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Research Notes: Acylcarnitine Profiles in Prader-Willi Syndrome

Background. Almost all cells in the body, including neurons, have hundreds to thousands of little organelles called mitochondria where carbohydrates, fats and amino acids are oxidized ("burned") to produce the energy the cell needs to carry out its functions such as contraction (muscle), sending signals for thinking and movement to happen, making hormones, and so forth. Carnitine is a key substance that transports fatty acids into mitochondria so they can be used to produce energy and also removes the residue ("ashes") left over from the oxidation process so they don't build up within the mitochondria. When carnitine binds with a fatty acid or other residue from oxidation, the result is called an acylcarnitine. There are different kinds of acylcarnitines, depending on what type of residue the carnitine is bound to. An acylcarnitine profile shows how much of each type of acylcarnitine there is in the blood or urine and is typically used to help diagnose certain inborn errors in metabolism. For example, the heel prick blood spot test performed under state-mandated newborn screening programs is in part an acylcarnitine profile designed to rapidly identify inborn errors of metabolism so that potentially life-saving treatment can be immediately begun.

None of the genes affected in Prader-Willi syndrome (PWS) are known to carry the instructions for making the enzymes needed for proper mitochondrial function and cellular energy metabolism. However, there is a binding site in the PWS region on chromosome 15 that a gene from another chromosome, nuclear respiratory factor-1 (NRF-1), is known to interact with. NRF-1 is known to play a role in the regulation of mitochondrial function and energy metabolism (that is, how the cells produce the energy they need to function). As a result, it is possible that mitochondrial function and energy metabolism are impaired in PWS. The fact that many of the known inborn errors of metabolism present with symptoms that are strikingly similar to those in PWS, including hypotonia, muscle weakness, lethargy, poor feeding during infancy and failure to thrive, developmental delays, cognitive impairment, respiratory problems including apnea, and dysmorphic facial features also suggests that impaired energy metabolism might be a factor in PWS. Such an impairment would not be any of the identified inborn errors of metabolism and so probably would not show up in any of the state-mandated newborn screening programs (which are highly automated via the use of pattern-matching computer algorithms) because any problem with energy metabolism that might exist in PWS would have its own distinct pattern of acylcarnitines. However, acylcarnitine profiles of those with PWS are likely to be useful for determining if, in fact, there are problems in energy metabolism in PWS and, if so, the nature of those problems.

The chart below is the acylcarnitine profile for a 11.5-month-old boy genetically diagnosed with PWS due to deletion and is provided here with the consent of the mother. At the time of the blood draw, the infant was healthy aside from PWS and had been fed an hour before (i.e., he was not in a fasting state). Analysis of the profile is complicated by the fact that the infant was receiving a modest amount of L-carnitine in his formula (2 mg/100 calories), as well as the lack of other laboratory findings (including whether or not ketosis was present). However, taking into account those limitations as well as the presence of low serum glucose (see Lab Results, "Harry") and the overall clinical profile, the acylcarnitine profile suggests:

  • significantly impaired glucose oxidation (glycolysis and pyruvate metabolism) with a resulting shift to fatty acid oxidation, which in turn is also possibly impaired and may be hypoketotic (given the encephalopathic symptomology characteristic of the presentation of PWS in the neonatal period and early infancy).
  • secondary carnitine deficiency, possibly secondary to impaired fatty acid beta- and omega-oxidation.
  • a metabolic state that mimics methylmalonic acidemia, propionic acidemia, and multiple carboxylase deficiency/biotinidase deficiency, as evidenced by increased formation of propionylcarnitine (C3).
  • a metabolic state that mimics short chain hydroxy acyl-CoA dehydrogenase deficiency, as evidenced by increased formation of hydroxybutyrylcarnitine (C4-OH).
  • a metabolic state that mimics isovaleric acidemia, as evidenced by increased formation of isovalerylcarnitine (C5).
  • a metabolic state that mimics 3-hydroxy-3-methylglutaryl-CoA lyase deficiency, as evidenced by increased formation of 3-OH-isovalerylcarnitine (C5-OH) and adipylcarnitine/methylglutarylcarnitine (C6-DC). Note that per Metagene, 3-hydroxy-3-methylglutaryl-CoA lyase deficiency is the only inborn error of metabolism in which the acylcarnitine profile shows elevated adipylcarnitine/methylglutarylcarnitine (C6-DC).
  • possible impairment of fatty acid omega-oxidation.
  • possible impaired metabolism of valine, isoleucine, methionine, threonine and leucine.
  • possible impaired metabolism of suberic acid, as evidenced by the increased formation of suberylcarnitine (C8-DC).

Note that the above tentative interpretation needs to be confirmed by acylcarnitine profiles from others with PWS and further testing (e.g., urinary organic acids).

The fact that the infant was not sick or fasting at the time of the blood draw is important to note because many inborn errors of metabolism do not become symptomatic unless the person is under metabolic stress such as can occur during even normally simple illnesses or in a fasting state (e.g., during night-time sleep or when there is a loss of appetite during illness), and the same may occur in PWS due to disturbed energy metabolism. The numerous reports of rapid cardiorespiratory deterioration and sepsis-like conditions in PWS patients during simple illnesses and following surgery suggest that metabolic decompensation secondary to impaired energy metabolism may occur in PWS, and analysis of acylcarnitine profiles from blood draws taken during illness and the perisurgical period may help in understanding the reasons for such events.

Acylcarnitine Species Value Reference
Range 		Interpretation
acetylcarnitine (C2) 18.61 <11.75 * High
* elevated C2 and C4-OH can be seen in ketosis, however the infant was not in a fasting state so the elevated C2 suggests a shift in the utilization of CoA from pyruvate oxidation to fatty acid oxidation
* although ketosis may be present, a likely explanation for at least part of the elevated C2 is carnitine's role as an acetyl group acceptor from acetyl-CoA and thus the formation of acetylcarnitine, a reaction catalyzed by carnitine acetyl-transferase. A high acetyl-CoA/CoA ratio due to an increased reliance on fatty acid oxidation inhibits pyruvate dehydrogenase (PDH) and therefore glucose oxidation. [ cite ]
* also note that elevated C2 is a prominent feature of 3-hydroxy-3-methylglutaryl-CoA lyase deficiency. [ cite ]
propionylcarnitine (C3) 0.55 <0.59 * High normal
* elevated in methylmalonic acidemias and propionic acidemia
* possibly elevated in multiple carboxylase deficiency/biotinidase deficiency
propenoylcarnitine (C3:1) <0.02 <0.02 Normal
isobutyrylcarnitine (C4) 0.20 <0.29 Normal
methylmalonylcarnitine (C4-DC) <0.02 <0.03 Normal
hydroxybutyrylcarnitine (C4-OH) 0.09 <0.03 * High
* elevated C4-OH and C2 can be seen in ketosis, however the infant was not in a fasting state which suggests a shift in the utilization of CoA from pyruvate oxidation to fatty acid oxidation
* elevated in short chain hydroxy acyl-CoA dehydrogenase deficiency
* elevated in SUCLA2 mutation leading to mitochondrial DNA depletion, succinyl-CoA synthase deficiency in the Kreb's cycle (a distal step of the methylmalonic acid pathway), and mild methylmalonic aciduria. [ cite ]
isovalerylcarnitine (C5) 0.22 <0.25 * High normal
* elevated in fasting
* elevated in isovaleric acidemia
tiglyl-methylcrotonylcarnitine (C5:1) <0.02 <0.02 Normal
3-OH-isovalerylcarnitine (C5-OH) 0.04 <0.03 * High
* elevated in 3-hydroxy-3-methylglutaryl-CoA lyase deficiency and multiple carboxylase deficiency/biotinidase deficiency
glutarylcarnitine (C5-DC) 0.02 <0.06 Normal
hexanoylcarnitine (C6) 0.08 <0.12 Normal
adipylcarnitine/methylglutarylcarnitine (C6-DC) 0.03 <0.02 * High
* beta-oxidation product of C-8
* suggests increased fatty acid omega-oxidation
* elevated in 3-hydroxy-3-methylglutaryl-CoA lyase deficiency
3-OH-hexanolylcarnitine (C6-OH) <0.02 <0.02 Normal
octanoylcarnitine (C8) 0.15 <0.25 Normal
octenoylcarnitine (C8:1) 0.42 0.81 Normal
suberylcarnitine (C8-DC) 0.02 <0.02 High
elevated in fasting
decanoylcarnitine (C10) 0.14 <0.39 Normal
decenolylcarnitine (C10:1) 0.02 <0.43 Low normal - suggestive of impaired medium chain fatty acid/capric acid uptake and oxidation.
dodecanolylcarnitine (C12) 0.05 <0.26 Low normal - suggestive of impaired medium chain fatty acid/lauric acid uptake and oxidation.
dodecenolycarnitine (C12:1) 0.02 <0.24 Low normal - suggestive of impaired medium chain fatty acid/lauric acid uptake and oxidation.
3-OH-dodecanolylcarnitine (C12-OH) <0.02 <0.02 Normal
tetradecanoylcarnitine (C14) 0.03 <0.05 Normal
tetradecenolycarnitine (C14:1) <0.02 <0.02 Normal
tetradecadienoylcarnitine (C14:2) 0.02 <0.12 Low normal - suggestive of impaired medium chain fatty acid/myristic acid uptake and oxidation.
3-OH-tetradecanoylcarnitine (C14-OH) <0.02 <0.02 Normal
3-OH-tetradecenoylcarnitine (C14:1-OH) <0.02 <0.02 Normal
hexadecanoylcarnitine (C16) 0.09 <0.15 Normal
3-OH-hexadecenolylcarnitine (C16:1) <0.02 <0.06 Normal
3-OH-hexadecenolylcarnitine (C16:1-OH) <0.02 <0.02 Normal
stearoylcarnitine (C18) 0.04 <0.09 Normal
oleoylcarnitine (C18:1) 0.16 <0.27 Normal
linoleoylcarnitine (C18:2) 0.09 <0.14 Normal
3-OH-oleoylcarnitine (C18:1-OH) <0.02 <0.02 Normal
3-OH-linoleoylcarnitine (C18:2-OH) <0.02 <0.02 Normal

Notes about the acylcarnitine profile:

  • The classic initial presentation of primary carnitine deficiency is hypoketotic hypoglycemic encephalopathy, accompanied by hepatomegaly, elevated liver transaminases, and hyperammonemia. Encephalopathy during the neonatal phase of PWS is obvious, but elevated liver transaminases and hyperammonemia have also both been reported in the neonatal presentation of PWS, which further suggests the existence of a functional or secondary carnitine deficiency in PWS.
  • Per eMedicine, patients with organic acidemias causing secondary carnitine deficiency may present with crises consisting of hypoglycemia, ketoacidosis, and hyperammonemia.
  • Per eMedicine, patients with respiratory chain defects or mitochondrial disorders and secondary carnitine deficiency may present with abnormal fatigability and lactic acidosis associated with exertion. These children also may present with encephalopathy and/or lipid storage myopathy and carnitine depletion. Carnitine deficiency has been observed in children with urea cycle defects and may exacerbate episodes of hyperammonemia.
  • From eMedicine:
    • "Secondary carnitine deficiency, which manifests with a decrease of carnitine levels in plasma or tissues, may be associated with genetically determined metabolic conditions, acquired medical conditions, or iatrogenic states.
      • "Disorders of the carnitine cycle or disorders of fatty acid beta-oxidation can cause secondary carnitine deficiency via several mechanisms. Block in fatty acid oxidation contributes to the accumulation of acyl-CoA intermediates. Transesterification with carnitine leads to the formation of acylcarnitine and the release of free CoA. These acylcarnitines are excreted readily in the urine. They inhibit carnitine uptake at the level of the carnitine transporter in renal cells, causing increased carnitine losses in the urine and systemic secondary depletion of carnitine.
      • "Other genetic conditions that are associated with Fanconi syndrome (eg, Lowe syndrome, cystinosis) may present with secondary carnitine deficiency because of increased renal losses of carnitine. Lysinuric protein intolerance is associated with an increased excretion of lysine in the urine, and the biosynthesis of carnitine needs lysine. Other metabolic disorders (eg, propionic acidemia, methylmalonic acidemia) also may present with secondary carnitine deficiency. Secondary carnitine deficiency also may be observed in respiratory chain defects.
      • Aminoacidopathies (eg, isovaleric acidemia, propionic acidemia, methylmalonic acidemia, glutaric acidemia type I, 3-hydroxymethylglutaryl-CoA lyase deficiency) also contribute to the accumulation of acyl-CoA intermediates at the site of the metabolic block. This occurs with the formation of acylcarnitine esters, which are transported out of the cell and excreted in the urine. The decreased threshold for carnitine excretion causes low total carnitine levels in plasma and tissue.
      • "Carnitine deficiency has been observed in children with urea cycle defects (eg, ornithine transcarbamylase deficiency, carbamoyl phosphate synthetase deficiency). It is unclear whether carnitine deficiency is related to the primary metabolic defect, to the concomitant liver disease observed in the initial presentation, or to benzoate therapy.
      • "Carnitine deficiency is observed in disorders of the mitochondrial respiratory chain, such as cytochrome c oxidase deficiency, in which the ATP depletion may compromise the energy-dependent carnitine uptake. An interference with carnitine transport occurs in tissues, including renal reabsorption, which explains the low plasma and tissue levels in these patients.
      • "Other inborn errors of metabolism or genetic disorders may cause secondary carnitine deficiency because of impairment of carnitine biosynthesis secondary to increased urinary losses of lysine, which occurs in lysinuric protein intolerance. Increased urinary loss of carnitine associated with Fanconi syndrome may be observed in syndromes such as cystinosis or Lowe syndrome (ie, X-linked oculocerebrorenal syndrome)."
  • Unfortunately, it is unknown whether or not ketosis was present at the time of the blood draw. However, it is known that the infant's formula contained L-carnitine and, as noted in the chart, a likely explanation for at least part of the elevated acetylcarnitine (C2) is carnitine's role as an acetyl group acceptor from acetyl-CoA and thus the formation of acetylcarnitine, a reaction catalyzed by carnitine acetyl-transferase. A high acetyl-CoA/CoA ratio due to an increased reliance on fatty acid oxidation inhibits pyruvate dehydrogenase (PDH) and therefore glucose oxidation, so the high C2 indicates that the exogenous carnitine is facilitating a shift back to more energy efficient glucose oxidation via a reduction of the acetyl-CoA/CoA ratio. [ cite ]
  • Propionylcarnitine (C3) is high normal and is also high in methylmalonic acidemias (MMA) in which metabolism of the amino acids isoleucine, methionine, threonine, and valine is impaired. In about 50% of the cases of MMA, the impairment is caused by a problem in the synthesis of adenosylcobalamin (AdoCbl, a derivative of vitamin B12) which works with methylmalonyl CoA mutase to convert methylmalonyl CoA to succinyl CoA, an intermediate (substrate) in the Kreb's cycle. Of interest in that regard is an anecdotal report that methylcobalamin injections per the autism protocol developed by James A. Neubrander, M.D. have been beneficial for a boy with PWS. (Note that succinyl-CoA is also needed within mitochondria for heme synthesis, the activation of acetoacetate by CoA transfer, and ketone body activation via succinyl-CoA: 3-ketoacid CoA-transferase [ cite 1, cite 2 ].) In the beta-oxidation of odd-chain fatty acids, the final products are propionyl CoA and acetyl CoA, with the propionyl CoA then being converted to succinyl CoA. Interestingly, isovalerylcarnitine (C5) is also high normal in the profile, which suggests a problem with the metabolizing of odd-chain fatty acids.
  • For clinicians treating those with PWS, eMedicine has an excellent review of the lab studies needed for the diagnosis of primary and secondary carnitine deficiencies:
    • "Immediately check blood glucose and urine ketones if a child presents to the emergency room with lethargy, seizures, apnea, or any episode of decreased consciousness. The absence or low amounts of ketones in the urine, combined with the episode of hypoglycemia in primary carnitine deficiency (as well as in other defects in the carnitine cycle or fatty acid oxidation), causes secondary carnitine deficiency.
    • "Obtain ammonia level, liver enzymes (ie, aspartate aminotransferase [AST], alanine aminotransferase [ALT], glutamyltransferase [GGT]), chemistry panel, uric acid, creatine kinase (CK), lactic acid, and coagulation tests.
      • "Ammonia levels can be moderately elevated, especially in primary carnitine deficiency and particularly if the child has a presentation similar to that of Reye syndrome.
      • "Transaminases usually are moderately elevated in primary carnitine deficiency.
      • "In some defects of the carnitine cycle that cause secondary carnitine deficiency (eg, CPT-II deficiency), a hepatocardiomuscular form can present with liver involvement. Other fatty acid oxidation disorders, such as LCHAD deficiency, can present with liver involvement.
      • "A chemistry panel may show evidence of metabolic acidosis.
      • "Hyperuricemia may be present in carnitine deficiency because carnitine competes for renal tubular excretion.
      • "Elevated serum CK levels may be observed in primary carnitine deficiency and in fatty acid oxidation disorders.
      • "Elevated lactate can be observed in respiratory chain defects or in LCHAD deficiency.
      • "Altered coagulation with prolonged prothrombin time may be found.
    • "Plasma carnitine: In primary carnitine deficiency, the carnitine level in plasma usually is less than 5% of normal, with acylcarnitines proportionately reduced. The ratio between acylcarnitine and free carnitine is normal. A feature of most fatty acid oxidation disorders is that they are associated with decreased plasma carnitine concentrations. This feature also is observed in other inborn errors of metabolism that cause secondary carnitine deficiency, such as organic acidemias caused by the formation of carnitine esters.
    • "Urine carnitine: This is only useful in primary carnitine deficiency in which the transporter in kidney cells has decreased capacity for reabsorption, causing increased carnitine excretion.
    • "Newborn screen: Recently, several patients with primary carnitine deficiency have been ascertained through newborn screening programs. In these cases, the acylcarnitine profile reveals a low level of free carnitine and all acylcarnitine species. However, plasma carnitine levels can be within the reference range if obtained too early, due to the transfer of carnitine through the placenta to the fetus.
    • "Urine organic acids: In primary carnitine deficiency, the urine organic acid analysis usually is normal. In cases of fatty acid oxidation disorders that cause secondary carnitine deficiency, inappropriate dicarboxylic aciduria occurs during periods of illness. Urinary organic acid profile usually is normal in these patients when they are well, except in cases of MCAD deficiency. In some disorders (eg, MCAD, LCHAD, short-chain acyl-CoA dehydrogenase [SCAD] deficiency) specific patterns can be seen. It is very important to collect this specimen during illness.
    • "Urine acylglycines: In MCAD deficiency, the urine contains increased amounts of glycine conjugates. The test also may be used in individuals with suspected glutaric aciduria type II or SCAD deficiency.
    • "Acylcarnitine profile and free fatty acids: Tandem mass spectrometry analyses of acylcarnitine profile and free fatty acids may be used to detect metabolic defects that cause secondary carnitine deficiency (eg, fatty acid oxidation disorders, organic acidemias) because acyl-CoA intermediates proximal to the block in fatty acid or amino acid oxidative pathway may be transesterified to carnitine. Modest amounts of long-chain 3-hydroxy fatty acids consistently are found in the plasma of patients with LCHAD deficiency, even if these patients are asymptomatic.
    • "In a fasting test, patients undergo a controlled and prolonged fast under strict medical supervision. Take blood samples at regular intervals to measure glucose, ketone bodies, and free fatty acids. Acylcarnitine profile may be obtained at the same time. Fasting may be continued in children for up to 24 hours, unless blood glucose drops to less than 3 mmol/L. An inadequate production of ketones with a high free fatty acid–to–ketone bodies ratio suggests a defect in long-chain fatty acid oxidation.
    • "Fatty acid oxidation studies in fibroblasts are used if a fatty acid oxidation defect is suspected clinically. The most appropriate first line of investigation in these patients is to study the entire fatty acid oxidation pathway. Methods involve (1) monitoring the rate of production of radioactive end products of fatty acid oxidation disorders for radiolabeled precursor fatty acids or (2) measuring by tandem mass spectrometry the disease-specific acylcarnitines produced when stable isotope fatty acid precursors are incubated with cells in the presence of excess L-carnitine.
    • "Enzyme assay, the criterion standard for demonstrating an enzyme defect, measures the activity in cultured fibroblasts or in some other tissue, such as muscle or liver. To account for the frequent finding of overlapping chain-length specificities, complex analysis using a mixture of different chain-length substrates and immunoprecipitation with antibodies to different enzymes is required.
    • "Carnitine transport assay in cultured fibroblasts specifically demonstrates the absence of active carnitine transport in cultured fibroblasts. This finding is specific for primary carnitine deficiency."

