Carnitine alters binding of aflatoxin to DNA and proteins in rat hepatocytes and cell-free systems.

The objective of this study was to determine effects of L-carnitine on aflatoxin B(1) (AFB(1))-DNA adduct formation in isolated rat hepatocytes, its dose response, specificity and mode of action. All experiments were conducted in either freshly isolated rat hepatocytes or cell-free systems. There was negative linear correlation between the dosage of carnitine and formation of [(3)H]AFB(1)-DNA adducts in the hepatocytes; however, the partitioning of AFB(1) into cellular compartments was not affected by carnitine. The attenuating effect of carnitine on AFB(1)-DNA adduct formation was also present in a cell-free system, but there was lack of specificity because acetylcarnitine and gamma-aminobutyric acid (GABA) were equally effective. Carnitine appears to interfere with bioactivation of AFB(1) and binding of AFB(1)-epoxide to DNA. On the contrary, carnitine enhanced the binding of AFB(1) and its epoxide to microsomal proteins, plasma proteins and bovine serum albumin. These results indicate that carnitine diverts AFB(1)-epoxide away from DNA by promoting binding to proteins. We conclude that modulation of AFB(1) binding to proteins and DNA by carnitine alters the carcinogenic and hepatotoxic potential of AFB(1) and poses concerns about the human AFB(1)-exposure data based on the AFB(1)-albumin adduct concentrations as a biomarker.

Increases in VO2max and metabolic markers of fat oxidation by caffeine, carnitine, and choline supplementation in rats.

We have previously shown that the combination of caffeine, carnitine, and choline supplementation decreased body fat and serum leptin concentration in rats and was attributed to increased fat utilization for energy. As a result, it was hypothesized that the supplements may augment exercise performance including physiological and biochemical indexes. Twenty 7-week-old male Sprague-Dawley rats were given free access to a nonpurified diet with or without supplementation of caffeine, carnitine, and choline at concentrations of 0.1, 5, and 11.5 g/kg diet, respectively. One half of each dietary group was exercised on a motor-driven treadmill for 3 weeks and maximal aerobic power (VO(2)max) was determined on the 18th day of exercise. Rats were killed 24-hr postexercise, and blood, regional fat pads, and skeletal muscle were collected. The VO(2)max was increased (P < 0.05) in the supplemented/exercised group; however, the respiratory quotient (RQ) was not affected. Postexercised concentrations of serum triglycerides were decreased but beta-hydroxybutyrate, acylcarnitine, and acetylcarnitine were increased in the supplemented animals. The changes in serum metabolites were complemented by the changes in the muscle and urinary metabolites. The magnitude of increase in urinary acylcarnitines (34-45-fold) is a unique effect of this combination of supplements. Cumulative evidence indicates enhanced beta-oxidation of fatty acids without a change in the RQ because acetyl units were excreted in urine as acetylcarnitine and not oxidized to carbon dioxide. For this phenomenon, we propose the term "fatty acid dumping." We conclude that supplementation with caffeine, carnitine, and choline augments exercise performance and promotes fatty acid oxidation as well as disposal in urine.

Caffeine, carnitine and choline supplementation of rats decreases body fat and serum leptin concentration as does exercise.

The effect of a combination of caffeine, carnitine and choline with or without exercise on changes in body weight, fat pad mass, serum leptin concentration and metabolic indices was determined in 20 male, 7-wk-old Sprague-Dawley rats. They were given free access to a nonpurified diet without or with caffeine, carnitine and choline at concentrations of 0.1, 5 and 11.5 g/kg diet, respectively. In a 2x2 factorial design, one-half of each dietary group was exercised, and the other half was sedentary. Body weight and food intake of all rats were measured every day for 28 d. Rats were killed and blood and tissue samples were collected and analyzed for biochemical markers. Food intake of the groups was not different, but the body weight was significantly reduced by exercise in both dietary groups. Fat pad weights and total lipids of epididymal, inguinal and perirenal regions were significantly reduced by the supplements as well as by exercise. Regardless of exercise, supplements significantly lowered triglycerides in serum but increased levels in skeletal muscle. Serum leptin concentrations were equally lowered by supplements and exercise. Serum leptin was correlated with body weight (r = 0.55, P< or =0.01), fat pad weight (r = 0.82, P< or =0.001) and serum glucose (r = 0.51, P< or =0.05). We conclude that the indices of body fat loss due to dietary supplements were similar to those due to mild exercise, and there were no interactive effects of the two variables.

