in the lens it was the same. The ratio of AC to FC in the retina was high, indicating a predominance of AC, while in lens it was low, indicating a higher concentration of FC. In contrast, L-CA in the cornea was present equally between the free and the esterified forms. The quantitative differences in the distribution of L-CA in various tissues of the camel eye reflect that it may be involved in providing a source of readily available energy.

Influence of sex and chronic haemodialysis treatment on total, free and acyl carnitine concentrations in human serum.

The influence of sex and haemodialysis treatment on serum total, free and acyl carnitine concentrations in healthy controls and chronic renal failure patients has been investigated. Patients on regular haemodialysis treatment generally displayed significantly decreased serum carnitine levels. The mean predialysis serum carnitine levels were not significantly different from the mean healthy control values. However, after dialysis a significant decrease in serum carnitine levels was observed compared to the predialysis and healthy control values. Moreover, serum ratio of acylated to free carnitine was significantly higher after haemodialysis as compared to both healthy controls and predialysis patients. Sex-related changes in serum total, free and acyl carnitine levels and ratios of acylated to free carnitine have been observed in healthy controls and patients on chronic haemodialysis treatment.

The effect of carnitine on ketogenesis in perfused livers from juvenile visceral steatosis mice with systemic carnitine deficiency.

Juvenile visceral steatosis (JVS) mice have been reported to have systemic carnitine deficiency, and the carnitine concentration in the liver of JVS mice was markedly lower than that of controls (11.6 +/- 2.6 versus 393.5 +/- 56.4 nmol/g of wet liver). To evaluate the role of carnitine in mitochondrial beta-oxidation in liver, we examined the effects of carnitine on ketogenesis in perfused liver from control and JVS mice. In control mice, ketogenesis was increased by the infusion of 0.3 mM oleate, but not by L-carnitine. In contrast, although ketogenesis in JVS mice was not increased by the infusion of oleate, it was increased 2.5-fold by the addition of 1000 microM L-carnitine. Addition of 50, 100, and 200 microM L-carnitine increased ketogenesis in a dose-dependent manner. The infusion of 0.3 mM octanoate or butyrate increased ketogenesis in a carnitine-independent fashion in both control and JVS mice. These findings suggest that endogenous long chain fatty acids from accumulated triglycerides may be used as substrates in the presence of carnitine in JVS mice. The relationship between ketogenesis and free carnitine concentration was examined in livers from JVS mice. Ketogenesis increased as free carnitine levels increased until concentrations exceeded about 100 nmol/g of wet liver (340 microM). The free carnitine concentration required for half-maximal ketone body production in liver of JVS mice was 45 microM (13 nmol/g of wet liver), which corresponds to a K(m) value of carnitine palmitoyltransferase I. We conclude that carnitine is a rate-limiting factor for beta-oxidation in liver only when the carnitine level in liver is very low.

Neonatal nutritional carnitine deficiency: a piglet model.

The clinical significance of nutritional carnitine deficiency remains controversial. To investigate this condition under controlled conditions, an animal model was developed using the parenterally alimented, carnitine-deprived newborn piglet. Forty-five piglets received total parenteral nutrition for 2-3 wk that was either carnitine-free or supplemented with 100-400 mg/L L-carnitine. Blood and a muscle biopsy were taken at the initial surgery. Carnitine balance studies were performed at 11-14 d of age. Blood, liver, heart, and skeletal muscle were taken at sacrifice for analysis of carnitine, electron microscopy, and oxidation studies. Carnitine-deprived piglets were in negative carnitine balance and had lower blood, urine, and tissue levels of carnitine than carnitine-supplemented animals. There was a positive correlation between excretion and plasma concentrations of free carnitine with an apparent renal threshold between 15 and 35 micromol/L. Plasma levels were correlated with liver and heart, but not muscle, concentrations of total acid-soluble carnitine. Carnitine-deprived piglets had evidence of lipid deposition in liver and skeletal muscle and tended to have a higher incidence of muscle weakness and cardiac failure. Basal rates of oxidation of [14C]palmitate to 14CO2 and 14C-acid-soluble products were lower in liver homogenates from carnitine-deprived piglets than in those from carnitine-supplemented animals and increased in a dose-dependent fashion with the addition of L-carnitine (0, 50, and 500 micromol/L) in vitro. In summary, carnitine deprivation in the neonatal piglet resulted in low carnitine status and morphologic/functional disturbances compatible with carnitine deficiency. The described animal model appears to be suitable for the investigation of neonatal nutritional carnitine deficiency.

Plasma Carnitine Kinetics during Orthotopic Liver Transplantation

Background: Carnitine is synthesized mainly in the liver and plays an essential role in the transport of fatty acids in liver mitochondria for subsequent oxidation and energy production. Methods: The plasma concentrations of free carnitine, acylcarnitine, total ketone bodies, lactate, pyruvate, and hepatocyte growth factor (HGF) were measured during liver transplantation. Results: The plasma free carnitine and acylcarnitine concentrations and the lactate to pyruvate ratio in patients with compromised grafts (group A) were significantly higher than those in patients with well-functioning grafts (group B) after reperfusion. The acylcarnitine concentration in group B decreased after incision, but it remained at a high level in group A. Significant correlations were found between the concentrations of HGF and free and acylcarnitine after reperfusion. Conclusion. The accelerated flux of carnitine in the graft may be associated with deterioration of energy metabolism in the graft. An increased acylcarnitine concentration may reflect impaired liver regeneration.