Clin Chim Acta. 2003 Nov.
A comparison of in vitro acylcarnitine profiling methods for the diagnosis of classical and variant short chain acyl-CoA dehydrogenase deficiency.
Young SP, Matern D, Gregersen N, Stevens RD, Bali D, Liu HM, Koeberl DD, Millington DS.
Department of Pediatrics, Duke University Medical Center, Durham, NC, USA.

BACKGROUND: Homozygosity and compound heterozygosity for the short chain acyl-CoA dehydrogenase (SCAD) gene sequence variants 625G-->A and 511C-->T are associated with ethylmalonic aciduria (EMA), a biochemical indicator of SCAD deficiency. The clinical and biochemical implications of these variants are not fully understood. The effect of these variants on the accumulation of butyrylcarnitine by fibroblasts in culture was studied. METHODS: In vitro acylcarnitine profiling in fibroblasts was carried out using [U-13C]-labeled or unlabeled palmitate in the presence of excess L-carnitine, with or without a medium chain acyl-CoA dehydrogenase (MCAD) inhibitor. Acylcarnitines were analyzed using tandem mass spectrometry. 625G/625G (wild type), 625G/625A and 625A/625A (variant) control fibroblasts were compared with fibroblasts from patients homozygous for inactivating SCAD mutations (SCAD deficient) and from patients with EMA who were homozygous or compound heterozygous for the SCAD variants. RESULTS: Variant control and patient fibroblasts accumulated moderate amounts of butyrylcarnitine compared with wild-type controls and in contrast to the significant amount of butyrylcarnitine accumulated by SCAD deficient fibroblasts, regardless of incubation conditions. CONCLUSIONS: Moderately reduced SCAD activity associated with SCAD variants can be detected using in vitro acylcarnitine profiling methods, which may be used as an indirect measure of SCAD activity.

Pediatr Res. 2003 Aug.
Rare disorders of metabolism with elevated butyryl- and isobutyryl-carnitine detected by tandem mass spectrometry newborn screening.
Koeberl DD, Young SP, Gregersen NS, Vockley J, Smith WE, Benjamin DK Jr, An Y, Weavil SD, Chaing SH, Bali D, McDonald MT, Kishnani PS, Chen YT, Millington DS.
Division of Medical Genetics, DUMC 3528, Bell Building Room 237, Trent Duke University Medical Center, Durham, NC, U.S.A.

Tandem mass spectrometry was adopted for newborn screening by North Carolina in April 1999. Since then, three infants with short-chain acyl-CoA dehydrogenase (SCAD) and one with isobutyryl-CoA dehydrogenase deficiency were detected on the basis of elevated butyrylcarnitine/isobutyrylcarnitine (C4-carnitine) concentrations in newborn blood spots analyzed by tandem mass spectrometry. For three SCAD-deficient infants, biochemical evaluation included a plasma acylcarnitine profile with markedly elevated C4-carnitine, urine organic acid analysis with markedly elevated ethylmalonic and 2-methylsuccinic acids, and markedly elevated [U-13C]butyrylcarnitine concentrations in medium from fibroblasts incubated with [U-13C]palmitic acid and excess l-carnitine, consistent with classic SCAD deficiency. Two of three infants diagnosed with classic SCAD deficiency remained asymptomatic; however, the third infant presented with seizures and a cerebral infarct at 10 wk of age. All three infants had putatively inactivating mutations in both alleles of the SCAD gene. The highly elevated plasma C4-carnitine levels in the three infants detected by newborn screening tandem mass spectrometry differentiated them from infants and children who were homozygous or compound heterozygous for one of two SCAD gene susceptibility variations; for the latter group the C4-carnitine levels were normal. Isobutyryl-CoA dehydrogenase deficiency in a fourth infant was confirmed after isolated elevation of C4-carnitine in the acylcarnitine profile.

J Chromatogr B Analyt Technol Biomed Life Sci. 2003 Jul 15.
Selective screening for fatty acid oxidation disorders by tandem mass spectrometry: difficulties in practical discrimination.
Shigematsu Y, Hirano S, Hata I, Tanaka Y, Sudo M, Tajima T, Sakura N, Yamaguchi S, Takayanagi M.
Department of Health Science, School of Nursing, Fukui Medical University, Matsuoka-cho, Fukui, Japan.

In a selective screening for fatty acid oxidation disorders by tandem mass spectrometry, we tested the diagnostic ratios and acylcarnitine concentrations in sera or blood spots, which were reported to be specific to very long-chain acyl CoA dehydrogenase deficiency, carnitine palmitoyltransferase I deficiency, and carnitine palmitoyltransferase II deficiency. While the acylcarnitine profiles in the majority of these patients were typical in the respective disorders, some overlapping of the indices was observed between these patients and the infants, who showed symptoms mainly related to hypoglycemia but did not have the disorders mentioned above. Although the diagnostic ratio of tetradecenoylcarnitine to dodecanoylcarnitine for very long-chain acyl CoA dehydrogenase deficiency seemed to minimize the overlapping in this study, additional measures including careful assessment of clinical data and enzyme assays may be necessary for the diagnosis in atypical cases.

J Chromatogr B Analyt Technol Biomed Life Sci. 2003 Jul 15.
Effect of supplementation with L-carnitine at a small dose on acylcarnitine profiles in serum and urine and the renal handling of acylcarnitines in a patient with multiple acyl-coenzyme A dehydrogenation defect.
Yoshino M, Tokunaga Y, Watanabe Y, Yoshida I, Sakaguchi M, Hata I, Shigematsu Y, Kimura M, Yamaguchi S.
Department of Pediatrics and Child Health, Kurume University School of Medicine, 67 Asahi-machi, Kurume, Japan.

We studied the effects of L-carnitine supplementation at a small dose on the profiles of acylcarnitines in serum and urine, as well as the renal handling of acylcarnitines, in a patient with multiple acyl-coenzyme A dehydrogenation defect. After supplementation with L-carnitine at a dose of 20 mg/kg/day, the concentration of each acylcarnitine measured both in the serum and in the urine had increased significantly, with the exception of that of an acylcarnitine with a carbon chain length (C) of 8 (C8 acylcarnitine). The magnitude of increase in the concentrations of the acylcarnitines in the serum was not associated with chain length, whereas in the urine, the magnitude tended to be greater in proportion to the shortness of the chain length. The fractional excretions of C2-C5 acylcarnitines exceeded 100%, indicating that they were produced in, or transported across, renal tubular epithelial cells and secreted into the urine. These results indicate that supplementation with a relatively small amount of L-carnitine can enhance the renal excretion of accumulated short-chain-length acylcarnitines through tubular excretion, in addition to basic glomerular filtration.

Metabolism. 2002 Mar.
Acylcarnitine profiles in fibroblasts from patients with respiratory chain defects can resemble those from patients with mitochondrial fatty acid beta-oxidation disorders.
Sim KG, Carpenter K, Hammond J, Christodoulou J, Wilcken B.
New South Wales Biochemical Genetics Service, Western Sydney Genetics Program, The Children's Hospital at Westmead, Sydney, Australia.

Mitochondrial fatty acid beta-oxidation (FAO) is coupled to the respiratory chain (RC). Functional defects of one pathway may lead to secondary alteration in flux through the other. We investigated the acylcarnitine profiles in cultured fibroblasts obtained from 14 healthy subjects, 31 patients with 8 different primary enzyme deficiencies of FAO, and 16 patients with primary RC defects including both isolated and multiple enzyme complex defects. Intact cells were incubated in media containing deuterium-labeled hexadecanoic acid and L-carnitine, and the acylcarnitines analysed using an electrospray tandem mass spectrometer. All FAO-deficient cell lines revealed disease-specific acylcarnitine profiles related to the sites of defects. Some cell lines from patients with RC defects showed profiles similar to those of controls, whereas others had abnormal profiles mimicking those found in FAO disorders. The acylcarnitine profiles of patients with RC enzyme defects were not predictable, and in some patients defects caused by mutations in either nuclear-encoded or mitochondrial DNA were associated with acylcarnitine abnormalities. While in vitro acylcarnitine profiling is useful for the diagnosis of FAO deficiencies, abnormal profiles do not exclusively indicate these disorders, and primary defects of the RC remain a possibility. Awareness of this diagnostic pitfall will aid in the selection of subsequent confirmatory tests and therapeutic options.

Mol Genet Metab. 1998 Dec.
Isolated isobutyryl-CoA dehydrogenase deficiency: an unrecognized defect in human valine metabolism.
Roe CR, Cederbaum SD, Roe DS, Mardach R, Galindo A, Sweetman L.
Institute of Metabolic Disease, Baylor University Medical Center, Dallas, Texas.

A 2-year-old female was well until 12 months of age when she was found to be anemic and had dilated cardiomyopathy. Total plasma carnitine was 6 microM and acylcarnitine analysis while receiving carnitine supplement revealed an increase in the four-carbon species. Urine organic acids were normal. In vitro analysis of the mitochondrial pathways for beta oxidation, and leucine, valine, and isoleucine metabolism was performed in fibroblasts using stable isotope-labeled precursors to these pathways followed by acylcarnitine analysis by tandem mass spectrometry. 16-2H3-palmitate was metabolized normally down to the level of butyryl-CoA thus excluding SCAD deficiency. 13C6-leucine and 13C6-isoleucine were also metabolized normally. 13C5-valine incubation revealed a significant increase in 13C4-isobutyrylcarnitine without any incorporation into propionylcarnitine as is observed normally. These same precursors were also evaluated in fibroblasts with proven ETF-QO deficiency in which acyl-CoA dehydrogenase deficiencies in each of these pathways was clearly identified. These results indicate that in the human, there is an isobutyryl-CoA dehydrogenase which exists as a separate enzyme serving only the valine pathway in addition to the 2-methyl branched-chain dehydrogenase which serves both the valine and the isoleucine pathways in both rat and human.

Biochem Biophys Res Commun. 1995 Aug 24.
Fatty acid beta-oxidation in peroxisomes and mitochondria: the first, unequivocal evidence for the involvement of carnitine in shuttling propionyl-CoA from peroxisomes to mitochondria.
Jakobs BS, Wanders RJ.
University of Amsterdam, Department of Paediatric Clinical Chemistry, The Netherlands.