Choline supplementation reduces urinary carnitine excretion in humans.

Two experiments were conducted to determine the effects of supplementary choline and/or pantothenate on the carnitine and lipid status of free-living humans. Analyses of carnitine and cholesterol fractions, triacylglycerols, and creatinine were determined in serum and/or urine. In experiment 1, adults receiving 13.5 mmol choline plus 1.4 mmol pantothenate/d had a significant decline in urinary carnitine excretion and renal clearance with nonesterfied carnitine (NEC) declining the most dramatically, 84%. Additionally, serum NEC and total carnitine concentrations decreased significantly. No changes were observed in any of the serum lipids examined. In experiment 2, subjects took 0.20 mmol and 0.02 mmol/kg choline or pantothenate, respectively. Choline, but not pantothenate, supplementation significantly decreased urinary carnitine excretion, renal clearance, and fractional clearance of NEC. We conclude that supplementary choline maintained serum carnitine concentrations by conserving urinary carnitine. Moreover, these observations merit additional investigation to determine metabolic and functional consequences of choline and carnitine interactions in humans.

A randomised, double-blind, placebo-controlled trial of L-carnitine in suspected acute myocardial infarction.

In a randomised, double-blind placebo-controlled trial, the effects of the administration of oral L-carnitine (2 g/day) for 28 days were compared in the management of 51 (carnitine group) and 50 (placebo group) patients with suspected acute myocardial infarction. At study entry, the extent of cardiac disease, cardiac enzymes and lipid peroxides were comparable between the groups, although both groups showed an increase in cardiac enzymes and lipid peroxides. At the end of the 28-day treatment period, the mean infarct size assessed by cardiac enzymes showed a significant reduction in the carnitine group compared to placebo. Electrocardiographic assessment of infarct size revealed that the QRS-score was significantly less in the carnitine group compared to placebo (7.4 +/- 1.2 vs 10.7 +/- 2.0), while serum aspartate transaminase and lipid peroxides showed significant reduction in the carnitine group. Lactate dehydrogenase measured on the sixth or seventh day following infarction showed a smaller rise in the carnitine group compared to placebo. Angina pectoris (17.6 vs 36.0%), New York Heart Association class III and IV heart failure plus left ventricular enlargement (23.4 vs 36.0%) and total arrhythmias (13.7 vs 28.0%) were significantly less in the carnitine group compared to placebo. Total cardiac events including cardiac deaths and nonfatal infarction were 15.6% in the carnitine group vs 26.0% in the placebo group. It is possible that L-carnitine supplementation in patients with suspected acute myocardial infarction may be protective against cardiac necrosis and complications during the first 28 days.

Choline supplementation alters carnitine homeostasis in humans and guinea pigs.

We have previously shown that supplementary choline causes significant decreases in urinary excretion of carnitine in humans. The objectives of the present work were to study this interaction in a different human population and on other body pools of carnitine in an animal model. In young adult women, daily choline supplementation (20 mg/kg body wt) resulted in a 75% lower urinary carnitine excretion than in controls, without significantly altering plasma carnitine concentrations. Supplementary choline was added to the ground diet of Sprague-Dawley rats (2.5 g/kg diet) and guinea pigs (3 g/kg diet). Choline supplementation had no effect on plasma concentrations or urinary excretion of carnitine in the rats. In guinea pigs, however, choline supplementation resulted in a significantly lower urinary excretion and higher plasma total carnitine concentrations. The skeletal muscle carnitine concentration was higher in the choline-supplemented guinea pigs, but not significantly higher in other tissues. These studies demonstrated that choline supplementation results in decreased urinary excretion of carnitine in young adult women, that guinea pigs are a suitable animal model for studying the effect of choline supplementation on carnitine status in humans, and that choline results in a conservation of carnitine in guinea pigs and perhaps in humans.