Reduction in blood free carnitine levels in association with changes in sodium valproate (VPA) disposition in epileptic patients treated with VPA and other anti-epileptic drugs.

Reduction in the blood free carnitine (FC) level as a side effect of sodium valproate (VPA) given epileptic patients was pharmacokinetically studied in connection with changes in the VPA disposition. The serum FC level in patients taking at least one of phenobarbital (PB), phenytoin (PHT) and/or carbamazepine (CBZ) in addition to VPA was significantly lower than that in the controls given only these other anti-epileptic drugs (AEDs). Patients medicated only with VPA also tended to have a lower serum FC level than the controls, although the difference was not significant. Among all the patients taking VPA with or without other AED(s), a significantly positive correlation was observed between the serum FC level and the value of dose and level ratio (L/D) of VPA, indicating that both the serum FC concentration and the L/D value of VPA were remarkably reduced in those patients receiving both medications. These results suggested that reduction in the blood FC level as a side effect of VPA reflected FC deficiency associated with the accelerated degradation of VPA in liver; such a condition appears to result from medication with VPA and other AED(s) which induce(s) enzyme(s) for the VPA metabolism.

Pivalate Affects Carnitine Status but Causes No Severe Metabolic Changes in Rat Liver

To investigate the carnitine deficiency induced by pivalate, rats had free access to drinking water with or without pivalate. Consumption of 20 mmol/L pivalate for 1 wk decreased the levels of both free and total carnitine in plasma to ∼20% of levels before treatment. After 4 wk, the concentrations of free carnitine in the liver, heart and muscle of pivalate-treated rats were ∼60–80% of the control, and in the kidney, 26% of the control. Fractional excretion of free carnitine (FEFC) in pivalate-treated rats was measured; however, the treatment for 3 or 8 d did not affect the values relative to those obtained before treatment. Treatment with pivalate for 4 wk did not affect plasma concentrations of glucose, ammonia and free fatty acids (FFA) in the rats; however, the concentration of 3-hydroxybutyrate (3-OHB) was higher, and the FFA/3-OHB ratio was lower than those of controls. In a liver perfusion study, ketogenesis from oleate and gluconeogenesis from lactate and pyruvate in rats treated with pivalate for 4 wk were not different from controls. These results suggest that administration of pivalate did not induce the excessive excretion of free carnitine in urine, and secondary carnitine deficiency induced by intake of 20 mmol/L pivalate for 4 wk did not cause severe metabolic changes in rat liver.

Carnitine metabolism in human muscle fiber types during submaximal dynamic exercise.

The effect of prolonged exhaustive exercise on free carnitine and acetylcarnitine concentrations in mixed-fiber skeletal muscle and in type I and II muscle fibers was investigated in humans. Needle biopsy samples were obtained from the vastus lateralis of six subjects immediately after exhaustive one-legged cycling at approximately 75% of maximal O2 uptake from both the exercised and nonexercised (control) legs. In the resting (control) leg, there was no difference in the free carnitine concentration between type I and II fibers (20.36 +/- 1.25 and 20.51 +/- 1.16 mmol/kg dry muscle, respectively) despite the greater potential for fat oxidation in type I fibers. However, the acetylcarnitine concentration was slightly greater in type I fibers (P < 0.01). During exercise, acetylcarnitine accumulation occurred in both muscle fiber types, but accumulation was greatest in type I fibers (P < 0.005). Correspondingly, the concentration of free carnitine was significantly lower in type I fibers at the end of exercise (P < 0.001). The sum of free carnitine and acetylcarnitine concentrations in type I and II fibers at rest was similar and was unchanged by exercise. In conclusion, the findings of the present study support the suggestion that carnitine buffers excess acetyl group formation during exercise and that this occurs in both type I and II fibers. However, the greater accumulation of acetylcarnitine in type I fibers during prolonged exercise probably reflects the greater mitochondrial content of this fiber type.

HPLC determination of carnitine and acylcarnitines in human plasma by means of fluorescence labeling using 2-(4-hydrazinocarbonylphenyl)-4,5-diphenylimidazole.