We have investigated how [1-14C]propionyl-CoA, which is the first product of the peroxisomal beta-oxidation of [1-14C] pristanic acid, is transported to mitochondria for further oxidation in human skin fibroblasts from patients with a defect in the mitochondrial carnitine/acylcarnitine translocase and carnitine-palmitoyltransferase II (CPT II) (EC 2.3.1.21), respectively. Oxidation of pristanic acid was found to be partially deficient in both types of mutant cells. More important, 14CO2 production was completely deficient in the carnitine/acylcarnitine translocase deficient cells but not in the carnitine-palmitoyltransferase II deficient cells. These results strongly suggest that formation of 14CO2 in the Krebs cycle from [1-14C]propionyl-CoA as generated in peroxisomes requires the active participation of the mitochondrial carnitine/acylcarnitine translocase. The results described in this paper provide the first evidence suggesting that propionyl-CoA leaves the peroxisome as a carnitine ester and strongly suggest that the commonly accepted concept that peroxisomal beta-oxidation is not dependent on carnitine is incorrect.

Acta Paediatr Jpn. 1994 Apr.
A study of urinary metabolites in patients with dicarboxylic aciduria for differential diagnosis.
Shimizu N, Yamaguchi S, Orii T.
Department of Pediatrics, Gifu University School of Medicine, Japan.

Dicarboxylic aciduria (DCA-uria) is a relatively common finding in the screening of organic acidemias by gas chromatography/mass spectrometry (GC/MS). A considerable number of patients with DCA-uria are involved in disturbances of mitochondrial and peroxisomal fatty acid beta-oxidation. The differential diagnosis of DCA-uria was investigated using a combination of organic acid analysis by GC/MS, carnitine determination, acylcarnitines by fast atom bombardment/mass spectrometry (FAB/MS) and acylglycines by stable-isotope dilution analysis. The relative distribution of urinary metabolites was examined in 46 patients with DCA-uria of different origins, including physiological ketosis of childhood, disorders of propionic acid metabolism, glutaric aciduria type II, Zellweger syndrome and patients who were clinically diagnosed as having Reye syndrome. Zellweger syndrome seemed to be distinguishable from other disorders by the high sebacic acid/adipic acid ratio of DCA-uria and increased excretion of 4-hydroxyphenyllactic acid and 2-hydroxysebacic acid. The mild form of glutaric aciduria type II was often missed by current organic acid analysis alone, but was readily diagnosed by acylcarnitine and acylglycine determination. The ratio of free/total carnitine was low in most of the DCA-uria patients except for two of five cases of Zellweger syndrome and one of three cases of Reye syndrome. The acylcarnitine analysis by FAB/MS showed adipyl-, suberyl-, sebacyl- or dodecanedioylcarnitine as major peaks in most of these patients, although these were not specific. Disease-specific peaks were detectable only in congenital organic acidemias such as glutaric aciduria type II, methylmalonic acidemia and propionic acidemia.

J Inherit Metab Dis. 1994.
Acylcarnitine profile in tissues and body fluids of biotin-deficient rats with and without L-carnitine supplementation.
Shigematsu Y, Bykov IL, Liu YY, Nakai A, Kikawa Y, Sudo M, Fujioka M.
Department of Pediatrics, Fukui Medical School, Japan.

Since biotin-deficient (BD) rats are a good animal model for human multiple carboxylase deficiency and have low plasma free carnitine levels, short-chain acylcarnitine profiles in biotin-deficient rats with L-carnitine supplementation (BDC rats) and BD rats were investigated by fast-atom bombardment and tandem mass spectrometry and gas chromatography/mass spectrometry. By the latter method, 3-hydroxyisovalerylcarnitine was identified in BD rats, and showed the greatest accumulation among short-chain acylcarnitines in tissues of BD rats, while the tissue levels of propionic acid were more markedly elevated than those of 3-hydroxyisovaleric acid. The tissue levels of 3-hydroxyisovaleryl-carnitine were significantly lower and those of propionyl-carnitine were somewhat higher in BDC rats than in BD rats, while the tissue levels of propionic acid and 3-hydroxyisovaleric acid in BDC rats were lower than those in BD rats. These changes were more apparent in kidney than in other tissues. The amounts of urinary excretion of acylcarnitines were markedly larger, and those of 3-hydroxyisovaleric acid were somewhat smaller in BDC rats than in BD rats, while those of propionic acid were very low in BD and BDC rats as compared with those of 3-hydroxyisovaleric acid. It seems that the relationship between the concentrations of 3-hydroxyisovalerylcarnitine and those of propionylcarnitine reflects the unique metabolism of the related metabolites in tissues, especially in kidney, which may be influenced by their urinary excretion and the availability of free carnitine. These data in biotin deficiency suggest that carnitine supplementation is possibly beneficial for patients with holocarboxylase synthetase deficiency who respond incompletely to biotin therapy.

Am J Hum Genet. 1993 May.\\ Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency: diagnosis by acylcarnitine analysis in blood.
Van Hove JL, Zhang W, Kahler SG, Roe CR, Chen YT, Terada N, Chace DH, Iafolla AK, Ding JH, Millington DS.
Department of Pediatrics, Duke University Medical Center, Durham, NC.

Medium-chain acyl-coenzyme A dehydrogenase (MCAD) deficiency is a disorder of fatty acid catabolism, with autosomal recessive inheritance. The disease is characterized by episodic illness associated with potentially fatal hypoglycemia and has a relatively high frequency. A rapid and reliable method for the diagnosis of MCAD deficiency is highly desirable. Analysis of specific acylcarnitines was performed by isotope-dilution tandem mass spectrometry on plasma or whole blood samples from 62 patients with MCAD deficiency. Acylcarnitines were also analyzed in 42 unaffected relatives of patients with MCAD deficiency and in other groups of patients having elevated plasma C8 acylcarnitine, consisting of 32 receiving valproic acid, 9 receiving medium-chain triglyceride supplement, 4 having multiple acyl-coenzyme A dehydrogenase deficiency, and 8 others with various etiologies. Criteria for the unequivocal diagnosis of MCAD deficiency by acylcarnitine analysis are an elevated C8-acylcarnitine concentration (> 0.3 microM), a ratio of C8/C10 acylcarnitines of > 5, and lack of elevated species of chain length > C10. These criteria were not influenced by clinical state, carnitine treatment, or underlying genetic mutation, and no false-positive or false-negative results were obtained. The same criteria were also successfully applied to profiles from neonatal blood spots retrieved from the original Guthrie cards of eight patients. Diagnosis of MCAD deficiency can therefore be made reliably through the analysis of acylcarnitines in blood, including presymptomatic neonatal recognition. Tandem mass spectrometry is a convenient method for fast and accurate determination of all relevant acylcarnitine species.

Biochem Pharmacol. 1991 Mar 15-Apr 1.
Inhibition of hepatic propionyl-CoA synthetase activity by organic acids. Reversal of propionate inhibition of pyruvate metabolism.
Krahenbuhl S, Brass EP.
Department of Medicine, Case Western Reserve University, Cleveland, OH.

Intracellular accumulation of propionyl-CoA is associated with impairment of important hepatic metabolic pathways. Since propionate absorbed from the intestine can be converted to propionyl-CoA in the liver, inhibition of propionyl-CoA synthesis from propionate and CoA may provide a strategy for decreasing toxicity from plasma propionate. Therefore, inhibition of propionyl-CoA formation by several organic acids was investigated. In isolated, solubilized mitochondria, octanoate, butyrate, salicylate and p-nitrobenzoate inhibited propionyl-CoA synthesis. Octanoate was the most potent inhibitor of propionyl-CoA synthetase activity and had a Ki of 58 microM. In isolated hepatocytes, octanoate inhibited propionate oxidation in a concentration-dependent manner. Consistent with previous studies, propionate (1.0 mM) inhibited the rates of 14CO2 formation from [1-14C]pyruvate (10 mM) to 55% of the control values in the hepatocyte system. Octanoate (0.8 mM) had no effect on [1-14C]pyruvate oxidation under control conditions, but increased 14CO2 formation from pyruvate to 88% of the control values in the presence of 1.0 mM propionate. Reversal of propionate inhibition of pyruvate oxidation by octanoate was associated with a 44% decrease in hepatocyte propionyl-CoA content. In contrast, while pyruvate oxidation rates were decreased to 53% of control rates in the presence of 10 mM propionylcarnitine, octanoate stimulated pyruvate oxidation under these conditions only to 67% of control levels. In conclusion, mitochondrial propionyl-CoA synthetase activity and hepatocyte propionyl-CoA accumulation can be inhibited by octanoate with consequent decreased propionate oxidation and toxicity in intact hepatocytes. The reversal by octanoate of propionate's inhibition of cellular metabolism may be useful in reducing tissue toxicity from circulating propionate.

Acta Paediatr Jpn. 1990 Aug.
Carnitine deficiency in inherited organic acid disorders and Reye syndrome.
Sugiyama N, Kidouchi K, Kobayashi M, Wada Y.
Department of Pediatrics, Nagoya City University, Medical School, Japan.

A large quantity of propionylcarnitine in the urine of patients with propionic acidemia and methylmalonic aciduria was demonstrated. The amount excreted depended on the administered L-carnitine dose from 25 to 75 mg/kg/day. A high level of propionylcarnitine was also detected in the amniotic fluid of fetuses at risk of methylmalonic aciduria. Glutaric aciduria type 1 was characterized by excessive urinary excretion of glutarylcarnitine. In a neonate with glutaric aciduria type 2, several specific acylcarnitines were detected in the urine. These included isovaleryl-, acetyl-, isobutyryl-, and butyrylcarnitine as major carnitine esters and glutaryl-, and octanoylcarnitine as minor components. However, the pattern of acylcarnitines excreted changed from isovalerylcarnitine (via leucine) to isobutyrylcarnitine (via valine) during early life. In patients diagnosed as Reye syndrome, tissue carnitine deficiency was not always recognized and no decrease in the free/total carnitine ratio was found in the liver or muscle. The clinical and pathophysiological manifestations seen in these disorders are considered to relate to mitochondrial activity. Therefore, it is necessary to measure acylcarnitine fractions in the urine in order to obtain more precise information about mitochondrial function because carnitine and acylcarnitine compounds may express the metabolic state of mitochondria.