Acetylcarnitine-mediated inhibition of ethanol oxidation in hepatocytes.

Carnitine-mediated prevention of ethanol-induced hepatic steatosis is related to the attenuation of ethanol metabolism by carnitine in the intact rat. Although carnitine retards ethanol oxidation in the intact animal, the in vitro activities of ethanol-metabolizing enzymes remain unaltered. Therefore, hepatocytes were targeted to understand the mechanism of carnitine effect on ethanol metabolism. Rat hepatocytes were isolated by a collagenase-perfusion technique and incubated in albumin-containing medium with ethanol in the presence or absence of added carnitine or related compounds. Ethanol oxidation was determined by the loss of ethanol as well as by the products formed. The rate of ethanol oxidation in the presence of carnitine was one-half the rate in the absence of carnitine (14 vs. 25 nmol.min-1.million-1 cells). It took 100 times the concentration of carnitine to equal the maximal inhibition produced by acetylcarnitine and the effect of acetylcarnitine was without a lag time. It is concluded that acetylcarnitine is the mediator of carnitine inhibition of ethanol oxidation.

Carnitine prolongs the half-life of ethanol in broilers.

The object was to determine if carnitine attenuated ethanol metabolism in broilers similar to that reported in the rats. Two groups (n = 5) of 5-week-old broilers were given for 10 days the feed with or without 0.5% L-carnitine supplement. A single oral dose of ethanol on day 8 was followed by serial blood samples which were analysed for ethanol. Another dose of ethanol was given on day 10 and 2 hr later, plasma and liver were collected and analysed for ethanol, total lipid, triglycerides and carnitine. The carnitine supplemented diet prolonged the half-life of ethanol due to attenuation of ethanol metabolism which is similar to that reported earlier in rodents. The increases in plasma and hepatic acylcarnitines indicate that supplementary carnitine lessens the load of free acyl groups in the liver by eventual oxidation or excretion.

Acetylcarnitine inhibits alcohol dehydrogenase.

Carnitine and acetylcarnitine are used as dietary supplements and as therapeutic agents. Carnitine attenuates ethanol metabolism in intact animals but the in vitro activities of alcohol dehydrogenase (ADH), microsomal ethanol oxidizing system (MEOS) or catalase are not significantly altered by carnitine. Since acetylcarnitine was a far more potent inhibitor of ethanol oxidation than carnitine in hepatocytes, the activities of rat liver ADH and MEOS were determined with or without acetylcarnitine. The activity of ADH, not MEOS, was significantly inhibited by acetylcarnitine at NAD: acetylcarnitine < or = 1. The inhibition is of a competitive nature where acetylcarnitine competes with NAD+ (Ki = 135 mumol.L-1). This finding is unique in that this is the first report of this function of acetylcarnitine and it is a novel interaction between two important nutrients, niacin and carnitine.

Opposite effects of dietary saturated and unsaturated fatty acids on ethanol-pharmacokinetics, triglycerides and carnitines.

