Charles J. Rebouche

Kinetics, Pharmacokinetics, and Regulation of l‐Carnitine and Acetyl‐l‐carnitine Metabolism

Abstract: In mammals, the carnitine pool consists of nonesterified l‐carnitine and many acylcarnitine esters. Of these esters, acetyl‐l‐carnitine is quantitatively and functionally the most significant. Carnitine homeostasis is maintained by absorption from diet, a modest rate of synthesis, and efficient renal reabsorption. Dietary l‐carnitine is absorbed by active and passive transfer across enterocyte membranes. Bioavailability of dietary l‐carnitine is 54‐87% and is dependent on the amount of l‐carnitine in the meal. Absorption of l‐carnitine dietary supplements (0.5‐6 g) is primarily passive; bioavailability is 14‐18% of dose. Unabsorbed l‐carnitine is mostly degraded by microorganisms in the large intestine. Circulating l‐carnitine is distributed to two kinetically defined compartments: one large and slow‐turnover (presumably muscle), and another relatively small and rapid‐turnover (presumably liver, kidney, and other tissues). At normal dietary l‐carnitine intake, whole‐body turnover time in humans is 38‐119 h. In vitro experiments suggest that acetyl‐l‐carnitine is partially hydrolyzed in enterocytes during absorption. In vivo, circulating acetyl‐l‐carnitine concentration was increased 43% after oral acetyl‐l‐carnitine supplements of 2 g/day, indicating that acetyl‐l‐carnitine is absorbed at least partially without hydrolysis. After single‐dose intravenous administration (0.5 g), acetyl‐l‐carnitine is rapidly, but not completely hydrolyzed, and acetyl‐l‐carnitine and l‐carnitine concentrations return to baseline within 12 h. At normal circulating l‐carnitine concentrations, renal l‐carnitine reabsorption is highly efficient (90‐99% of filtered load; clearance, 1‐3 mL/min), but displays saturation kinetics. Thus, as circulating l‐carnitine concentration increases (as after high‐dose intravenous or oral administration of l‐carnitine), efficiency of reabsorption decreases and clearance increases, resulting in rapid decline of circulating l‐carnitine concentration to baseline. Elimination kinetics for acetyl‐l‐carnitine are similar to those for l‐carnitine. There is evidence for renal tubular secretion of both l‐carnitine and acetyl‐l‐carnitine. Future research should address the correlation of supplement dosage, changes and maintenance of tissue l‐carnitine and acetyl‐l‐carnitine concentrations, and metabolic and functional changes and outcomes.

Tissue distribution of carnitine biosynthetic enzymes in man

The distribution in human tissues of enzymes which convert epsilon-N-trimethyl-L-lysine to L-carnitine was studied. Existing methodology was modified and new procedures were developed to measure enzyme activities. Epsilon-N-Trimethyl-L-lysine was converted to gamma-butyrobetaine in three enzymatic steps (hydroxylation at carbon 3, aldol cleavage between carbons 2 and 3 to yield glycine and gamma-trimethylaminobutyraldehyde, and subsequent oxidation of the aldehyde) in all tissues studied (liver, brain, kidney, heart and skeletal muscle), but gamma-butyrobetaine was hydroxylated to form L-carnitine only in liver, kidney and brain. Gamma-Butyrobetaine hydroxylase (4-trimethylaminobutyrate, 2-oxoglutarate: oxygen oxidoreductase (3-hydroxylating), EC 1.14.11.1) activity in liver was dependent on the age of the subject. The activity rose from 12% in infants to 100% of the adult mean by age 15 years. No age dependence could be demonstrated for the other three enzymes studied.

Ascorbic acid and carnitine biosynthesis.

It has been suggested that early features of scurvy (fatigue and weakness) may be attributed to carnitine deficiency. Ascorbate is a cofactor for two alpha-ketoglutarate-requiring dioxygenase reactions (epsilon-N-trimethyllysine hydroxylase and gamma-butyrobetaine hydroxylase) in the pathway of carnitine biosynthesis. Carnitine concentrations are variably low in some tissues of scorbutic guinea pigs. Ascorbic acid deficiency in guinea pigs resulted in decreased activity of hepatic gamma-butyrobetaine hydroxylase and renal but not hepatic epsilon-N-trimethyllsine hydroxylase when exogenous substrates were provided. It remains unclear whether vitamin C deficiency has a significant impact on the overall rate of carnitine synthesis from endogenous substrates. Nevertheless, results of studies of enzyme preparations and perfused liver in vitro and of scorbutic guinea pigs in vivo provide compelling evidence for participation of ascorbic acid in carnitine biosynthesis.

