Determination of indole-3-pyruvic acid levels in Arabidopsis thaliana by gas chromatography-selected ion monitoring-mass spectrometry

Primary systemic carnitine deficiency (SCD) is a rare hereditary disorder transmitted by an autosomal recessive mode of inheritance. The disorder includes cardiomyopathy, muscle weakness, hypoketotic coma with hypoglycemia, and hyperammonemia. In this study, we conducted a linkage analysis of a Japanese SCD family with a proband-a 9-year-old girl-and 26 members. The serum and urinary carnitine levels were determined for all members. The entire genome was searched for linkage to the gene locus for SCD, by use of a total of approximately 300 polymorphic markers located approximately 15-20 cM apart. In the family, there were two significantly different phenotypes, in terms of serum free-carnitine levels: low serum free-carnitine level (29.5+/-5.0 microM; n=14) and normal serum free-carnitine level (46.8+/-6.2 microM; n=12). There was no correlation of urinary free-carnitine levels with the low serum-level phenotype (putative heterozygote), but in normal phenotypes (wild type) urinary levels decreased as the serum levels decreased; renal resorption of free carnitine appeared to be complete in wild-type individuals, when the serum free-carnitine level was <36 microM. Linkage analysis using an autosomal dominant mode of inheritance of heterozygosity revealed a tight linkage between the disease allele and D5S436 on chromosome 5q, with a two-point LOD score of 4.98 and a multipoint LOD score of 5.52. The haplotype analysis revealed that the responsible genetic locus lies between D5S658 and D5S434, which we named the "SCD" locus. This region was syntenic with the jvs locus, which is responsible for murine SCD. Phylogenic conversion of the SCD locus strongly suggests involvement of a single gene, in human SCD.     

Gene-dose effect on carnitine transport activity in embryonic fibroblasts of JVS mice as a model of human carnitine transporter deficiency

Recently, the marked decline in renal carnitine reabsorption has been thought to account fotr the systemic carnitine deficiency in juvenile visceral steatosis (JVS) mice. We have conducted a kinetic analysis using embryonic fibroblasts derived from normal, heterozygous, and homozygous jvs mice and found that the high-affinity carnitine transporter (Km = 5.5 microM), which shows Na+ and temperature dependency and stereospecificity, is defective in homozygous jvs mice. Moreover, a gene dose-dependent decrease of carnitine transport activity, which was due to a decrease in the number of the transporter molecules, was found in heterozygous jvs mice. Similar phenomena have been observed in human primary carnitine deficiency. Therefore, JVS mice may be useful for understanding this extremely rare human hereditary disorder.     

Weanling and adult rats differ in fatty acid and carnitine metabolism during sepsis

Increased oxidation of fat is an important host response to sepsis, and carnitine is essential for long-chain fatty acid oxidation. Because neonates have low levels of carnitine, their ability to respond to a septic insult may be impaired. The purpose of this study was to compare fatty acid and carnitine metabolism in septic weanling (60 to 85 g) and septic adult (285 to 310 g) rats. Sepsis was induced in weanling and adult male Sprague-Dawley rats by cecal ligation and puncture (CLP). The rats were killed 16 hours after CLP or sham operation, and serum glucose, lactate, beta-hydroxybutyrate, fatty acid, carnitine, liver fatty acid, and tissue carnitine levels were measured. The data suggest that during sepsis weanling rats may be more dependent on fatty acid oxidation than adult rats are, as evidenced by their elevated serum fatty acid and acylcarnitine levels, and relative hypoglycemia and hyperketonemia. In addition, although total serum carnitine levels were increased in both adult and weanling septic rats, tissue carnitine levels of weanling rats became significantly depleted during sepsis, unlike in adult rats. This study supports further investigation regarding the role of exogenous carnitine in newborn sepsis.     