Carnitine and acylcarnitines are important substances involved in the oxidation and metabolism of fatty acids. An HPLC method is presented for the quantitative analysis of these compounds. The method is based on the detection of fluorescent derivatives of carnitine and short- and medium-chain acylcarnitines labeled with 2-(4-hydrazinocarbonylphenyl)-4,5-diphenylimidazole (HCPI). The labeling of carnitine and acylcarnitines with HCPI was performed at room temperature for 1 h using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide as a condensing reagent. The analytes prepared using cation-exchange cartridges were separated on an octadecylsilyl silica gel (ODS) column by a gradient elution system of acetonitrile/Tris-HCI buffer and determined using a synthetic internal standard. Fluorescence detection was performed at 475 nm with excitation at 340 nm. The detection limits for carnitine, acetyl-, propionyl-, hexanoyl- and octanoylcarnitine ranged from 0.24 to 1.97 nmol per ml human plasma, at a signal-to-noise ratio of 3. The within-day and between-day precision of the assay for carnitine and acylcarnitines in plasma samples had relative standard deviations (RSDs) lower than 10.3%. The concentration of free carnitine, acylcarnitines and total carnitine in human plasma could be successfully determined by the proposed method.

Free and esterified carnitine in continuous ambulatory peritoneal dialysis patients

Free, acetyl-, medium- and long-chain acylcarnitine and total plasma carnitine concentrations were measured in eight continuous ambulatory peritoneal dialysis (CAPD) patients and eight age- and sex-matched healthy controls. Daily loss of carnitine was also quantified in both groups, by analysis of urine and dialysis fluid. Plasma total carnitine concentration in CAPD patients was not significantly different from controls (42.8 +/- 1.6 and 43.1 +/- 2.3 mumol/liter, respectively). However, the plasma free carnitine concentration of CAPD patients was significantly lower than that of controls (28.5 +/- 1.4 and 36.2 +/- 2.5 mumol/liter, respectively; P < 0.05). No difference in the daily loss of total carnitine was found between CAPD patients and controls (269.7 +/- 30.0 and 240.5 +/-33.0 mumol/liter, respectively), but the daily loss of free carnitine was significantly greater in CAPD patients (175.8 +/- 17.3 and 105.8 +/- 16.4 mumol/liter, respectively; P < 0.05). The ratio of total acylcarnitine (acetyl-, medium- and long-chain acylcarnitine) to free carnitine was significantly greater in plasma of CAPD patients than in controls (P < 0.01) and was lower in daily fluid losses (P < 0.001). These ratio differences suggests that an alteration in acyl group metabolism is occurring in CAPD patients. This may be attributable to an accumulation of medium- and long-chain acylcarnitine in liver of CAPD patients which would be exchanged for plasma free carnitine and/or to a differential loss of free and acylcarnitine across the peritoneal cavity.

Transient reduction of human left ventricular mass in carnitine depletion induced by antibiotics containing pivalic acid.

To study the effect of induced carnitine depletion on myocardial structure and function. 7 healthy adult volunteers given 1200 mg pivmecillinam per day for 7-8 weeks were studied by echocardiography before and after 7-8 weeks of treatment and a 15 months follow up after the treatment period. Teaching hospital. Carnitine concentration in serum, urine, and muscle and echocardiographic measurements. After 7-8 weeks of treatment the median free serum carnitine concentration was reduced to 7% and the median total muscle carnitine concentration to 46% of the pretreatment levels. The median diastolic interventricular septum thickness decreased by 14% (mean 26%, P = 0.028) and the median left ventricular mass by 10% (mean 20%, P = 0.018). Fifteen months later these dimensions had increased but not completely returned to pretreatment values. Extended treatment with pivalic acid containing antibiotics causes carnitine depletion which may lead to changes in cardiac structure.

Abnormalities of carnitine metabolism in chronic fatigue syndrome

Carnitine may be involved in the pathogenesis of the chronic fatigue syndrome (CFS). However, no information about the cellular metabolism of carnitine in CFS patients is currently available. Therefore, we aimed to measure the levels of carnitine (total, free and short-chain) in both peripheral blood lymphocytes (PBLs) and sera from patients with CFS. The serum levels of total, free and short-chain were comparable in CFS patients, considered as the whole group, to those in healthy control subjects, even though a trend indicating slightly reduced serum concentrations of free carnitine was observed in male patients with CFS. In contrast, the concentrations of total, free and short-chain carnitine in PBLs from patients with CFS were significantly lower than in cells from healthy controls. Our study indicates that patients with CFS require exogenous carnitine supplementation. The low carnitine concentrations in PBLs from patients with CFS probably reflect the carnitine deficiency occurring in other tissues, including the skeletal muscles. The low cellular concentrations of carnitines may help to explain both the immunological abnormalities and the impaired energy metabolism in skeletal muscles.

Effect of Short-Term Treatment with Pivalic Acid Containing Antibiotics on Serum Carnitine Concentration - A Risk Irrespective of Age

Treatment with pivalic acid containing prodrugs has been shown to cause carnitine depletion by loss of pivaloyl carnitine in urine. A 7-day standard pivmecillinam treatment of adults lead to a marked decrease of the free serum carnitine concentration (44.6 to 12.9 μmol/liter), whereas no change was seen in those given norfloxacine (40.0 to 40.5 μmol/liter). In some patients irrespective of age the free serum carnitine concentration was decreased to levels (around 10 μmol/liter) at which an impaired ketone-body production may occur. Therefore, there is reason for cautious use of this type of drug irrespective of the age of the patients.