Acta Paediatr Jpn. 1990 Feb.
Organic acid and acylcarnitine profiles of glutaric aciduria type I.
Matsumoto M, Matsumoto I, Shinka T, Kuhara T, Imamura H, Shimao S, Okada T.
Division of Human Genetics, Kanazawa Medical University, Ishikawa, Japan.

Urinary organic acid and acylcarnitine profiles from a 2-month-old boy were studied by gas chromatography-mass spectrometry and fast atom bombardment mass spectrometry. The patient excreted large amounts of glutaric acid and significant amounts of 3-hydroxyglutaric acid, glutaconic acid and glutarylcarnitine, and his serum glutaric acid level was markedly elevated. Thus he was chemically diagnosed as having glutaric aciduria type I (GAI). In addition to the above metabolites previously described in GAI, significantly increased excretion of 2-ketoglutaric acid, succinic acid, adipic acid, adipylcarnitine, suberic acid and azelaic acid was found. 2-Ketoadipic acid methylsuccinic acid and ethylmalonic acid were also detectable, suberylcarnitine was not increased, and dehydroadipylcarnitine was decreased in his urine. These results suggest that excess glutaryl-CoA causes the competitive inhibition of the dehydrogenation of adipyl-CoA to dehydroadipyl-CoA and results in an increase of adipic acid and adipylcarnitine and a decrease of dehydroadipylcarnitine. It is also suggested that oxidative decarboxylation of 2-ketoglutaric acid to succinyl-CoA is inhibited by high levels of glutaryl-CoA, and that the dehydrogenation of succinic acid to fumaric acid is inhibited owing to the increased glutaric acid derived from excess glutaryl-CoA. These results indicate that gas chromatography-mass spectrometry is the most appropriate and accurate method for the differential chemical diagnosis of GAI and glutaric aciduria type II.

J Nutr. 1989 Aug.
Effect of carnitine on propionate metabolism in the vitamin B-12-deficient rat.
Brass EP, Ruff LJ.
Department of Medicine, University of Colorado Health Sciences Center, Denver.

Acyl-CoA thioesters are generated during the oxidation of organic acids in mammalian systems. Vitamin B-12 deficiency is associated with decreased L-methylmalonyl-CoA mutase activity, and consequent accumulation of propionyl-CoA and methylmalonyl-CoA. The formation of propionylcarnitine from propionyl-CoA and carnitine provides an alternative pathway to remove propionyl-CoA from cells. Hepatocytes isolated from vitamin B-12-deficient rats metabolized propionate (1 mM) to CO2 and glucose at only 23% and 12%, respectively, of the rates observed in hepatocytes from control animals. In contrast, no difference was seen in rates of pyruvate metabolism by hepatocytes from control and vitamin B-12-mdeficient rats. Addition of carnitine (10 mM) to hepatocyte incubations increased the rate of propionylcarnitine formation 10- to 20-fold without altering conversion of propionate to CO2 or glucose. The rate of propionylcarnitine formation was not affected by vitamin B-12 deficiency. When carnitine (10 mM) was added, propionylcarnitine generation represented 65-71% of total propionate utilization in hepatocytes isolated from vitamin B-12-deficient rats. Gluconeogenesis from [1-14C]pyruvate was inhibited by 1 mM propionate in hepatocytes from vitamin B-12-deficient rats. No effect of 1 mM propionate on glucose formation from pyruvate was seen using hepatocytes from control rats. Intraperitoneal administration of L-carnitine resulted in a significant increase in urinary propionylcarnitine excretion from vitamin B-12-deficient rats, but not from control animals. The results demonstrate that exogenous carnitine can significantly enhance propionyl-group utilization via the formation of acylcarnitines under the conditions of impaired acyl-CoA metabolism associated with vitamin B-12 deficiency.

Biochem J. 1988 Oct 1.
Carnitine metabolism in the vitamin B-12-deficient rat.
Brass EP, Stabler SP.
Department of Medicine, University of Colorado School of Medicine, Denver.
[ Free full text ]

In vitamin B-12 (cobalamin) deficiency the metabolism of propionyl-CoA and methylmalonyl-CoA are inhibited secondarily to decreased L-methylmalonyl-CoA mutase activity. Production of acylcarnitines provides a mechanism for removing acyl groups and liberating CoA under conditions of impaired acyl-CoA utilization. Carnitine metabolism was studied in the vitamin B-12-deficient rat to define the relationship between alterations in acylcarnitine generation and the development of methylmalonic aciduria. Urinary excretion of methylmalonic acid was increased 200-fold in vitamin B-12-deficient rats as compared with controls. Urinary acylcarnitine excretion was increased in the vitamin B-12-deficient animals by 70%. This increase in urinary acylcarnitine excretion correlated with the degree of metabolic impairment as measured by the urinary methylmalonic acid elimination. Urinary propionylcarnitine excretion averaged 11 nmol/day in control rats and 120 nmol/day in the vitamin B-12-deficient group. The fraction of total carnitine present as short-chain acylcarnitines in the plasma and liver of vitamin B-12-deficient rats was increased as compared with controls. When the rats were fasted for 48 h, relative or absolute increases were seen in the urine, plasma, liver and skeletal-muscle acylcarnitine content of the vitamin B-12-deficient rats as compared with controls. Thus vitamin B-12 deficiency was associated with a redistribution of carnitine towards acylcarnitines. Propionylcarnitine was a significant constituent of the acylcarnitine pool in the vitamin B-12-deficient animals. The changes in carnitine metabolism were consistent with the changes in CoA metabolism known to occur with vitamin B-12 deficiency. The vitamin B-12-deficient rat provides a model system for studying carnitine metabolism in the methylmalonic acidurias.

Introduction

Carnitine, in addition to its well-established role as an obligate for the mitochondrial oxidation of long-chain fatty acids, interacts with other metabolic pathways by generating acylcarnitines from the corresponding acyl-CoAs. Under normal physiological conditions, the generation of short-chain acylcarnitines (acyl-group chain length less than 10 carbon atoms) from short-chain acyl-CoAs operates near steady state, with the distribution of total carnitine between free (unesterified) carnitine and the short-chain acylcarnitines reflecting the metabolic state of the tissue. Under pathophysiological conditions of acyl-CoA accumulation secondary to a metabolic defect, the generation of acylcarnitines provides a mechanism to remove the acyl group and liberate free CoA. This reaction may be critical for maintaining normal cellular function under conditions where organic acids (and the corresponding acyl-CoAs) accumulate. This mechanism is thought to be in part responsible for the efficacy of carnitine in the treatment of some hereditary organic acidurias characterized by acyl-CoA accumulation secondary to an enzyme deficiency, such as in propionic acidaemia and the methylmalonic acidurias. The resulting increase in urinary acylcarnitine excretion may lead to insufficient endogenous carnitine to meet metabolic requirements.

Understanding of the inter-relationship between the changes in endogenous carnitine metabolism, the consequences of the acyl-CoA buildup and overall cellular function is limited by a lack of experimental data. Evidence obtained in vitro demonstrates that high concentrations of unusual organic acids such as propionate can disrupt a number of metabolic pathways, including gluconeogenesis, fatty acid oxidation and pyruvate oxidation. For propionate, many of the cellular effects can be related to the accumulation of propionyl-CoA and methylmalonyl-CoA. Under conditions where propionate interferes with cellular metabolism, addition of carnitine results in a partial normalization of metabolism associated with a large increase in propionylcarnitine production. The use of carnitine in the treatment of patients with organic acidurias has resulted in clinical improvement of the patients and increased urinary excretion of the specific acylcarnitine corresponding to the accumulating acyl-CoA.

The current studies were initiated to establish an animal model in vivo of impaired acyl-CoA utilization to permit controlled studies of the inter-relationships between carnitine metabolism, the underlying metabolic defect and overall cellular function. In the vitamin B-12 (cobalamin)-deficient rat, the activity of L-methylmalonyl-CoA mutase is decreased. This results in the secondary accumulation of propionyl-CoA and methylmalonyl-CoA generated during the normal metabolism of several amino acids and odd-chain-length fatty acids. This situation is directly analogous to the human methylmalonic acidurias. The current studies demonstrate that in the vitamin B-12-deficient rat there is a marked increase in tissue short-chain acylcarnitine contents and in the urinary excretion of acylcarnitines, including significant amounts of propionylcarnitine, associated with the metabolic abnormalities of vitamin B-12 deficiency.

[...]

Results

Characterization of vitamin B-12-deficient rats

Rats fed on control or vitamin B- 12-deficient diets maintained on the pair-feeding schedule gained weight at similar rates. Urinary excretion of methylmalonic acid was monitored as an index of the metabolic defect associated with vitamin B- 12 deficiency. After 11 weeks, methylmalonic acid excretion averaged 0.24 + 0.03 and 10.3 + 5.6 ,mol/day in the control (n = II) and vitamin B-12-deficient (n = 11) animals respectively. After 21 weeks on the vitamin B-12-deficient diet, the animals demonstrated a profound vitamin B-12 deficiency, as evidenced by a 230-fold increase in urinary methylmalonic acid excretion, a 740 decrease in hepatic vitamin B-12 content and a 21% decrease in hepatic holo-L-methylmalonyl-CoA mutase activity.

Urinary carnitine excretion

Over the course of 21 weeks on the special diet, the vitamin B-12-deficient group developed an increase in urinary excretion of short-chain acylcarnitines as compared with the control animals. This difference was demonstrated by both a higher daily elimination of acylcarnitines and an increase in the percentage of total urinary carnitine present as acylcarnitine. The increase in urinary acylcarnitines associated with vitamin B-12 deficiency was evident after 11 weeks on the deficient diet, and after 21 weeks acylcarnitines accounted for 300 of total urinary carnitine in the deficient animals, compared with 150 in the control group. In contrast, urinary excretion of total carnitine was not different in the two groups. The increased urinary excretion of acylcarnitines in the vitamin B- 12-deficient animals was correlated with the urinary methylmalonic acid excretion. Acylcarnitines are generated from the corresponding acyl-CoA, and therefore the specific acylcarnitines excreted in the vitamin B-12-deficient rats should correspond to the acyl-CoAs which accumulate secondarily to the metabolic defect in vitamin B-12 deficiency. Acetylcarnitine and propionylcarnitine were therefore specifically quantified in the urine of control and vitamin B- 12-deficient rats after 21 weeks on the special diets. A h.p.l.c. method was used to resolve acetylcarnitine and propionylcarnitine for postchromatographic quantification [24]. In control rats, acetylcarnitine accounted for most (58 of the total urinary acylcarnitine in the vitamin B-12-deficient rats. The increased content of propionylcarnitine in the urine of the vitamin B-12-deficient rats was verified by using fast-atom-bombardment mass spectrometry, in both absolute (comparison with external standard) and relative (comparison with acetylcarnitine) terms.

