Duna Penn

Carnitine blood concentrations and fat utilization in parenterally alimented premature newborn infants.

To investigate the relationships among carnitine intake, carnitine blood concentrations, and the ability to utilize exogenous fat, total carnitine, free carnitine, acylcarnitine, beta-hydroxybutyrate, free fatty acid and triglyceride plasma concentrations were measured in 26 parenterally alimented appropriate-for-gestational-age premature infants before and at the end of a four-hour infusion of Intralipid, 1 gm/kg body weight. There was an increase in plasma levels of AC, BOB, FFA, and TG, but a decrease of FC, TC was unaffected by the infusion, but strongly correlated with calculated carnitine intake. At the end of the fat infusion, AC and BOB were positively correlated, and FFA negatively correlated with TC. The results demonstrate the proportion of AC to FC to be an additional indicator of fatty acid utilization and suggest that decreased carnitine intake in premature infants may impair fatty acid oxidation and ketogenesis.

Impaired fatty acid oxidation in children on valproic acid and the effect of L‐carnitine

Fatty acid oxidation was studied in 12 patients (aged 3 to 19 years) receiving valproic acid (VPA), predominantly as monotherapy, before and after 1 month of L-carnitine supplementation (50 mg/kg/day po) in order to determine whether L-carnitine plays a role in preventing the hepatotoxic effects of this drug. Five of these patients were also studied prior to VPA treatment. Only one patient taking VPA had an abnormally low plasma free carnitine. Acyl-/free carnitine ratios were elevated in five patients on VPA and normalized after L-carnithe supplementation. Mean plasma concentrations of free fatty acids, β-OH-butyrate, and cumulative excretion of 13CO2 after administration of 1-13C-octanoic acid were not changed by VPA or L-carnitine treatment. Urinary dicarboxylic acids, acylglycines, and octanoylcarnitine were elevated during VPA therapy and unaltered by L-carnitine. These results suggest that, in patients at low risk for VPA-induced hepatotoxicity (patients aged >2 years and taking VPA as monotherapy), VPA causes metabolic abnormalities resembling those found in inborn errors of mitochondria1 β-oxidation which are not corrected by L-carnitine.

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.

Carnitine and Total Parenteral Nutrition of the Neonate

The newborn is dependent upon fat for energy production. Fatty acid oxidation requires the cofactor carnitine. The preterm infant is born with limited carnitine reserves. During total parenteral nutrition (TPN) plasma and tissue carnitine concentrations decrease indicating that rates of carnitine biosynthesis are inadequate. The ability of the premature infant to oxidize fatty acids is related to the carnitine status. Several studies suggest an improvement of fatty acid oxidation after a fat challenge if TPN is supplemented with L-carnitine. Nitrogen balance may also be improved but this needs confirmation. It remains to be established whether routine L-carnitine supplementation is needed in neonatal TPN.

Detection of inborn errors of fatty acid oxidation from acylcarnitine analysis of plasma and blood spots with the radioisotopic exchange-high-performance liquid chromatographic method

Sixty-one plasma samples from patients with inborn errors of fatty acid oxidation and from control subjects were analyzed in a blinded fashion for acylcarnitines by the radioisotopic exchange-high-performance liquid chromatographic method. All samples from patients with medium-chain acyl-coenzyme A dehydrogenase (MCAD) deficiency (n = 30), some of which had been stored in a frozen state for several years, showed a prominent octanoylcarnitine peak. In all blood spots from 11 patients with MCAD deficiency, octanoylcarnitine was also detected. Control plasma specimens and blood spots contained small amounts of octanoylcarnitine; however, the octanoylcarnitine/acetylcarnitine ratio differentiated patients with MCAD deficiency. Longer-chain acylcarnitines were found in plasma of all three patients with defects in long-chain fatty acid oxidation. Plasma and blood spots from a patient with multiple acyl-coenzyme A dehydrogenase deficiency contained C4-acylcarnitine, hexanoylcarnitine, octanoylcarnitine, and decanoylcarnitine. The results suggest that the method may be highly sensitive in detecting MCAD deficiency and other defects in fatty acid oxidation from plasma or blood spots.

Analysis of carnitine esters by radio-high performance liquid chromatography in cultured skin fibroblasts from patients with mitochondrial fatty acid oxidation disorders

Acylcarnitines are important diagnostic markers for inborn errors of fatty acid oxidation, but their analysis in body fluids may not always be reliable. Recently, disease-specific acylcarnitine profiles generated by cultured skin fibroblasts were reported to facilitate the diagnosis by localizing a specific enzymatic defect in the mitochondrial β-oxidation pathway. Using a novel methodologic approach, fibroblasts from 16 patients with inborn errors of fatty acid oxidation and 13 control subjects were preincubated with L-[3H]carnitine to label the intracellular carnitine pool. Cells were subsequently incubated with unlabeled palmitic acid and, after methanol extraction of cells and media, labeled free carnitine and acylcarnitines were analyzed by radio-HPLC. Quantitation was based on the integrated radioactivity of individual peaks relative to the total radioactivity recovered. In control cell lines, all saturated acylcarnitines were detected, and reference values were established. With the exception of one cell line deficient in electron transfer flavoprotein, all mutant cell lines showed abnormal and disease-specific relative concentrations of acylcarnitines. Advantages of the method include use of a small number of cells, no need for trypsinization and permeabilization of cells before incubation, simple extraction without purification of the specimen before HPLC, and relatively inexpensive equipment. The method allows a focused approach to the subsequent, more laborious confirmation of a particular disease by direct enzymatic and/or molecular analysis. It remains to be established whether the method can replace widely used global measurements of fatty acid oxidation rates in vitro that do not provide specific information about the enzyme deficiency involved.