3-hydroxy-3-methylglutaric aciduria and recurrent Reye-like syndrome

INTRODUCTION: 3-hydroxy-3-methylglutaryl-coenzyme A lyase deficiency (HMG-CoA lyase) is an inborn error of ketogenesis and Leucine catabolism. HMG-CoA lyase catalyses the final step in leucine degradation, converting HMG-CoA to acetyl-CoA and acetoacetic acid. Clinical manifestations include hepatomegaly, lethargy or coma and apnoea. Biochemically there is a characteristic absence of ketosis with hypoglycemia, acidosis, hipertransaminasemia and variable hyperammoniemia. The urinary organic acid profile includes elevated concentrations of 3-hydroxy-3-isovaleric, 3-hydroxy-3-methylglutaric, 3-methylglutaconic and 3-methylglutaric acids. CLINICAL CASE: Here, we report the case of a 17-year-old girl who presented in both ten months and five years of age a clinical picture characterized by lethargy leading to apnea and coma, hepatomegaly, hypoglycemia, metabolic acidosis, hyperammoniemia, elevated serum transaminases and absence of ketonuria. Diagnostic of Reye syndrome was suggested by hystopathologic finding of hepatic steatosis and clinical and biochemical data. As of 11 years old, laboratory investigations revealed carnitine deficiency and characteristic aciduria. Confirmatory enzyme diagnosis revealing deficiency of HMG-CoA lyase was made in cultured fibroblasts. CONCLUSION: Our report constitutes an example of the presentation of HMG-CoA lyase deficiency as recurrent Reye-like syndrome.     

Carnitine deficiency in inborn errors of metabolism

Several conditions, considered as inborn errors of metabolism, involve severe deficiencies in carnitine in both plasma and muscle. In the absence of evidence suggesting primary carnitine deficiency due to a biosynthetic enzymatic defect in the liver, the various diseases with carnitine deficiency are related to genetic defects in organic acid metabolism leading to blocked mitochondrial beta oxidation. We describe a 4.5-year-old boy and 2 female infants with glutaric aciduria type I, isovaleric acidemia, and long-chain acid dehydrogenase deficiency, in whom severe carnitine deficiency was apparent. In all 3, long-term carnitine treatment proved to be vital and eliminated most of the symptoms.     

Influence of multiple-dose administration of cefetamet pivoxil on blood and urinary concentrations of carnitine and effects of simultaneous administration of carnitine with cefetamet pivoxil

Cefetamet pivoxil (CEMT-PI), a drug of pivaloyloxymethyl group, was investigated for its impact on the carnitine blood homeostasis and renal excretion upon administering CEMT-PI alone, and CEMT-PI simultaneously with carnitine. 500 mg of CEMT-PI (group A) and 500 mg of CEMT-PI and an equimolar amount (200 mg of carnitine) of levocarnitine chloride (group B) were administered twice a day for 7 and 1/2 consecutive days to 5 healthy volunteers (group A) and 3 healthy volunteers (group B). No serious side effects nor abnormal values in physical and laboratory tests were observed throughout the study in both groups. During the treatment period, plasma total carnitine decreased slowly down to 25.5 microM (group A) and 38.8 microM (group B) and plasma free carnitine reached steady state levels at 17.7 microM (group A) and 29.2 microM (group B) on day 5. These concentrations represent 45 and 37% in group A, 66 and 58% in group B of the average pre-treatment baseline levels. Plasma pivaloylcarnitine quickly reached plateau levels of 6.12 microM (group A) and 4.05 microM (group B) on day 4. After treatment stop, plasma total and free carnitine returned to the pretreatment baseline level within 5 days (group A) and 3 days (group B), and plasma pivaloylcarnitine was detectable until day 7 of the treatment-free follow up in both groups. Although carnitine was given concurrently at a dose equimolar to the ingested amount of pivalic acid in group B, the plasma total and free carnitine exhibited a decrease. This was considered attributable to the fact that the bioavailability of carnitine is as low as 16% when administered orally, which is considerably less compared to the 55% bioavailability of cefetamet pivoxil.     