Plasma and tissue carnitine pools

Groups of vitamin B-12-deficient and control animals were killed after 24 weeks on the diet, and their plasma, liver and skeletal-muscle carnitine pools were measured. The vitamin B-12-deficient animals demonstrated decreased plasma concentrations of carnitine and total carnitine as compared with the control animals. No differences in the concentrations of plasma short- or long-chain acylcarnitines were seen. Thus the percentage of total acid-soluble carnitine (the sum of carnitine and short-chain acylcarnitine) present as acylcarnitines was increased in the vitamin B-12-deficient rats (22%) as compared with the control rats (18 %).

Comparison of the liver carnitine pools in the two groups of rats showed decreases in carnitine and total carnitine content in the vitamin B-12-deficient rats. The percentage of hepatic total acid-soluble carnitine present as acylcarnitine was increased in the vitamin B-12-deficient rats (59 ). The skeletal-muscle contents of carnitine, short- and long-chain acylcarnitine and total carnitine were equivalent in the two groups. Propionylcarnitine contents in skeletal muscle and liver of control rats were low (12+3 and 16 +4 nmol/g respectively; n = 6). Skeletal-muscle propionylcarnitine content was 51 + 29 nmol/g (n = 7) in the vitamin B-12-deficient rats, accounting for 32 of the muscle short-chain acylcarnitines. Hepatic propionylcarnitine content in the vitamin B-12-deficient rats was 12+ 3 nmol/g (n = 7), and not different from control values.

Effect of fasting on carnitine metabolism in the vitamin B-12-deficient rat

During fasting, significant changes occur in carnitine metabolism in the rat, including a redistribution of the total carnitine pool into acylcarnitines. After 24 weeks of pair-feeding, a group of control and vitamin B-12-deficient rats were fasted for 48 h. Over the 48 h fast, the plasma/3-hydroxybutyrate concentration rose from 0.12+0.01 mm in the fed state (n = 6) to 0.80+ 0.07 mm (n = 7) in the control rats, and from 0.11+0.01 mM (n=7) to 1.13+0.34 mM (n=5) in the vitamin B-12-deficient rats. With fasting, the urinary excretion of carnitine, acylcarnitines and total carnitine decreased as compared with the fed state, with an increase in the percentage of total carnitine as acylcarnitine. In the vitamin B-12-deficient rats the larger excretion of acylcarnitines as compared with controls seen in the fed state was maintained, and resulted in an increased total carnitine excretion during fasting. In plasma, the free carnitine concentration fell by 180% in control animals during fasting, but it rose by 170% in the vitamin B-12-deficient rats. Plasma short-chain acylcarnitines increased by 86% in control rats and by 136% in the vitamin B-12-deficient group.

Increases in short-chain acylcarnitines were also seen in liver during fasting. The percentage of hepatic total acid-soluble carnitine present as short-chain acylcarnitine was higher in the vitamin B-12-deficient rats (53%) as compared with control animals (43%). In skeletal muscle from control rats, the short-chain acylcarnitine content fell with fasting by 22%. In contrast, an increase of 68% was seen in the short-chain acylcarnitine content of skeletal muscle in vitamin B-12-deficient rats during the 48 h fast. Thus fasting accentuated the redistribution of total carnitine towards short-chain acylcarnitines in the vitamin B-12-deficient rats.

Discussion

Methylmalonic aciduria in the vitamin B-12-deficient rat was associated with a redistribution of urine and tissue total carnitine content towards short-chain acylcarnitines, and away from free carnitine. Propionylcarnitine became a significant constituent of the carnitine pool in vitamin B-12 deficiency. The increase in short-chain acylcarnitines seen in vitamin B-12 deficiency was maintained or accentuated under the metabolic stress of starvation ketosis.

In the rat, vitamin B-12 is a required cofactor for two enzymes. The first, methionine synthase, is responsible for methionine generation from homocysteine. The second, L-methylmalonyl-CoA mutase, converts L-methylmalonyl-CoA into succinyl-CoA for further metabolism. In the vitamin B-12-deficient rat L-methylmalonyl-CoA and propionyl-CoA accumulate secondarily to the decreased L-methylmalonyl-CoA mutase activity. This accumulation in turn leads to a large increase in urinary methylmalonic acid excretion. This is qualitatively identical with the defects in the methylmalonic acidurias. In the current studies urinary excretion of methylmalonic acid was used as a marker of the metabolic defect with the development of vitamin B-12 deficiency. After 21 weeks on the experimental diet, the methylmalonic acid excretion in the vitamin B-12-deficient rats was 230 times the control value. Hepatic vitamin B-12 contents and holo-L-methylmalonyl-CoA mutase activity also confirmed the vitamin B-12 deficiency.

Increased generation of acylcarnitines from acyl-CoAs may be reflected as either an increase in the absolute amount of acylcarnitine or as a redistribution of the carnitine pool (regardless of total carnitine content) towards acylcarnitines. The changes observed in the carnitine pool with the development of vitamin B-12 deficiency were consistent with both of these concepts. Urinary excretion of acylcarnitines was doubled in the vitamin B-12-deficient rats as compared with controls.

The percentage of total acid-soluble carnitine present as acylcarnitine was also higher in urine, plasma and liver from the vitamin B-12-deficient animals as compared with the controls. Overall, the increased urinary excretion of acylcarnitines correlated well with the degree of metabolic impairment resulting from the vitamin B-12 deficiency as assessed by methylmalonic acid excretion. The use of the pair-feeding schedule eliminated energy intake or age, both of which alter carnitine metabolism, as possible etiologies for the differences observed in the two groups of animals.

The nature of the acyl-CoA accumulation is also reflected in the specific acyl moieties present in the acylcarnitine pool. Propionylcarnitine excretion was increased 11-fold in vitamin B-12-deficient rats as compared with control animals. Propionylcarnitine has also been identified as a major acylcarnitine in the urine of patients with methylmalonic aciduria. The increase in urinary acylcarnitine excretion with vitamin B-12 deficiency was approximately 300 nmol/day, or which 100 nmol/day could be accounted for by propionylcarnitine. This is consistent with the production of other, as yet unidentified, acylcarnitines in vitamin B-12 deficiency. Acyl-CoAs other than propionyl-CoA or methylmalonyl-CoA might also accumulate and form acylcarnitines secondarily to disruption of normal intermediary metabolism by the propionyl-CoA build-up.

Burton & Frenkel identified propionylcarnitine in the liver of vitamin B-12-deficient rats. In the most severely vitamin B-12-deficient animals in the current study, propionylcarnitine was estimated to represent 45% and 30% of total short-chain acylcarnitine in skeletal muscle and liver respectively. The large variability in the degree of methylmalonic aciduria attained in the current studies minimized the changes in mean tissue propionylcarnitine content. Despite the large degree of methylmalonic aciduria in our animals, and the dramatic changes in the CoA pool previously described in vitamin B-12 deficiency, the magnitude of the redistribution of tissue carnitines towards acylcarnitines was relatively small. Although the reasons for this are unclear, the poor conversion of branched-chain acyl-CoAs (such as methylmalonyl-CoA, a major acyl-CoA in vitamin B-12 deficiency) into the corresponding acylcarnitine, may have minimized the changes in the carnitine pool.

Starvation is associated with an increase in short-chain acylcarnitines. This phenomenon was maintained or enhanced in the vitamin B-12-deficient rats. For example, although skeletal-muscle short-chain acylcarnitine content fell in the control animals with fasting, consistent with previous observations, short-chain acylcarnitine content of skeletal muscle increased by 68% with fasting in the vitamin B-12-deficient group. This increase in short-chain acylcarnitines may be associated with the increased utilization of branched-chain amino acids by muscle during starvation.

The biosynthesis of carnitine requires the methylation of lysine contained in protein to form trimethyl-lysine. The impairment of methionine formation from homocysteine in vitamin B-12 deficiency might therefore interfere with carnitine biosynthesis. As most of the rat's carnitine is in skeletal muscle, the normal total carnitine content in the skeletal muscle of the vitamin B-12-deficient rats after 24 weeks on the diets, along with the normal total urinary carnitine elimination, suggests that there is no major impairment in carnitine biosynthesis in these animals. Thus the increase in short-chain acylcarnitines seen in vitamin B-12 deficiency reflect changes in the CoA pool rather than a broad disturbance in carnitine homoeostasis.

The vitamin B-12-deficient rat demonstrates changes in carnitine metabolism in both the fed and fasted states as compared with control animals. These changes correspond both qualitatively and quantitatively to predictions based on the accumulation of specific acyl-CoAs in these animals. The vitamin B-12-deficient rat provides a potential model system for studying the effects of changes in acylcarnitine generation on intermediary metabolism in the methylmalonic acidurias, with possible extrapolation to other organic acidaemias and diseases associated with acyl-CoA accretion.

Clin Chim Acta. 1988 Jun 30.
Urinary C6-C12 dicarboxylic acylcarnitines in Reye's syndrome.
Tracey BM, Cheng KN, Rosankiewicz J, Stacey TE, Chalmers RA.
Perinatal and Child Health, MRC Clinical Research Centre, Harrow, Middlesex, UK.

C6-C12 dicarboxylic acylcarnitines have been identified for the first time in urine from a 2-year-old girl presenting with Reye's syndrome. The acylcarnitines were extracted by ion-exchange chromatography and analysed, both underivatised and as methyl esters using high-resolution fast-atom-bombardment mass spectrometry and B/E-linked scanning. The acylcarnitines were quantified by capillary gas chromatography of the acids extracted after hydrolysis of the acylcarnitine esters. Dodecandioylcarnitine was present in the highest concentration (35.9 mmol/mol creatinine) which exceeded the urinary free dodecandioic acid concentration. The adipic, suberic and sebacic acylcarnitine concentrations were less than 10% of the respective free acid concentrations. It is possible that beta-oxidation of dicarboxylic acids is partially inhibited in Reye's syndrome leading to accumulation of precursor dodecandioyl CoA which is metabolised to dodecandioylcarnitine. The accumulation of these metabolic intermediates may be significant in the pathogenesis of Reye's syndrome.

