Janos Kerner

Pivampicillin-promoted excretion of pivaloylcarnitine in humans

Pivampicillin treatment of seven children (five boys and two girls) for 7 days significantly reduced the amounts of total acid-soluble carnitine, free carnitine, and long-chain acylcarnitines and increased the amounts of acid-soluble acylcarnitine in plasma. The fasting plasma levels of 3-hydroxybutyrate at the end of treatment were 15% of the control value. The levels of free fatty acids were decreased, whereas triglyceride levels were unaffected, indicating impaired fat metabolism. Daily urinary excretion of total carnitine was four to five times higher than controls after the first day of treatment, although the amounts of free carnitine and acetylcarnitine were decreased. The urinary acylcarnitines were isolated and characterized by gas chromatography/electron impact mass spectrometry and fast-atom bombardment mass spectrometry. Pivaloylcarnitine was the predominant urinary acylcarnitine; it represented greater than 96% of the increased excretion of total carnitine and 75-80% of the total conjugated pivalic acid. The renal clearance of acylcarnitines was comparable to that of creatinine, indicating no reabsorption of pivaloylcarnitine. These data suggest a detoxification function of carnitine for pivalic acid in humans.

Differential excretion of xenobiotic acyl-esters of carnitine due to administration of pivampicillin and valproate

The fate of supplemental carnitine was studied in human subjects treated with drugs known to cause carnitine deficiency. Six children were treated with pivampicillin and equimolar L-carnitine for 7 days. On the last day of treatment, the plasma levels of total and free carnitine were decreased, but acylcarnitine levels were increased. A 12-fold increase in urinary excretion of acylcarnitines was found; it increased from 188.5 +/- 82.7 to 2218.4 +/- 484.1 mumole/day, and 84% was pivaloylcarnitine. Free carnitine excretion was reduced. Ten epileptic children on chronic valproate treatment received equimolar carnitine for a 2-week period. Plasma carnitine levels were elevated on the last day of treatment. A 3.4-fold increase in urinary acylcarnitines was found, but most of the excreted carnitines were free (64.5-fold increases). These data show that pivalate is readily converted to carnitine esters, in contrast to the limited conversion of valproate to acylcarnitines in humans.

Effect of etomoxiryl-CoA on different carnitine acyltransferases.

The effects of etomoxiryl-CoA on purified carnitine acyltransferases and on carnitine acyl-transferases of rat heart mitochondria and rat liver microsomes were determined. At nanomolar concentrations, the data agreed with that of other investigators who have shown that etomoxiryl-CoA must be binding to a high affinity site with specific inhibition of mitochondrial carnitine palmitoyltransferase (CPTo). Micromolar amounts of etomoxiryl-CoA inhibited both short- and long-chain carnitine acyltransferases. The concentrations of etomoxiryl-CoA required for 50% inhibition of the different carnitine acetyltransferases and microsomal and peroxisomal carnitine octanoyltransferase were in the low micromolar range. Mixed-type and uncompetitive inhibition kinetics were obtained, depending on the source of purified enzyme. When purified rat heart CPT was incubated with etomoxiryl-CoA, it increased the K0.5 and decreased the Hill coefficient for acyl-CoA. Both proteins and phospholipids of mitochondria and microsomes formed covalent adducts of [3H]etomoxir, with the predominant labeling in phospholipids. None of the purified enzymes formed covalent adducts when incubated with [3H]etomoxiryl-CoA, in contrast to intact mitochondria or microsomes. The major 3H-labeled protein for rat heart mitochondria had a molecular weight of 81,000 +/- 4000, and the major proteins from microsomes had a molecular weight of 51,000-57,000. Malonyl-CoA prevented most of the tritum incorporation into the 81,000 Da protein of mitochondria, but it had little effect on incorporation of tritiated etomoxir into the 51,000-57,000 Da proteins of microsomes. When 50 microM etomoxiryl-CoA was added to microsomes and to mitochondria that had been incubated with radioactive etomoxiryl-CoA, much of the radioactive etomoxir disappeared from the major microsomal proteins, but virtually none was displaced from the mitochondrial protein. Thus, at least two different types of covalent etomoxir complexes were formed. This pulse-chase experiment showed that the mitochondrial protein-etomoxir complex was not turned over, consistent with other data showing that etomoxir inhibited carnitine palmitoyltransferase. In contrast, the major protein-etomoxir complex in microsomes was turned over during the pulse-chase experiment.

Analysis of acylcarnitines in normal human urine with the radioisotopic exchange-high performance liquid chromatography (HPLC) method.

The identification of camitine esters in the urine is important in the diagnosis of metabolic diseases which alter camitine metabolism [l]. Their quantitation may also give clues to the physiological role of carnitine in the metabolism of branched chain amino acids and fatty acids, the influence of nutrition on these processes and the possible function of camitine in the conjugation of potentially toxic endogenous or exogenous organic acids. The excretion of total, free and acylcamitine [2-41 and that of acetylcamitine [5] in healthy humans has been measured in several studies. However, the analysis of individual camitine esters in normal human urine has been incomplete due to a lack of methods sensitive and specific enough to detect low concentrations of these compounds. In the following, we report the quantitation of camitine esters in normal human urine using a modification of the radioisotopic exchange-HPLC method originally described by Kemer and Bieber for tissue extracts and colostrum WI.