Carnitine palmitoyltransferase activities: effects of serum albumin, acyl-CoA binding protein and fatty acid binding protein

The carnitine palmitoyltransferase activity of various subcellular preparations measured with octanoyl-CoA as substrate was markedly increased by bovine serum albumin at low microM concentrations of octanoyl-CoA. However, even a large excess (500 microM) of this acyl-CoA did not inhibit the activity of the mitochondrial outer carnitine palmitoyltransferase, a carnitine palmitoyltransferase isoform that is particularly sensitive to inhibition by low microM concentrations of palmitoyl-CoA. This bovine serum albumin stimulation was independent of the salt activation of the carnitine palmitoyltransferase activity. The effects of acyl-CoA binding protein (ACBP) and the fatty acid binding protein were also examined with palmitoyl-CoA as substrate. The results were in line with the findings of stronger binding of acyl-CoA to ACBP but showed that fatty acid binding protein also binds acyl-CoA esters. Although the effects of these proteins on the outer mitochondrial carnitine palmitoyltransferase activity and its malonyl-CoA inhibition varied with the experimental conditions, they showed that the various carnitine palmitoyltransferase preparations are effectively able to use palmitoyl-CoA bound to ACBP in a near physiological molar ratio of 1:1 as well as that bound to the fatty acid binding protein. It is suggested that the three proteins mentioned above affect the carnitine palmitoyltransferase activities not only by binding of acyl-CoAs, preventing acyl-CoA inhibition, but also by facilitating the removal of the acylcarnitine product from carnitine palmitoyltransferase. These results support the possibility that the acyl-CoA binding ability of acyl-CoA binding protein and of fatty acid binding protein have a role in acyl-CoA metabolism in vivo.     

Opportunities for promoting palliative medicine in cancer research

OBJECTIVE: To study the effect of cisplatin on plasma concentrations and urinary excretion of carnitine in ten patients with different malignancies treated with chemotherapy. METHODS: Carnitine concentrations were determined using a radioenzymatic assay and other metabolites by routine methods of clinical chemistry. Renal clearances were calculated by dividing urinary excretions by the respective plasma concentrations. RESULTS: Before treatment, all patients had a normal plasma carnitine concentration. During treatment with cisplatin, the plasma total carnitine concentration increased by approximately 30% and normalized 7 days after stopping therapy. Urinary excretion of total carnitine increased by a factor of 10 during cisplatin administration and also normalized 7 days after cessation of chemotherapy. This increase was due to excretion of both free carnitine and acylcarnitine and averaged approximately 1 mmol carnitine per day. Similarly, urinary clearance of total carnitine was increased during therapy with cisplatin by a factor of approximately 8 and returned to normal 7 days after chemotherapy. In comparison, patients with similar malignancies treated with radiotherapy showed no significant increase in renal carnitine excretion. Similar to urinary excretion of carnitine, excretion of glucose and phosphate, two metabolites also reabsorbed by the proximal tubule of the nephron, was increased during therapy with cisplatin. There was a strong linear correlation between urinary excretion of free carnitine and acylcarnitines. CONCLUSIONS: Treatment with cisplatin is associated with a tenfold increase in renal carnitine excretion, most likely due to inhibition of carnitine reabsorption by the proximal tubule of the nephron. Well-nourished patients support this loss of carnitine even after repeated cycles of chemotherapy without developing hypocarnitinaemia. However, cachectic patients with decreased dietary carnitine uptake may develop carnitine deficiency when treated repeatedly with chemotherapies including cisplatin.     

Clear correlation of genotype with disease phenotype in very-long-chain acyl-CoA dehydrogenase deficiency

Very-long-chain acyl-CoA dehydrogenase (VLCAD) catalyzes the initial rate-limiting step in mitochondrial fatty acid beta-oxidation. VLCAD deficiency is clinically heterogenous, with three major phenotypes: a severe childhood form, with early onset, high mortality, and high incidence of cardiomyopathy; a milder childhood form, with later onset, usually with hypoketotic hypoglycemia as the main presenting feature, low mortality, and rare cardiomyopathy; and an adult form, with isolated skeletal muscle involvement, rhabdomyolysis, and myoglobinuria, usually triggered by exercise or fasting. To examine whether these different phenotypes are due to differences in the VLCAD genotype, we investigated 58 different mutations in 55 unrelated patients representing all known clinical phenotypes and correlated the mutation type with the clinical phenotype. Our results show a clear relationship between the nature of the mutation and the severity of disease. Patients with the severe childhood phenotype have mutations that result in no residual enzyme activity, whereas patients with the milder childhood and adult phenotypes have mutations that may result in residual enzyme activity. This clear genotype-phenotype relationship is in sharp contrast to what has been observed in medium-chain acyl-CoA dehydrogenase deficiency, in which no correlation between genotype and phenotype can be established.     