Clin Chim Acta. 1988 Apr 29.
Urinary acylcarnitines in a patient with neonatal multiple acyl-CoA dehydrogenation deficiency, quantified by a carboxylic acid analyzer with a reversed-phase column.
Kidouchi K, Niwa T, Nohara D, Asai K, Sugiyama N, Morishita H, Kobayashi M, Wada Y.
Department of Pediatrics, Nagoya City Higashi General Hospital, Japan.

A quantitative analysis for urinary acylcarnitines in a patient with neonatal multiple acyl-CoA dehydrogenation deficiency is described. This method (liquid chromatography) can quantify twelve acylcarnitines including glutarylcarnitine and 3 isomeric acylcarnitines (butyryl-1, valeryl- and octanoylisomer) in urine. Before and up to the 15th hour of DL-carnitine therapy, isovalerylcarnitine was the largest single component existing in urinary acylcarnitines. Its excretion increased approximately 10 times within 1 day of DL-carnitine therapy. However, the acetyl-, the isobutyryl- and the butyrylcarnitine values increased gradually. From the 8th day of the therapy, the isobutyrylcarnitine value exceeded the isovalerylcarnitine. The patient's dominant urinary specific acylcarnitine derived from amino acids oxidation deficiency was changed from isovalerylcarnitine(leucine) to isobutyrylcarnitine(valine) during the early period of DL-carnitine therapy. Glutarylcarnitine was a minor component in the urine. Its degree of increase was as small as that of octanoylcarnitine. 2-Methylbutyrylcarnitine and propionylcarnitine were not detected.

Pediatr Res. 1985 May.
Diagnostic and therapeutic implications of medium-chain acylcarnitines in the medium-chain acyl-coA dehydrogenase deficiency.
Roe CR, Millington DS, Maltby DA, Bohan TP, Kahler SG, Chalmers RA.

The medium-chain acyl-coA dehydrogenase deficiency is one of several metabolic disorders presenting clinically as Reye syndrome. Evidence is presented for a characteristic organic aciduria that distinguishes this disorder from Reye syndrome and other masqueraders characterized by dicarboxylic aciduria. The key metabolites, suberylglycine and hexanoylglycine, are excreted in high concentration only when the patients are acutely ill. More significantly, using novel techniques in mass spectrometry, the medium-chain defect is shown to be characterized by excretion of specific medium-chain acylcarnitines, mostly octanoylcarnitine, without significant excretion of a normal metabolite, acetylcarnitine, in four patients with documented enzyme deficiency. Similar studies on the urine of two patients reported with Reye-like syndromes of unidentified etiology have suggested the retrospective diagnosis of medium-chain acyl-coA dehydrogenase deficiency. Administration of L-carnitine to medium-chain acyl-coA dehydrogenase deficiency patients resulted in the enhanced excretion of medium-chain acylcarnitines. Octanoylcarnitine is prominent in the urine both prior to and following L-carnitine supplementation. The detection of this metabolite as liberated octanoic acid, following ion-exchange chromatographic purification and mild alkaline hydrolysis, provides a straightforward diagnostic procedure for recognition of this disorder without subjecting patients to the significant risk of fasting. In view of the carnitine deficiency and the demonstrated ability to excrete the toxic medium-chain acyl-coA compounds as acylcarnitines, a combined therapy of reduced dietary fat and L-carnitine supplementation (25 mg/kg/6 h) has been devised and applied with positive outcome in two new cases.

Pediatr Res. 1984 Dec.
Urinary excretion of l-carnitine and acylcarnitines by patients with disorders of organic acid metabolism: evidence for secondary insufficiency of l-carnitine.
Chalmers RA, Roe CR, Stacey TE, Hoppel CL.

Concentrations of l-carnitine and acylcarnitines have been determined in urine from patients with disorders of organic acid metabolism associated with an intramitochondrial accumulation of acyl-CoA intermediates. These included propionic acidemia, methylmalonic aciduria, isovaleric acidemia, multicarboxylase deficiency, 3-hydroxy-3-methylglutaric aciduria, methylacetoacetyl-CoA thiolase deficiency, and various dicarboxylic acidurias including glutaric aciduria, medium-chain acyl-CoA dehydrogenase deficiency, and multiple acyl-CoA dehydrogenase deficiency. In all cases, concentrations of acylcarnitines were greatly increased above normal with free carnitine concentrations ranging from undetectable to supranormal values. The ratios of acylcarnitine/carnitine were elevated above the normal value of 2.0 +/- 1.1. l-Carnitine was given to three of these patients; in each case, concentrations of plasma and urine carnitines increased accompanied by a marked increase in concentrations of short-chain acylcarnitines. These acylcarnitines have been examined using fast atom bombardment mass spectrometry in some of these diseases and have been shown to be propionylcarnitine in methylmalonic aciduria and propionic acidemia, isovalerylcarnitine in isovaleric acidemia, and hexanoylcarnitine and octanoylcarnitine in medium-chain acyl-CoA dehydrogenase deficiency. The excretion of these acylcarnitines is compatible with the known accumulation of the corresponding acyl-CoA esters in these diseases. In this group of disorders, the increased acylcarnitine/carnitine ratio in urine and plasma indicates an imbalance of mitochondrial mass action homeostasis and, hence, of acyl-CoA/CoA ratios. Despite naturally occurring attempts to increase endogeneous l-carnitine biosynthesis, there is insufficient carnitine available to restore the mass action ratio as demonstrated by the further increase in acylcarnitine excretion when patients were given oral l-carnitine.

J Clin Invest. 1984 June.
L-carnitine enhances excretion of propionyl coenzyme A as propionylcarnitine in propionic acidemia.
C R Roe, D S Millington, D A Maltby, T P Bohan, and C L Hoppel.
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Treatment with L-carnitine greatly enhanced the formation and excretion of short-chain acylcarnitines in three patients with propionic acidemia and in three normal controls. The use of fast atom bombardment mass spectrometry and linked scanning at constant magnetic (B) to electric (E) field ratio identified the acylcarnitine as propionylcarnitine in patients with propionic acidemia. The normal children excreted mostly acetylcarnitine. Propionic acidemia and other organic acidurias are characterized by the intramitochondrial accumulation of short-chain acyl-Coenzyme A (CoA) compounds. The substrate specificity of the carnitine acetyltransferase enzyme and its steady state nature appears to facilitate elimination of propionyl groups while restoring the acyl-CoA:free CoA ratio in the mitochondrion. We suggest that L-carnitine may be a useful therapeutic approach for elimination of toxic acyl CoA compounds in several of these disorders.

Clin Chim Acta. 1983 Aug 15.
General (medium-chain) acyl-CoA dehydrogenase deficiency (non-ketotic dicarboxylic aciduria): quantitative urinary excretion pattern of 23 biologically significant organic acids in three cases.
Gregersen N, Kolvraa S, Rasmussen K, Mortensen PB, Divry P, David M, Hobolth N.

Urinary analysis of the pattern of 23 organic acid metabolites derived from fatty acids in three patients with general (medium-chain) acyl-CoA dehydrogenase deficiency was performed. Although there exist quantitative differences in the excreted amounts of the different metabolites in the three patients the qualitative picture was the same. The excretion of adipic, suberic and sebacic acids was substantial, whereas that of dodecanedioic acid was within or just above control limit. The monounsaturated C6-C10-dicarboxylic acid excretion was only marginally or not increased. 5-OH-hexanoic acid and hexanoylglycine were excreted in excessive amounts, whereas 7-OH-octanoic acid, 9-OH-decanoic acid, octanoylglycine and decanoylglycine were excreted in limited amounts. The excreted amounts of 6-OH-hexanoic, 8-OH-octanoic and 10-OH-decanoic acids were not or only marginally elevated compared to controls. In one of the patients the excretion of ethylmalonic and methylsuccinic acids was enhanced, whereas the excretion of these two acids in the two other patients was comparable to that in controls. The urinary excretion of hexanoic, octanoic, decanoic and dodecanoic acids was just a little above the control limit, whereas the esterified hexanoic and octanoic acids were excreted in appreciable amounts. It is argued that the microsomal omega- and omega-1-oxidation systems are involved in the dicarboxylic and omega-1-OH-monocarboxylic acids formation at C10 and C12 level and that the C8-C6-dicarboxylic and omega-1-OH-monocarboxylic acids are formed from higher chained acids by beta-oxidation in both mitochondria and peroxisomes.

Pediatr Res. 1982 Oct.
C6-C10-dicarboxylic aciduria: investigations of a patient with riboflavin responsive multiple acyl-CoA dehydrogenation defects.
Gregersen N, Wintzensen H, Christensen SK, Christensen MF, Brandt NJ, Rasmussen K.

The abnormal metabolites-adipic, suberic, and sebacic acids-were detected in large amounts in the urine of a boy during a Reye's syndrome-like crisis. Substantial amounts of 5-OH-caproic acid, caproylglycine, glutaric acid, and 3-OH-butyric acid and moderately elevated amounts of ethylmalonic acid, methylsuccinic acid, 3-OH-isovaleric acid, and isovalerylglycine were also found. These metabolites were consistently present in urine samples collected in the boy's habitual condition after the attack. 1-[14C]-Palmitic acid was oxidized at a normal rate, whereas U-[14C]-Palmitic acid was oxidized at a reduced rate in cultured skin fibroblasts from the patient, thus indicating a defect at the level of medium- and/or short-chain fatty acid oxidation. Riboflavin medication (100 mg three times a day) significantly reduced the excreted amounts of pathologic metabolites, suggesting a flavineadeninedinucleotide-related acyl-CoA dehydrogenation defect as the cause of the disease. Carnitine in plasma was low in the patient (6 mumole/liter, controls 26-74 mumole/liter), suggesting carnitine deficiency as a secondary effect of the acyl-CoA dehydrogenation deficiency. The present patient, who presented with a Reye's syndrome-like attack, suffers from impaired dehydrogenation of acyl-CoA resulting in accumulation of acyl-CoA in the cells. Attacks with similar symptoms are seen in other acyl-CoA dehydrogenation deficiencies, such as glutaric aciduria types I and II, other types of C6-C10-dicarboxylic acidurias and isovaleric acidemia. Reduced flow through the acyl-CoA dehydrogenation steps may therefore be an ethiologic factor in Reye's syndrome. Several of the accumulated acyl-CoA's are toxic and may be responsible for some of the symptoms. The low carnitine level in plasma and the elevated esterified carnitine excretion in the present patient indicate that acyl-CoA accumulation may cause a functional carnitine deficiency by sequestration of carnitine as acyl-carnitines. As the inborn defect, systemic carnitine deficiency may exhibit symptoms like those of Reye's syndrome, it may be speculated whether functional carnitine deficiency in patients with accumulated acyl-CoA is another causal factor in the development of the symptoms during attacks.