Effect of malonyl-CoA on the kinetics and substrate cooperativity of membrane-bound carnitine palmitoyltransferase of rat heart mitochdonria

The effect of malonyl-CoA on the kinetic parameters of carnitine palmitoyltransferase (outer) the outer form of carnitine palmitoyltransferase (palmitoyl-CoA: L-carnitine O-palmitoyltransferase, EC 2.3.1.21) from rat heart mitochondria was investigated using a kinetic analyzer in the absence of bovine serum albumin with non-swelling conditions and decanoyl-CoA as the cosubstrate. The K0.5 for decanoyl-CoA is 3 microM for heart mitochondria from both fed and fasted rats. Membrane-bound carnitine palmitoyltransferase (outer) shows substrate cooperativity for both carnitine and acyl-CoA, similar to that exhibited by the enzyme purified from bovine heart mitochondria. The Hill coefficient for decanoyl-CoA varied from 1.5 to 2.0, depending on the method of assay and the preparation of mitochondria. Malonyl-CoA increased the K0.5 for decanoyl-CoA with no apparent increase in sigmoidicity or Vmax. With 20 microM malonyl-CoA and a Hill coefficient of n = 2.1, the K0.5 for decanoyl-CoA increased to 185 microM. Carnitine palmitoyltransferase (outer) from fed rats had an apparent Ki for malonyl-CoA of 0.3 microM, while that from 48-h-fasted rats was 2.5 microM. The kinetics with L-carnitine were variable: for different preparations of mitochondria, the K0.5 ranged from 0.2 to 0.7 mM and the Hill coefficient varied from 1.2 to 1.8. When an isotope forward assay was used to determine the effect of malonyl-CoA on carnitine palmitoyltransferase (outer) activity of heart mitochondria from fed and fasted animals, the difference was much less than that obtained using a continuous rate assay. Carnitine palmitoyltransferase (outer) was less sensitive to malonyl-CoA at low compared to high carnitine concentrations, particularly with mitochondria from fasted animals. The data show that carnitine palmitoyltransferase (outer) exhibits substrate cooperativity for both acyl-CoA and L-carnitine in its native state. The data show that membrane-bound carnitine palmitoyltransferase (outer) like carnitine palmitoyltransferase purified from heart mitochondria exhibits substrate cooperativity indicative of allosteric enzymes and indicate that malonyl-CoA acts like a negative allosteric modifier by shifting the acyl-CoA saturation to the right. A slow form of membrane-bound carnitine palmitoyltransferase (outer) was not detected, and thus, like purified carnitine palmitoyltransferase, substrate-induced hysteretic behavior is not the cause of the positive substrate cooperativity.

Impaired thermoregulation in cold-exposed rats with hypothalamic obesity

Rats with obesity-producing, hypothalamic knife cuts were fed a high fat diet and placed in the cold (2 degrees C) for six days starting 3, 11, or 24 days after surgery. Between surgery and cold exposure, knife-cut rats consumed 90% to 122% more energy and gained more weight (32 +/- 4, 112 +/- 5, and 241 +/- 9 g) than sham-operated rats (15 +/- 2, 34 +/- 2, and 58 +/- 3 g). When exposed to cold, sham-operated rats increased (22% to 30%) energy intake whereas knife-cut rats decreased (5% to 51%) intake. After 24 hours at 2 degrees C body temperatures of knife-cut rats were 1.2, 0.7, and 0.7 degrees less than those of control rats; body temperatures continued to decrease to 2.9, 3.0 and 2.5 degrees less than control rats after six days at 2 degrees C. Fasting for 12 hours at 2 degrees C caused a further reduction in body temperature to 4.9, 4.8, and 5.9 degrees less than in control rats. Cold exposure increased urinary excretion of norepinephrine and epinephrine (indicators of sympathoadrenal activity) in all rats. Guanosine diphosphate (GDP) binding to brown adipose tissue (BAT) mitochondria (an indicator of the thermogenic capacity of the tissue) was similar in cold-exposed, knife-cut, and sham-operated rats. Cold acclimation before hypothalamic knife-cut surgery prevented the cold-induced decrease in body temperatures of knife-cut rats.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

AMIHO ACID METABOLISM IN CHRONIC VALPROATE TREATMENT AT TWO LEVELS OF CARNITINE INTAKE

While valproate (VPA) is known to cause changes of nitrogen containing metabolites, the amino acid (AA) metabolism and its alterations during carnitine (C) therapy have not been studied. Ten VPA treated C deficient children equimolar C were given over a 14 days period. Before C treatment the fasting plasma levels of ammonia, taurine, aspartate, hydroxyproline, glutamate, proline, glycine, alanine, methionine were elevated, the levels of leucine and ornithine were depressed in VPA treated children as compared to controls (p<0.05), their levels remained unchanged after C therapy. The elevated ammonia and glutamate with normal glutamine levels show impaired glutamate-glutamine cycle. After a standard meal the plasma levels of AA exhibited different elevation before and after C treatment. The urinary output of AA was lower in the VPA treated group, output of 8 individual AA increased after the C treatment, showing that the C may influence the AA metabolism, yet most changes are C independent and are caused probably by the VPA per se.

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.