Alterations in energy metabolism of hypertrophied rat cardiomyocytes: influence of propionyl-L-carnitine

Alterations in energy metabolism, reduced fatty acid oxidation, and cardiac carnitine content have been implicated in the evolution from compensated to decompensated cardiac hypertrophy. We determined high-energy nucleotide levels in hypertrophied quiescent cardiomyocytes isolated from rat hearts 4 weeks after banding of abdominal aorta. In hypertrophied quiescent cardiomyocytes, a decrease in ATP content (p = 0.03), and ratios of ATP/total adenine nucleotides and of ATP/ADP were observed, together with an increase in ADP. In addition, palmitate, but not glucose oxidation, was markedly reduced in hypertrophied myocytes. In the presence of 25 microM propionyl-L-carnitine (PLC) or L-carnitine (LC), palmitate oxidation was significantly stimulated in hypertrophied myocytes. The ATP/ADP ratio was significantly increased only with PLC. This effect was not due to an enhanced PLC uptake, since total PLC uptake was 50% lower than that of LC. Changes in the energy generating system of quiescent myocytes occur early in pressure overload hypertrophy, and these alterations can be attenuated by PLC.     

Diagnosis of fatty infiltration of the liver on contrast enhanced CT: limitations of liver-minus-spleen attenuation difference measurements

Fatty acid oxidation has been studied with the tritium release assay in cultured fibroblasts from patients with defects in beta-oxidation and in the mitochondrial respiratory chain. Cells from all patients with beta-oxidation defects and cells from 10 of 16 patients with respiratory chain defects showed an impairment of fatty acid oxidation. The result of the tritium release assay is not only dependent on the proper function of the beta-oxidation cycle but is also influenced by the reoxidation of reduced cofactors. The assay can thus be used to study the expression of respiratory chain defects in cultured fibroblasts.     

Transplantation of teres major muscle for infraspinatus muscle in irreparable rotator cuff tears

Fatty acid oxidation defects can cause recurrent rhabdomyolysis or chronic progressive muscle weakness. Diagnosis is often possible on blood using tandem mass spectrometry or molecular genetic techniques. Riboflavin and carnitine are effective in some cases of multiple acyl-CoA dehydrogenase deficiency and primary carnitine deficiency, respectively. Controlled trials are needed to evaluate other proposed forms of treatment.     

Measles-mumps-rubella and hepatitis B vaccination uptake in adolescents: a survey in metropolitan Melbourne

Serum levels of free, acyl, and total carnitine were determined in 32 patients with seizures, before and after 3, 6, and 12 months of treatment with valproic acid (17 patients), carbamazepine (10 patients), or phenobarbital (5 patients). In all three treated groups, both free and total carnitine levels showed a significant decline with respect to pretreatment levels. This decline was most marked and most consistent in patients treated with valproic acid. In 35% of the patients in this group, carnitine deficiency (ie, total carnitine < 30 micromol/L) was observed by month 12. In none of the three groups were serum carnitine levels significantly correlated with the serum concentration of the drug. These findings suggest a need to monitor serum carnitine levels in children treated with any of these drugs.     

On carnitine requirements in full term and low birth weight neonates

The concentration of total carnitine in the blood serum of 15 newborns between days 5-21 of life was determined. The concentration of carnitine in low-birth-weight newborns is decreased in comparison with normal weight newborns; this deficiency may increase as a consequence of lack of carnitine provided with food or concomitant infection. The authors suggest that supplementation with carnitine be provided to this group of patients so as to prevent possible metabolic and clinical consequences.     