Urinary medium-chain acylcarnitines in medium-chain acyl-CoA dehydrogenase deficiency, medium-chain triglyceride feeding and valproic acid therapy: sensitivity and specificity of the radioisotopic exchange/high performance liquid chromatography method.

ABSTRACTS: To determine the sensitivity and specificity of detecting urinary medium-chain acylearnitines for the diagnosis of MCAD deficiency, 114 urine specimens from 75 children with metabolic diseases and controls were analyzed in a blinded fashion using a radioisotopic ex-change/HPLC method. All 47 patients with MCAD deficiency were correctly diagnosed using the criterion hexanoylcarnitine or octanoylcarnitine peak areas larger than those of other medium-chain acylcarnitines. The majority of them were tested during the asymptomatic state without L-carnitine loading. Four patients with other defects of fatty acid oxidation and three patients receiving vatproic acid had a similar acylcarnitine exeretion pattern. To farther examine the specificity of the method, eight infants receiving a diet enriched with medium-chain triglycerides and 13 additional patients receiving valproic acid were studied. Most of these also tested positive for MCAD deficiency by the above criterion. Analysis by a new gas chromatographic-mass spectrometric procedure revealed that octanoylearnite, not valproylcarnitine, was the most abundant medium-chain carnitine ester excreted by a patient treated with valproic acid. Quantitation of urinary hexanoylcarnitine and octanoylcarnitine showed considerable overlap among patients with MCAD deficiency and those receiving valproic acid or a medium-chain triglyceride-enriched diet. MCAD deficiency can be reliably detected in urine specimens by this method without the need for prior carnitine loading. However, other defects in fatty acid oxidation must be differentiated from MCAD deficiency, and a history of medium-chain triglyceride or valproic acid administration must be considered if the diagnosis of MCAD deficiency is sought through analysis of urinary acylcarnitines.

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 μmol/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 μmol/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.

Carnitine deprivation adversely affects cardiovascular response to bacterial endotoxin (LPS) in the anesthetized neonatal pig

Sepsis and endotoxemia are important stressors for the neonate. Newborn infants receiving total parenteral nutrition are routinely deprived of carnitine. To investigate whether carnitine deprivation affects the neonates ability to respond to endotoxin, 19 newborn piglets received parenteral nutrition for 2–3 weeks that was either carnitine free (CARN-) or supplemented (CARN+) with L-camitine (400 mg/L). Cardiovascular performance, i.e., heart rate; blood pressure (BP); cardiac output (CO); systemic vascular resistance (SVR), and metabolic response, i.e., plasma glucose; lactate; tumor necrosis factor α; tissue nitric oxide; and urinary nitrites, were studied serially in anesthetized piglets for 3 h after endotoxin (lipopolysaccharide (LPS), 250 μg/kg intravenous bolus) or vehicle administration. Plasma and tissue carnitine values were lower in CARN- than in CARN+ piglets. Prior to LPS, no differences were found for most parameters (excepting lower diastolic BP and SVR in CARN- animals). Systolic, diastolic, and mean BP fell after LPS but recovered by the end of the experiment. Nadirs were lower in CARN- than in CARN + piglets. CO tended to be higher in CARN- than in CARN+ animals and fell after LPS. SVR fell after LPS and was lower in CARN- than in CARN+ piglets. LPS-treated animals transiently increased urinary flow. By all measures (plasma tumor necrosis factor α, glucose and lactate, tissue nitric oxide, and urinary nitrite excretion), LPS provocation was similar for both groups. Chronologically, BP changes were more closely related to SVR than to CO. Our findings suggest that carnitine deprivation diminishes tissue carnitine concentrations and adversely affects cardiovascular response to LPS, in part mediated by the peripheral vasculature.

Carnitine deprivation adversely affects cardiac performance in the lipopolysaccharide- and hypoxia/reoxygenation-stressed piglet heart.

Sepsis and hypoxia are important stressors for the neonate. Newborn infants receiving total parenteral nutrition are routinely deprived of carnitine and develop low carnitine plasma and tissue levels. Because of its high metabolic rate and dependence on fatty acids for energy, the newborn heart may be particularly vulnerable to stress in the face of an inadequate carnitine supply. To investigate whether carnitine deprivation affects cardiac performance under stress, 23 neonatal piglets received parenteral nutrition for 2-3 weeks that was either carnitine free (CARN -) or supplemented (CARN +) with L-carnitine (400 mg/L). Bacterial endotoxin (lipopolysaccharide (LPS), 250 microg/kg intravenous bolus) or saline vehicle was administered to anesthetized piglets 3 h prior to study of isolated perfused hearts. Left ventricular systolic pressure (LVSP), left ventricular end diastolic pressure, and left ventricular developed pressure (LVDP) were measured in vitro under aerobic, hypoxic, and reoxygenation conditions in all animals. Plasma and tissue carnitine values were lower in CARN - than in CARN + piglets. In hearts from LPS-treated animals prior to hypoxia, there was no difference in ventricular compliance between CARN - and CARN + groups. LVSP and LVDP were lower in CARN - than CARN + hearts. During hypoxia, LVSP and LVDP fell, but left ventricular end diastolic pressure increased in hearts from both LPS- and saline- treated piglets. Reoxygenation led to poorer recovery in CARN - than CARN + hearts from LPS-treated animals, but not from saline controls. During hypoxia/reoxygenation, lactate efflux initially rose and then fell, while carnitine efflux increased continually. Acetyl- and medium-chain acylcarnitines were detected in the coronary effluent. Our findings suggest that carnitine deprivation diminishes heart carnitine concentrations and impairs cardiac recovery from combined endotoxic and hypoxic stress. Possible mechanisms include reduced acyl buffering and/or impaired transport of fatty acyl groups into mitochondria.