Primary systemic carnitine dehciency. II. Renal handling of carnitine

Renal tubular reabsorption rates, reabsorptive maxima, and apparent renal plasma excretory thresholds for carnitine were determined in four children with primary systemic carnitine deficiency (SCD), in two of the mothers of these children, in one patient with muscle carnitine deficiency (MCD), and in seven controls. In SCD the observed values were well below those found in six of seven controls, but one control, a healthy 20-year-old woman with normal muscle carnitine level, also exhibited a renal carnitine leak. In the two mothers of patients with SCD and in the case of MCD some of the parameters of the renal handling of carnitine were slightly altered. Tubular secretion of short-chain acylcarnitines was noted in patients and controls at high plasma free carnitine levels. Augmented excretion of short-chain acylcarnitines occurred at lower plasma free carnitine levels in the patients with SCD than in the other subjects. Free and short-chain acyl-carnitines may compete for the same renal reabsorptive site. A renal defect cannot fully account for primary SCD but may contribute to the carnitine depletion in this disorder.

Cow milk feeding in infancy: Further observations on blood loss from the gastrointestinal tract

Because feeding of cow milk causes normal infants to lose increased amounts of occult blood from the gastrointestinal tract, we conducted a prospective trial to measure intestinal blood loss quantitatively and to monitor iron nutritional status. Fifty-two infants entered the trial at 168 days of age and were assigned at random to receive either cow milk or a milk-based formula. Initially, 31 infants had been breast-fed and 21 had been fed formulas. With the feeding of cow milk, the proportion of guaiac-positive stools increased from 3.0% at baseline to 30.3% during the first 28 days of the trial (p less than 0.01), whereas the proportion of positive stools remained low (5.0%) with the feeding of formula. The proportion of guaiac-positive stools among cow milk-fed infants declined later, but for the entire trial it remained significantly (p less than 0.01) elevated. Stool hemoglobin concentration increased markedly with the introduction of cow milk, rising from a mean (+/- SD) of 622 +/- 527 micrograms/gm dry stool at baseline to 3598 +/- 10,479 micrograms/gm dry stool during the first 28 days of ingestion of cow milk. Among infants fed formula, stool hemoglobin did not increase and was significantly (p less than 0.01) less than in the cow milk group. Among infants fed cow milk, the increase in hemoglobin concentration tended to be greater for those who had initially been fed human milk than for those who had initially been fed formulas. Iron nutritional status was not significantly different between the two feeding groups. However, one infant became iron deficient after 4 weeks of ingesting cow milk. We conclude that cow milk feeding leads to increased intestinal tract blood loss in a large proportion of normal infants and that the amount of iron lost is nutritionally important.

Lactoferrin in the Preterm Infants' Diet Attenuates Iron-Induced Oxidation Products

Free radical injury is thought to play a significant role in the pathogenesis of several disease processes in low birth weight premature infants including retinopathy of prematurity and necrotizing enterocolitis. Because iron is a known catalyst in free radical–mediated oxidation reactions, the objectives of the present in vitro studies were to determine whether after exposure to air 1) iron present in infant formula, or that added to human milk or formula as medicinal iron or as iron contained in human milk fortifier, increases free radical and lipid peroxidation products; and 2) recombinant human lactoferrin added to formula or human milk attenuates iron-mediated free radical formation and lipid peroxidation. Before adding medicinal iron to formula and human milk, significantly more ascorbate and α-hydroxyethyl radical production and more lipid peroxidation products (i.e. thiobarbituric acid reactive substances, malondialdehyde, and ethane) were observed in formula. After the addition of medicinal iron to either formula or human milk, further increases were observed in free radical and lipid peroxidation products. When iron-containing human milk fortifier was added to human milk, free radicals also increased. In contrast, the addition of apo-recombinant human lactoferrin to formula or human milk decreased the levels of oxidative products when medicinal iron or human milk fortifier was present. We speculate that the presence of greater concentration of iron and the absence of lactoferrin in formula compared with human milk results in greater in vitro generation of free radicals and lipid peroxidation products. Whether iron-containing formula with lactoferrin administered enterally to preterm infants will result in less free radical generation in vivo has yet to be established.