Trifunctional enzyme deficiency: adult presentation of a usually fatal beta-oxidation defect

Disorders of mitochondrial fatty acid oxidation are a common cause of exercise-induced rhabdomyolysis and myoglobinuria. We report three adult patients from a family with symptoms of recurrent exercise-induced rhabdomyolysis. This presentation closely resembles adult-type carnitine palmitoyltransferase II deficiency except that these patients had an associated peripheral neuropathy. Investigation of fatty acid oxidation in the patients revealed a deficiency of the mitochondrial trifunctional enzyme of beta-oxidation, a newly described fatty acid oxidation disorder with multiorgan involvement and a usually fatal outcome in early childhood. Our cases therefore represent a new phenotype of the disease, which is characterized by recurrent rhabdomyolysis and peripheral neuropathy, but without involvement of other organs, and which is associated with prolonged survival beyond the fourth decade. A low-fat/high-carbohydrate diet proved beneficial in one of the patients, drastically reducing the frequency of rhabdomyolytic episodes. Our findings suggest that mitochondrial trifunctional enzyme deficiency should be considered in patients with recurrent episodes of myoglobinuria and peripheral neuropathy presenting in later life.     

L-Carnitine: therapeutic applications of a conditionally-essential amino acid

A trimethylated amino acid roughly similar in structure to choline, carnitine is a cofactor required for transformation of free long-chain fatty acids into acylcarnitines, and for their subsequent transport into the mitochondrial matrix, where they undergo beta-oxidation for cellular energy production. Mitochondrial fatty acid oxidation is the primary fuel source in heart and skeletal muscle, pointing to the relative importance of this nutrient for proper function in these tissues. Although L-carnitine deficiency is an infrequent problem in a healthy, well-nourished population consuming adequate protein, many individuals within the population appear to be somewhere along a continuum, characterized by mild deficiency at one extreme, and tissue pathology at the other. Conditions which seem to benefit from exogenous supplementation of L-carnitine include anorexia, chronic fatigue, coronary vascular disease, diphtheria, hypoglycemia, male infertility, muscular myopathies, and Rett syndrome. In addition, preterm infants, dialysis patients, and HIV+ individuals seem to be prone to a deficiency of L-carnitine, and benefit from supplementation. Although available data on L-carnitine as an ergogenic aid is not compelling, under some experimental conditions pretreatment has favored aerobic processes and resulted in improved endurance performance.     

Carnitine affects octanoate oxidation to carbon dioxide and dicarboxylic acids in colostrum-deprived piglets: in vivo analysis of mechanisms involved based on CoA- and carnitine-ester profiles

Newborn, colostrum-deprived piglets (n = 21) were used to study the effects of L-carnitine supplementation on the in vivo oxidation of [1-14C]octanoate to CO2 and dicarboxylic acids. Pigs were fitted with arterial and bladder catheters and were infused with octanoate (supplying 35-100% of piglets' energy expenditure) and with or without valproate for a period of 24 h. After achieving steady-state octanoate oxidation, carnitine was coinfused [50 mumol/kg 0.75 prime plus 20 mumol/h.kg 0.75)], and deviations in the octanoate oxidation rate, dicarboxylic acid excretion rate, and carnitine metabolism were monitored. At the end of the 24-h infusion, samples of liver and muscle were analyzed for carnitine- and CoA-esters by HPLC. Carnitine stimulated octanoate oxidation by 7% (P < 0.05) and decreased dicarboxylic acid excretion by 45% (P < 0.05). Carnitine supplementation increased (P < 0.05) concentrations of carnitine and acetyl carnitine in hepatic tissue (three- and 55-fold, respectively) and plasma (seven- and 11-fold); whereas, muscle-carnitine concentration doubled upon carnitine supplementation, but acetyl carnitine concentration remained unaltered. Urinary excretion of acetyl and free carnitine also increased with carnitine supplementation, but accounted for < 10% of carnitine infused. Hepatic total CoA and CoA esters increased with carnitine supplementation, whereas muscle acetyl-CoA decreased. Valproate had only marginal effects on octanoate metabolism. These data confirm the hypothesis that carnitine effects the in vivo oxidation of octanoate in colostrum-deprived piglets and suggest that the effects may be mediated by aiding the export of excess acetyl groups from muscle or by enhancing uptake of octanoate into liver mitochondria.     

