Attila Sandor

Enhanced uptake of carnitine by perfused rat liver following starvation

Previously, the release of carnitine from the perfused rat liver was found to be protein-mediated, dependent on the nutritional state but not on metabolic energy. Further, it was shown to exceed the physiological demand by about 10-fold (Sandor et al. (1985) Biochim. Biophys. Acta 835, 83-91). In the present study the uptake of carnitine by perfused rat liver has been investigated. The liver tissue and the perfusate were in equilibrium when the carnitine concentration in the perfusate was close to 45 microM, physiological in the rat plasma. Under this condition, when no net carnitine transport occurred, an unidirectional uptake of L-[3H]carnitine was observed. Quantitatively, the uptake rate was 355 +/- 60 (S.D.) nmol/h per 100 g body weight at 45-50 microM perfusate concentration. This uptake capacity balances the previously reported excessive release (Sandor et al., op. cit.). On this basis we propose that a futile release/uptake cycle operates in carnitine transport across the liver cell membrane. Liverse of 24-h starved rats took up L-[3H]carnitine at 56% higher rate from the perfusate (75 microM) than livers of fed rats. Kinetic analysis revealed that fasting caused a decrease in Km value from 4.22 mM to 2.59 mM, whereas Vmax remained practically unchanged, average 0.95 mumol/min per 100 g body weight. D-[3H]Carnitine was transported at the same rate as L-carnitine and underwent the effect of fasting as well. The uptake was partially inhibited by 1 mM 2,4-dinitrophenol and 5 mM KCN, showing its dependency on metabolic energy. If Li+ replaced Na+ a strong inhibitory effect (to 20% of control) was observed, which suggests a co-transport of carnitine with Na+. Mersalyl, an SH reagent, had no effect on the uptake, whereas it practically abolished the release of carnitine from the perfused livers. This observation suggests that the inward and outward transport of carnitine are mediated by two different proteins.

On Carnitine Content of the Human Breast Milk

Summary: The concentration of total carnitine in human breast milk remained at a constant mean level near 62.9 range: 56.0–69.8/nmoles/ml during the first 21 days postpartum. The carnitine level fell significantly to 35.2 ± 1.26 nmoles/ml until the 40–50th day. The carnitine concentrations did not depend on the secreted volume of milk. In comparison, fresh and commercial pasteurized cows milk contained 206.2 (range: 192–269) nmoles/ml and 160.0 (range: 158–200) nmoles/ml carnitine, respectively. In the sera of nursing mothers, the concentration of total carnitine was lower on the first day after delivery (27.2 ± 1.19 nmoles/ml) but returned to normal by the 21st day postpartum (38.8 ± 2.97 nmoles/ml).Speculation: Recently it has been reported that human and animal newborns need exogenous carnitine (lo), which probably plays an important role in neonatal ketogenesis. The natural carnitine source for newborns is the mothers milk. Determining the relationship between days postpartum and carnitine concentration in breast milk makes possible an estimate of when the mothers milk is the optimal carnitine source.

Butyrobetaine is equal to L-carnitine in elevating L-carnitine levels in rats

This work shows that butyrobetaine administered to rats in a single dose can be highly effective in elevating L-carnitine levels in all tissues. This ability of butyrobetaine was compared to that of L-carnitine. In an experiment with tracer dose of the compounds, 12 h following administration of [3H]butyrobetaine plasma and tissues contained radioactivity exclusively in L-carnitine and in similar amounts as in the other group of animals receiving L-[3H]carnitine. This was observed both after intraperitoneal and oral administration of the compounds. In the loading experiments 100 mumol [3H]butyrobetaine was administered orally to one group and 100 mumol L-[3H]carnitine to the other group of animals and 12 h later it was found that butyrobetaine caused the same increments in L-carnitine as L-carnitine administration. The increments in the organs of the butyrobetaine-treated group (in decreasing order) were as follows: kidney, 1227 nmol/g vs. 652 nmol/g; liver, 469 nmol/g vs. 258 nmol/g; muscle, 1043 nmol/g vs. 881 nmol/g; plasma, 79.4 nmol/ml vs. 39.3 nmol/ml. Butyrobetaine (100 mumol) caused similar increments when it was administered intraperitoneally. Based on these results butyrobetaine can be considered as a potential agent for L-carnitine supplementation therapy.

12 L-CARNITINE REPLACEMENT THERAPY IN CHRONIC VALPROATE TREATMENT

Valproate (VPA) is known to cause carnitine (C) deficiency, 10 children receiving chronic VPA treatment were given equimolar C (1.2 mg C/mg VPA) concomitantly for 14 days. The plasma level of β hydroxybutyrate was lower in VPA treated children than in the control subjects (31.8-7.4 vs 90.0±21.4 nmol/ml, means±SEM, p (p0.05), Which remained unchanged after the C treatment (29.7±7.1), showing that the C was not able in itself to improve the plasma ketone level. The plasma level of FFA, triglycerides and cholesterol remained unaffected by C treatment, the level of HDL cholesterol decreased in the supplemented group. The daily excreted total N was not affected by C treatment (6.0 ±0.5, 7.3±0.3 and 7.3±1.0 g/day; day 0, 14 and control subjects) with no changes of excreted urea and ammonia suggesting,that the organism does not utilize proteins (and/or amino aoids) as alternative fuels instead of ketone bodies during VPA treatment.