OBJECTIVE: The objective of this study was to determine the effects of saturated fatty acid (SFA) and unsaturated fatty acid (UFA) diets on ethanol pharmacokinetics. Hepatic ADH and plasma carnitines were also evaluated as possible indicators of the mechanism involved. METHODS: Sprague-Dawley male rats were fed modified AIN76 diets containing 10% coconut oil (SFA) or corn oil (UFA) for 120 days. A single dose (3 g/kg bw) of ethanol (13% solution) was orally administered using a gastric canula on day 30, 90, 105 and 120. Tail vein blood samples were collected at various intervals following ethanol dose and were analyzed for blood-ethanol concentration (BEC). In an analogous trial rats were given these diets for 70 days and blood samples were collected on day 35 and 63 for triglycerides, cholesterol and carnitine determination. The animals were killed on day 70 to collect liver for ADH determination. RESULTS: Compared to the UFA group, the SFA group exhibited significantly higher BEC, larger area under the curve, longer half-life of ethanol, and lower rates of ethanol elimination. Plasma carnitines were also higher in the SFA vs UFA group. However, hepatic ADH activity was not different between the groups. CONCLUSION: Dietary SFA protects liver from alcohol injury by retarding ethanol metabolism, and carnitine may be involved.

Effects of diet restriction and carnitine on carbon tetrachloride toxicity in the rat.

Male Sprague Dawley rats were fed a semisynthetic diet ad libitum, 55% of ad libitum (45% restriction), or supplemented with 1% L-carnitine for 68 days. The LD50 dose of CCl4 was orally administered, and mortality and hepatic lipids were determined. The CCl4-induced mortality was significantly increased by feed restriction but not by carnitine. Carnitine prevented hepatic lipid accumulation caused by CCl4 under these conditions.

Wheat gluten-based diet retarded ethanol metabolism by altering alcohol dehydrogenase and not carnitine status in adult rats.

The objective of this study was to determine the effect of a lysine-deficient diet on carnitine status in adult rats and subsequently on ethanol metabolism. Adult male rats were fed either the AIN-76 diet (NS), the AIN-76 diet with wheat gluten (WG) replacing casein, the WG diet plus 0.8% L-lysine (LS), or the LS diet plus 0.5% L-carnitine (CS) for 30 days. On the 31st day the rats were given an oral dose of ethanol and blood-ethanol concentrations (BEC) were monitored for the next 8 hours. One week later the rats were given a second dose of ethanol and urine was collected until killed, 3 hours post-ethanol administration (PEA). Besides growth retardation and hypoproteinemia, BEC were significantly elevated in the WG group compared to the other group at hours 3-8 PEA. There were no significant differences in BEC between the LS and CS groups; however, their BEC were significantly higher than that of the NS group. The BEC were inversely related to liver alcohol dehydrogenase (ADH) activities which were significantly lower in WG, LS and CS groups than in the NS group. Plasma, liver and urine carnitine values were significantly higher in the CS group than in the NS, WG and LS groups, wherein the values were similar. It is concluded that the WG diet reduced ADH activity and attenuated ethanol metabolism without significantly altering blood, liver and urinary carnitines in the adult rat.

Suppression of aflatoxin B1-induced lipid abnormalities and macromolecule-adduct formation by L-carnitine.

The fatty liver and hypolipidemia caused by aflatoxin B1 (AFB1) were studied in male Sprague-Dawley rats fed Purina Rat Chow with or without L-carnitine supplement for 6 weeks. In Experiment 1, the rats (n = 20) were divided into four groups, i.e., nonsupplemented control (NSC), nonsupplemented AFB1 (NSA), carnitine supplemented control (CSC), and carnitine supplemented AFB1 (CSA). The NSA and CSA groups were given an oral dose of [3H]AFB1 (1 mg/kg) 6 hr before kill. In Experiment 2 (n = 10) there were only NSA and CSA groups and they were killed 24 hr post-AFB1 administration. Hepatic and plasma concentrations of total lipid, triglycerides, AFB1-macromolecules adducts and urinary excretion of AFB1 were determined. Carnitine supplementation ameliorated AFB1-induced hepatic steatosis and hypolipidemia. Supplementary carnitine reduced covalent binding of AFB1 to hepatic DNA, RNA, and protein. The carnitine effect was more pronounced after 24 hr than after 6 hr of AFB1 treatment. We conclude that supplementary carnitine suppressed AFB1-induced fatty liver and AFB1-macromolecule adduct formation in the rat.