Carnitine transport in cultured muscle cells and skin fibroblasts from patients with primary systemic carnitine deficiency

Summaryl-Carnitine transport was studied in cultured muscle cells and skin fibroblasts of patients with primary systemic carnitine deficiency and control subjects. In both cell culture types, two systems for carnitine transport were identified. The kinetic parameters for carnitine transport were remarkably similar in cultured muscle cells and skin fibroblasts. Normal rates and kinetic properties of carnitine transport were observed for both cell lines from patients with systemic carnitine deficiency. These studies do not rule out a defect in carnitine transport in vivo.

Quantitative estimation of absorption and degradation of a carnitine supplement by human adults

Results of kinetic and pharmacokinetic studies have suggested that dietary carnitine supplements are not totally absorbed, and are in part degraded in the gastrointestinal tract of humans. To determine the metabolic fate of dietary carnitine supplements in humans, we administered orally a tracer dose of [methyl-3H]L-carnitine with a meal to five normal adult males, who had been adapted to a high-carnitine diet plus carnitine supplement (2 g/d) for 14 days. Appearance of [methyl-3H]L-carnitine and metabolites in serum, and urinary and fecal excretion of radiolabeled carnitine and metabolites was monitored for 5 to 11 days following administration of the test dose. Maximum concentration of [methyl-3H]L-carnitine in serum occurred at 2.0 to 4.5 hours after administration of the tracer, indicating relatively slow absorption from the intestinal lumen. Total radioactive metabolites excreted in urine and feces ranged from 47% to 55% of the ingested tracer. Major metabolites found were [3H]trimethylamine N-oxide (8% to 49% of the administered dose; excreted primarily in urine) and [3H]gamma-butyrobetaine (0.44% to 45% of the administered dose; excreted primarily in feces). Urinary excretion of total carnitine was 16% to 23% of intake. Fecal excretion of total carnitine was negligible (less than 2% of total carnitine excretion).

Kinetic compartmental analysis of carnitine metabolism in the dog.

This study was undertaken to quantitate the dynamic parameters of carnitine metabolism in the dog. Six mongrel dogs were given intravenous injections of L-[methyl-3H]carnitine and the specific radioactivity of carnitine was followed in plasma and urine for 19-28 days. The data were analyzed by kinetic compartmental analysis. A three-compartment, open-system model [(a) extracellular fluid, (b) cardiac and skeletal muscle, (c) other tissues, particularly liver and kidney] was adopted and kinetic parameters (carnitine flux, pool sizes, kinetic constants) were derived. In four of six dogs the size of the muscle carnitine pool obtained by kinetic compartmental analysis agreed (+/- 5%) with estimates based on measurement of carnitine concentrations in different muscles. In three of six dogs carnitine excretion rates derived from kinetic compartmental analysis agreed (+/- 9%) with experimentally measured values, but in three dogs the rates by kinetic compartmental analysis were significantly higher than the corresponding rates measured directly. Appropriate chromatographic analyses revealed no radioactive metabolites in muscle or urine of any of the dogs. Turnover times for carnitine were (mean +/- SEM): 0.44 +/- 0.05 h for extracellular fluid, 232 +/- 22 h for muscle, and 7.9 +/- 1.1 h for other tissues. The estimated flux of carnitine in muscle was 210 pmol/min/g of tissue. Whole-body turnover time for carnitine was 62.9 +/- 5.6 days (mean +/- SEM). Estimated carnitine biosynthesis ranged from 2.9 to 28 mumol/kg body wt/day. Results of this study indicate that kinetic compartmental analysis may be applicable to study of human carnitine metabolism.

Freeze‐fracture electronmicroscopic analysis of plasma membranes of cultured muscle cells in Duchenne dystrophy

Depletion of intramembranous particles has been reported in the muscle fiber plasma membrane in Duchenne dystrophy. We searched for a similar abnormality in plasma membranes of cultured muscle cells obtained from six patients with Duchenne dystrophy and six controls. A precise method for phase microscopic to freeze-fracture electronmicroscopic correlation was devised to identify the cell of origin of each replicated membrane face. For both control and patient cells, P-face particle density was higher in myotubes than in mononuclear cells. However, there were no significant differences between control and dystrophic cells when comparing p faces of myotubes, E faces of myotubes, p faces of single cells, or E faces of single cells. The frequency distribution of particle diameters was similar in p faces of control and Duchenne myotubes. Other structural features of the freeze-fractured plasma membrane also were similar.