Role of carnitine in promoting the effect of antidiabetic biguanides on hepatic ketogenesis.

Abstract Effects of biguanides on carnitine content of rat and guinea pig liver and on capacity of rat liver slices for ketogenesis were studied. In acute experiments, fed. 24-hour and 48-hour fasted male rats were given a single dose ofbuformin and the carnitine and acetylearnitine level in the tissues were determined 1 or 3 hr afterwards. The same was performed on fed guinea pigs. In all the 1-hr groups we found an increase ranging from 30 to 50 per cent in hepatic carnitine level. In chronic experiments rats were treated with buformin or metformin for 6 days. The carnitine content, carnitine acetyltransferase and carnitine palmitoyltransferase activities were determined. The respective carnitine levels in the buformin- and metformin-treated groups were 4 times and 2.5 times the control value expressed on a per gram basis. In addition, carnitine acetyltransferase activity, given as mU/mg mitochondrial protein, increased 2-fold in the buformin-treated group. The increase in carnitine content strongly suggests that liver has enhanced capacity for oxidation of fatty acids and consequently for production of ketone bodies. The latter has been verified in the chronic experiments by the following observations: (1) The buformin administration increased the total ketone body content of the freeze-clamped liver specimens to 210 per cent of the control value. The calculated mitochondrial NAD + /NADH ratio was reduced from 10.6 to 5.96 in the same specimens. (2) the liver slices from treated animals formed 30–40 per cent more ketone bodies than those from control ones during the 30-min and 60-min incubations. (3) The ketone body associated radioactivity deriving from Na-[1 14 -C] palmitate accounted for 90.5 per cent of water soluble radioactivity in slices from treated animals, whereas it accounted for 66.8 per cent in slices from control ones.

CARNITINE SUPPLEMENTATION IN PREMATURE INFANTS

The possible effects of carnitine supplementation on nitrogen metabolism were studied on AGA preterm infants(birthweight 980-1750g) maintained on mixed nutrition (50% pooled milk 50% formula daily). Started by various postnatal ages (mean 25.5 days) 15 infants received L-carnitine supplemented formula (600 nmol/ml over endogenous content)during 7 days, another 10 served as controls. Plasma carnitines increased whereas alanine (0.21±0.02, 0.19±0.03, 0.25±0.02 mmol/l; day 0, 7, and 14, means±SEM, p<0.05) and glutamine (0.37±0.06, 0.31±0.05, 0.4l±0.05 mmol/l p<0.05) decreased with a fall of urea level. Urinary urea (2.6±0.23, 2.25±0.18, 2.31±0.21 mmol/kg/day, p < 0.05) and ammonia (1.07-0.1, 0.87±0.1, 1.4±0.1 mmol/kg/day, p<0.05) decreased suggesting lowered amino acid degradation. Surprisingly, 7 days after the supplementation, excretion of acylcarnitines remained high (5.9±1.5, 13.1±2.5, 10.3±1.3 umol/day, p< 0.05) which was not seen for the free fraction.

26 ORAL L-CARNITINE SUPPLEMENTATION IN LOW-BIRTH-WEIGHT NEONATES MAINTAINED ON POOLED MILK

As the carnitine /C/ content of human milk declines during lactation, it is doubtful, that its amount is adequate to statisfy the daily needs of premature infants fed with pooled milk. Effects of supplementation have been studied in 20 AGA premature infants /weight at birth 1200 to 1800 g; gest. age 28 to 34 wk/. Throughout 7 days, started at postnatal ages of 10 to 33 days, infants were fed exclusively with pooled human milk containing 300 nmol/ml C as added supplement. Total, free and acyl C were significantly elevated in the plasma at the end of study with an increase in beta hydroxybutyrate /22.9±2.5 vs 38.4±3.9 umol/1, means±SEM, p<0.05/ and a decrease in triglyceride /1.67±0.08 vs 1.29±0.07 nmol/1, p < 0.001/ and urea levels /1.72±0.09 vs 1.36±0.07 nmol/1, p< 0.005/. In the urine both fractions of C significantly increased at the end of study as compared to presupplementary control days. Urinary excretion of total nitrogen /77.3±6.7 vs 62.4±2.4 mg/kg/day, p<0.05/ and urea /1.88±0.11 vs 1.25±0.09 mnol/kg/day, p<0.005/ decreased. The present data suggest, that improved carnitine availability resulted in increased fat oxidation, utilization of amino acids/proteins decreased. It is suggested, that the nutritional value of pooled milk for low-birth-weight infants should be reconsidered because its low carnitine content.