Effects of L-carnitine on carbon tetrachloride-induced changes in serum and liver lipids and acylcarnitines.

Effects of 0.5% L-carnitine-supplemented diet on carbon tetrachloride-induced alterations of hepatic and serum lipids were examined in male rats. Treatment with carbon tetrachloride significantly increased hepatic total lipids, triglycerides, and total carnitine, but these were not significantly altered by 0.5% L-carnitine supplementation. However, in plasma, supplementary carnitine significantly decreased nonesterified fatty acids and increased acylcarnitines. These suggest that carnitine may soften hepatic lipid load by releasing acylcarnitines in blood.

Clearance of exogenous carnitine in bovine semen: Potential diagnosis of epididymal function and patency of the ductus deferens.

Carnitine content in the ejaculate depends mainly on the capability of the epididymis wall to transfer carnitine from the blood and on the patency of ejaculatory ductus systems. An elevation of carnitine in semen subsequent to an intravenous injection of carnitine is expected. Intravenous injections of carnitine (L-isomer and DL-isomers) caused a significant (P <0.05) elevation (more than 10-fold) in blood carnitine. However, carnitine injection failed to increase net secretion of carnitine into the ejaculate and blood elimination half-life was 2.3 hours. Mean concentrations of carnitine in the electroejaculate (3.0 nmoles/ml) were significantly lower than in the ejaculate following natural mating (180 nmoles/ml). Vasectomy decreased net carnitine per ejaculate to about 1/5 the prevasectomy value, when ejaculate was collected following natural mating. However, vasectomy did not affect carnitine concentrations in semen collected by electroejaculation. Twenty-one percent of the carnitine in semen originated in the accessory glands and 79% in the epididymides. Carnitine in the electroejaculate was originated almost exclusively in the accessory glands. It was concluded that the diagnostic value of carnitine in semen is limited. Some considerations are: secretion of carnitine is not organ specific, there are large individual variations, there is a negative effect of electroejaculation, and a carnitine loading dose technique is not feasible. However, there is a diagnostic potential in using carnitine assay to detect epididymides occlusion, but only when ejaculate is collected by an artificial vagina.

Alterations of serum and urinary carnitine profiles in cancer patients: hypothesis of possible significance.

The present study examined the serum and urinary carnitine concentrations of 21 cancer patients with metastatic disease and 13 healthy age-matched controls by taking three consecutive samples during an 8-week period. The serum concentrations of all fractions of carnitine were significantly lower in the female cancer patients than in the female controls. The concentrations of urinary carnitine fractions were relatively higher in the total cancer population; however, only acid-insoluble acylcarnitine (AIAC) was statistically significant. The renal clearance of acid-soluble acylcarnitine (ASAC) and AIAC was significantly greater in cancer subjects than in controls. Significant inverse relationships were established between the ASAC and AIAC clearances and their respective serum concentrations. The renal tubular reabsorption of AIAC was significantly less in cancer patients than in control subjects as indicated by the fractional excretion of carnitine. The increased clearance of acylcarnitine and excretion of large amounts of AIAC are proposed to be a response to chemotherapy and represent a loss of energy to the cancer patient.

Maternal, cord, and neonatal carnitine correlations and lipid profiles of various birthweight infants.

The carnitine, acylcarnitine, and triglyceride concentrations were determined in the average for gestational age neonates of various birthweights and gestational ages and their corresponding maternal and umbilical cord plasma. Forty-two infants were divided into one of four groups based on birthweight: Group 1, less than 1000 gm; group II, 1001 to 1510 gm; group III, 1511 to 2500 gm; and group IV were term infants who served as the reference group. Results indicated that there was an overall decrease of total carnitine and nonesterified carnitine with advancing gestational age (r = -0.4418, p less than 0.01). Furthermore, there were three distinct phases of plasma carnitine concentrations from 24 weeks to term. The plasma carnitine profile of the neonates less than 1500 gm was strongly correlated (r = 0.73) to their maternal plasma carnitine profile and that of term neonate to their umbilical cord plasma. Acylcarnitine and triglycerides were not significantly correlated.