The attenuated elevation of cytoplasmic calcium concentration following the uptake of low density lipoprotein in type C Niemann-Pick fibroblasts

In Niemann-Pick disease type C fibroblasts, the deficiency of cholesterol esterification has been reported. In this experiment, we detected the attenuated elevation of cytoplasmic calcium concentration following low density lipoprotein uptake in the fibroblasts. Moreover, we administered calcium channel agonist (0.5 microM YC-170) and calcium channel antagonists to the fibroblasts. YC-170 improved the attenuated elevation of calcium concentration and the deficient cholesterol esterification by 40% and 90% respectively. Calcium channel antagonists decreased the cholesterol esterification in normal and affected fibroblasts. These data indicated that the attenuated elevation of cytoplasmic calcium concentration was strongly related to the etiology of this disease.     

Retrospective biochemical screening of fatty acid oxidation disorders in postmortem livers of 418 cases of sudden death in the first year of life

OBJECTIVE: Fatty acid oxidation (FAO) disorders are frequently reported as the cause of sudden and unexpected death, but their postmortem recognition remains difficult. We have devised a biochemical protocol in which informative findings in liver tissue are microvesicular steatosis, elevated concentrations of C8-C16 fatty acids, glucose depletion, and low carnitine concentration. STUDY DESIGN: We analyzed 27 cases representing five FAO disorders and compared the results with those obtained in a retrospective blinded analysis of 418 cases of sudden infant death (313 SIDS, 45 infections, and 34 accidents and abuse). RESULTS: All cases of accidents and abuse correctly tested negative. Among the others, 25 (6%) showed at least two abnormal findings. Of these, 14 closely matched the biochemical profiles seen in specific FAO disorders. These included 2 cases with medium-chain acyl-CoA dehydrogenase deficiency, 4 cases consistent with glutaric acidemia type 2, 4 cases with either very long-chain acylcoenzyme A dehydrogenase deficiency or long-chain 3-hydroxy-acyl-coenzyme A dehydrogenase deficiency, and 4 cases predicted to be affected with carnitine uptake defect. CONCLUSION: The results of this study support the view that approximately 5% of all cases of sudden infant death are likely caused by an FAO disorder.     

Short-chain acyl-CoA dehydrogenase deficiency: a cause of ophthalmoplegia and multicore myopathy

OBJECTIVE: To determine an underlying genetic defect within the differential diagnosis of congenital multicore myopathy. BACKGROUND: A 13.5-year-old girl presented with congenital-onset facial and neck weakness, slowly progressive severe limb girdle and axial myopathy, respiratory weakness, cardiomyopathy, progressive joint contractures, lumbar lordosis, progressive external ophthalmoplegia with ptosis, and cataracts. Muscle biopsy at 3 years revealed type I fiber predominance and hypotrophy, multicores with a focal decrease in mitochondria and oxidative enzymes, and internal nuclei. METHODS AND RESULTS: Serum carnitine was decreased (total, 18.2 micromol/L; free, 11.7 micromol/L). Urine organic acids intermittently revealed very large amounts of ethylmalonic and methylsuccinic acids intermittently, with elevated butyrylglycine, 2-methylbutyrylglycine, and tiglylglycine. Fibroblast acylcarnitine profiles revealed marked butyrylcarnitine elevation. Electron-transferring flavoprotein-linked reduction enzymatic assay of fibroblasts with butyryl-coenzyme A (CoA) as substrate, after immunoinactivation of medium-chain acyl-CoA dehydrogenase activity, revealed a complete absence of short-chain acyl-CoA dehydrogenase (SCAD) activity. No SCAD protein was detectable with Western blot analysis. CONCLUSIONS: This patient expands the clinical phenotype of SCAD deficiency and emphasizes the need for its consideration in the differential diagnosis of progressive external ophthalmoplegia and congenital multicore myopathy.