Scand J Clin Lab Invest Suppl. 1982.
C6-C10-dicarboxylic aciduria: biochemical considerations in relation to diagnosis of beta-oxidation defects.
Gregersen N, Kolvraa S, Mortensen PB, Rasmussen K.

By means of gas chromatographic methods substantial amounts of the C6-C10-dicarboxylic acids, i.e. adipic, suberic and sebacic acids, have been found in the urine from children with unexplained attacks of lethargy and hypotonia, presumably related to episodes of fever and/or insufficient food intake. The course have once been fatal and is often characterized by severe hypoglycemia without ketonuria. Systematic gas chromatographic/mass spectrometric determinations of selected organic acid metabolites in the urine, together with enzymatic measurements in fibroblasts and clinical data from 4 patients of this category, have shown that the biochemical basis of this syndrome can be inborn errors of the beta-oxidation of fatty acids, localized to the medium-chain acyl-CoA dehydrogenation system. The biosynthesis of adipic, suberic and sebacic acids was studied using ketotic rats as the model, since ketosis in rats and humans is accompanied by excessive urinary excretion of adipic and suberic acids. A probable pathway for the production of the three dicarboxylic acids was found to be an initial omega-oxidation of the medium-chain C10-C14-monocarboxylic acids followed by beta-oxidation of the resulting medium-chain dicarboxylic acids. It is argued that the source of the omega-oxidizable monocarboxylic acids in ketosis most probably is the fat deposites, and it is speculated that the patients with beta-oxidation defects supplement this source with beta-oxidation intermediate medium-chain monocarboxylic acids, accumulated as a result of the defect. The ratio between the excreted amounts of adipic acid and sebacic acid in the urine from the patients with beta-oxidation defects is less than 50. This is in contrast to the ratio in urine from ketotic patients, where it is greater than 100. Adipic acid/sebacic acid ratio-measured by means of a gas chromatographic analysis-is therefore suggested as a tool in the diagnosis of dicarboxylic acidurias. Based on the clinical picture and the pattern of a series of organic acids in the urinary metabolic profile our four patients can be divided in two types of dicarboxylic aciduria. The two types have different therapeutic implications.

J Biol Chem. 1981 Jun 10.
Carnitine:acylcarnitine translocase of rat heart mitochondria. Competition for carnitine uptake by carnitine esters.
Idell-Wenger JA.
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The kinetic behavior of the carnitine:acylcarnitine translocase was studied in isolated rat heart mitochondria. The kinetic parameters, Km(apparent) and Vmax, for carnitine were determined by measuring the rates of influx of [14C]carnitine using two different methods to quench the exchange reaction. The range of the Km(app) was 0.38-1.50 mM and the Vmax was 0.20-0.34 nmol/mg . min by both methods. Carnitine esters of acetyl isobutyryl, and octanoyl groups were competitive with carnitine for uptake and Ki values for these esters were 1.1, 2.6, and 0.10 mM, respectively. The Km(app) for carnitine was increased in the presence of these carnitine esters, while the Vmax for carnitine was unchanged. Distribution of radiolabel from free [14C]carnitine into acetylcarnitine, isobutyrylcarnitine, and octanoylcarnitine during the incubations was examined by thin layer chromatography and was negligible. The Km values for carnitine and the Ki value for acetylcarnitine are within the concentration ranges of these compounds in the intact heart (Idell-Wenger, J. A., Grotyohann, L. W., and Neely, J. R. (1978) J. Biol. Chem. 253, 4310-4318). the Ki values for isobutyrylcarnitine and octanoylcarnitine may also be within their concentration ranges in vivo, but exact concentrations in heart muscle are not known. These data support the concept that carnitine esters of short (acetylcarnitine), branched (isobutyrylcarnitine), and medium (octanoylcarnitine) chain acyl groups compete with free carnitine for transport into the mitochondria under physiological conditions.

Clin Chim Acta. 1980 Mar 28.
Non-ketotic C6-C10-dicarboxylic aciduria: biochemical investigations of two cases.
Gregersen N, Rosleff F, Kolvraa S, Hobolth N, Rasmussen K, Lauritzen R.

Two boys, who are not related, with hypoglycemia and C6-C10-dicarboxylic aciduria were investigated. Besides substantial amounts of adipic, suberic and sebacic acids, the urinary metabolic profile of organic acids contained 5-OH-caproic acid and caproylglycine. During acute attacks the concentrations of adipic, suberic and sebacic acids were 300--530, 160--200 and 35--200 micrograms/mg creatinine, respectively, and the excretions of 5-OH-caproic acid and caproylglycine were 75-330 and 41-260 micrograms/mg creatinine, respectively. It is argued that the biosynthesis of adipic acid passes through an omega-oxidation, that the production of 5-OH-caproic acid is caused by an omega-1-oxidation, and that caproylglycine formation passes through a glycine-N-acylase catalysed conjugation of accumulated caproic acid in the patients. Suberic acid and sebacic acid are in the same way omega-oxidation products of accumulated caprylic acid and capric acid, respectively. From the excretion pattern presented it is hypothesized that the patients suffer from a defect in the dehydrogenation of fatty acids in the beta-oxidation pathway. The biological significance of the findings is discussed.

Acta Paediatr Scand. 1979 Sep.
The excretion of C6-C10-dicarboxylic acids in the urine of newborn infants during starvation. Evidence for omega-oxidation of fatty acids in the newborn.
Gregersen N, Ingerslev J.

The excretion of C6-C10-dicarboxylic acids, i.e. adipic, suberic and sebacic acids, was measured during the three first days of life in 3 fasting newborns, 2 newborns fed with isocaloric glucose and 2 newborns given mothers'-milk. On the second and third day of life the starved children excreted 27-84 mmol adipic acid/mol creatinine, 6-22 mmol suberic acid/mol creatinine and 4-7 mmol sebacic acid/mol creatinine. The excretion of C6-C10-dicarboxylic acids in the neonates given glucose or mothers'-milk was, for the first three days of life, 0-9 mmol adipic acid/mol creatinine, 0-10 mmol suberic acid/mol creatinine and 0-4 mmol sebacic acid/mol creatinine. The latter amounts are equivalent to the excretion of dicarboxylic acids in older children. It is argued that the detected dicarboxylic acids are formed by omega-oxidation of long-chain monocarboxylic acids followed by beta-oxidation, and that the excreted amounts reflect omega-oxidation activity. It is speculated that the substantial omega-oxidation activity in the starving newborn serve to provide succinyl-CoA-substrate for the citric acid cycle and for gluconeogenesis.

Clin Chim Acta. 1979 May 16.
Dicarboxylic aciduria: the response to fasting.
Truscott RJ, Hick L, Pullin C, Halpern B, Wilcken B, Griffiths H, Silink M, Kilham H, Grunseit F.

The urine of a child who presented with an episode of a disease resembling Reye's syndrome was found to contain large quantities of the dicarboxylic acids adipic and suberic acids, as well as the glycine conjugate of suberic acid, suberyl glycine. A variety of other dicarboxylic acids, both saturated and unsaturated, were also found in the urine at the time of the attack. It was found that the excretion of these unusual metabolites could be markedly increased by fasting for periods of greater than 10 h. These results indicate that the patient may have a defect in fatty acid oxidation which becomes clinically significant during periods of prolonged fasting.

Clin Chim Acta. 1976 Aug 2.
Suberylglycine excretion in the urine from a patient with dicarboxylic aciduria.
Gregersen N, Lauritzen R, Rasmussen K.

Suberylglycine (HOOC(CH2)6CONHCH2COOH) was found in the urine from a patient with C6-C10-omega-dicarboxylic aciduria and unexplained episodes of lethargy and unconsciousness. The total excretion of adipic, suberic and sebacic acid ranged from 0.77 to 1.3 mg/mg creatinine after episodes of acute attack of the disease. Suberylglycine, identified by gas chromatography/mass spectrometry, was repeatedly found in the urine samples. The amount of this conjugate ranged from 0.2 to 0.5 mg/mg creatinine. The precursors of the dicarboxylic acids are suggested to be long chain monocarboxylic acids, oxidized through omega- and beta-oxidation to adipic, suberic and sebacic acid. Suberylglycine is subsequently formed by glycine-N-acylase catalyzed conjugation.

J Nutr. 1979 Jan.
The effect of long-term fasting on the branched chain acylcarnitines and branched chain carnitine acyltransferases.
Choi YR, Fogle PJ, Bieber LL.
[ Free full text ]

The effect of fasting for 8 days on the levels of carnitine acyltransferases in heart, liver, liver mitochondria, skeletal muscle, skeletal muscle mitochondria, kidney, and testes in young adult male rats was determined. The specific activities of acetyl-, octanyl-, isobutyryl-, and isovaleryl-carnitine acyltransferase in mitochondria isolated from the livers of fasted animals were significantly higher than the levels of the transferases isolated from livers of fed animals. Similar results were obtained with the 500 x g supernatant fluids from liver. In contrast, the specific activities of carnitine acyltransferases of 500 x g supernatant fractions isolated from heart, skeletal muscle, kidney, and testes were the same for fed as fasted animals. The total carnitine content of liver, muscle, heart, and kidney was less in animals fasted for 8 days than in fed animals, but the amount/g of organ was higher in the animals fasted for 8 days. The amount of specific short-chain acylcarnitines in liver, muscle, and heart was determined for both fed and fasted animals. The amount of isobutyrylcarnitine and isovalerylcarnitine increased significantly in muscle from fasted animals. These data are consistent with the previous suggestion that carnitine may have a role in the metabolism of the branched-chain amino acids.


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