Plasma carnitine alterations in premature infants receiving various nutritional regimes.

Plasma carnitine, carnitine esters, and triglyceride concentrations were determined in 36 appropriate-forgestational-age (AGA) infants at various stages of prematurity throughout hospitalization to determine the effect of a carnitine-free and carnitine-containing diet on plasma carnitine and triglyceride concentrations. The infants were entered into one of three experimental groups based on birth weight: group I less than 1.0 kg; group II 1.0-1.51 kg; and group III 1.52-2.5 kg. Throughout the study subjects were placed on appropriate nutritional regimes which included hyperalimentation (HA), intravenous (iv) fat emulsion (Intralipid), Portagen, Enfamil-24 Premature Formula, Enfamil-20, and breastmilk. Blood samples were drawn from each infant at birth, days 1-5,7 then weekly, also before and after each nutritional intervention to determine carnitine and triglyceride concentrations. Results showed that plasma total carnitine and nonesterified carnitine decreased in all groups when the infants were maintained on a carnitine-free diet (HA, Intralipid, Portagen). In general, the carnitine levels continued to decrease until a carnitine-containing diet was initiated. Once a carnitine-containing diet was begun, plasma total carnitine (TC) and nonesterified carnitine (NEC) levels increased at fairly similar rates in all groups. However, an inverse relationship between carnitine and triglyceride (TG) concentrations were not seen in these infants. This would indicate that most premature infants require exogenous carnitine to maintain the plasma concentration of carnitine. However, a decreased concentration of plasma carnitine was not correlated with an elevated TG level under the conditions of this study.

The serum carnitine status of cancer patients.

Carnitine is necessary for the translocation of fatty acids into the mitochondria, and the relative concentration of carnitine and acylcarnitine in the serum are known to reflect metabolic states. A survey of serum carnitine concentrations was made in 54 cancer and 81 noncancer patients for the purpose of determining the carnitine profile. The total carnitine, nonesterified carnitine, and acid-insoluble acylcarnitine concentrations of cancer patients were similar to noncancer patients and within the normal range; however, the acid-soluble acylcarnitine concentration was significantly lower in cancer patients than in controls (6.7 vs 11.5 nmol/ml). When percentages and ratios were calculated for the relative proportions of acylcarnitines, large variations were found to occur among cancer types. The acylcarnitine ratio (the sum of acid-soluble and acid-insoluble acylcarnitine divided by nonesterified carnitine) ranged from 0.17 in leukemia to 0.30 in breast cancer cases. Since the acylcarnitine concentration and ratio are reflective of the metabolic state, the depressed acylcarnitine ratio in cancer patients may be due to decreased production, increased utilization, or increased excretion of acid-soluble acylcarnitine. Elevated concentrations of nonesterified carnitine and total carnitine were observed in two patients, and some of the lowest acylcarnitine concentrations and ratios were observed in advanced cancer cases. The therapeutic regimen and/or the neoplastic process itself may be responsible for the observed differences in the serum carnitine profile.

Attenuation of ethanol metabolism by supplementary carnitine in rats.

The purpose of this study was to examine the effect of supplementary D,L-carnitine on blood-ethanol levels and ascertain if the effect was a result of altered absorption or metabolism of ethanol. Mature male Sprague-Dawley rats were fed Purina Rat Chow as such or supplemented with various levels of D,L-carnitine. First it was established that supplementing carnitine at 1% (w/w) level produced steady state concentrations of carnitine in blood after 3 days of feeding. When a single dose of ethanol was given orally after 5 days of carnitine supplementation, the blood levels of ethanol remained significantly elevated for 2-8 hours in the carnitine supplemented animals. Time course of blood-ethanol concentrations revealed that carnitine did not affect the rates of ethanol absorption and therefore, the effect must be due to the attenuation of ethanol clearance from the blood.