PubMed Health⌕ Search

Biomedical subjects

C R Roe

Publications and source records attributed to C R Roe.

At least 37 records · Page 2Linked to original sources
PubMed1998

Studies on the oxidation of phytanic acid and pristanic acid in human fibroblasts by acylcarnitine analysis.

The alpha-oxidation of phytanic acid and the beta-oxidation of pristanitc acid were investigated in cultured fibroblasts from controls and patients affected with different peroxisomal disorders using deuterated substrates. Formation of [omega-2H6]4,8-dimethylnonanoylcarnitine ([omega-2H6]C11-carnitine) from [omega-2H6]phytanic acid and [omega-2H6]pristanic acid was used as marker for these processes. Analysis was performed by tandem mass spectrometry. In normal cells, formation of [omega-2H6]C11-carnitine from both [omega-2H6]phytanic acid and [omega-2H6]pristanic acid was observed. When peroxisome-deficient fibroblasts were incubated with these substrates, [omega-2H6]C11-carnitine was not detectable or, in two cases, very low, which results from deficiencies in both peroxisomal alpha- and beta-oxidation. In cells with an isolated beta-oxidation defect at the level of the peroxisomal bifunctional protein, formation of [omega-2H6]C11-carnitine could also not be detected. Cells with an isolated defect in the alpha-oxidation of phytanic acid, obtained from patients affected with Refsum disease (McKusick 266500) or rhizomelic chondrodysplasia punctata (McKusick 215100), did not form [omega-2H6]C11-carnitine from [omega-2H6]phytanic acid. The observed formation of [omega-2H6]C11-carnitine from [omega-2H6]pristanic acid in these cells is in accordance with a normal peroxisomal beta-oxidation in these disorders. This study shows that separate incubation of fibroblasts with [omega-2H6]phytanic acid and [omega-2H6]pristanic acid, followed by acylcarnitine analysis in the medium by tandem mass spectrometry, can be used for screening cell lines for deficiencies in the peroxisomal alpha- and beta-oxidation pathways. Phytanic acid (3,7,11,15-tetramethylhexadecanoic acid) and pristanic acid (2,6,10,14-tetramethylpentadecanoic acid) are branched-chain fatty acids that are constituents of the human diet. As phytanic acid possesses a beta-methyl group, it cannot be degraded by beta-oxidation. Instead, phytanic acid is first degraded by alpha-oxidation, yielding pristanic acid, which is subsequently degraded by beta-oxidation (Figure 1). Phytanic acid alpha-oxidation is thought to occur partly, and pristanic acid beta-oxidation exclusively, in peroxisomes (see Wanders et al 1995 for review). Accumulation of phytanic acid and pristanic acid is found in blood and tissues of patients affected with generalized peroxisomal disorders. In this type of disorder, no morphologically distinguishable peroxisomes are present in tissues, resulting in accumulation of metabolites that are normally metabolized in these organelles (see Wanders et al 1995 for review). The group of generalized peroxisomal disorders consists of three diseases, differing in clinical presentation. Patients suffering from the most severe disease, Zellweger syndrome (McKusick 214100), have symptoms from birth on and usually do not live beyond their first year of life. Neonatal adrenoleukodystrophy (N-ALD, McKusick 202370) has a milder presentation, whereas infantile Refsum disease (IRD, McKusick 266510) is the mildest form among the generalized peroxisomal disorders. Not only in these generalized peroxisomal disorders, but also in some isolated peroxisomal beta-oxidation defects, elevated levels of phytanic acid and pristanic acid are found (ten Brink et al 1992a). The elevated phytanic acid levels are considered to be caused by product inhibition of alpha-oxidation by accumulating pristanic acid. This is reflected in a highly elevated pristanic acid to phytanic acid ratio in plasma from patients suffering from bifunctional protein deficiency or peroxisomal thiolase deficiency (ten Brink et al 1992a). Elevated phytanic acid concentrations are also found in plasma from patients affected with classical Refsum disease and rhizomelic chondrodysplasia punctata (RCDP). As pristanic acid beta-oxidation is not disturbed in these disorders, pristanic acid levels are normal (ten Brink et al 1992

Carnitine↗
PubMed1998

Phytanic acid and pristanic acid are oxidized by sequential peroxisomal and mitochondrial reactions in cultured fibroblasts.

The relationship between peroxisomal and mitochondrial oxidation of the methyl branched fatty acids, phytanic acid and pristanic acid, was studied in normal and mutant human skin fibroblasts with established enzyme deficiencies. Tandem mass spectrometry was used for analysis of the acylcarnitine intermediates. In normal cells, 4,8-dimethylnonanoylcarnitine (C11:0) and 2,6-dimethylheptanoylcarnitine (C9:0) accumulated after incubation with either phytanic acid or pristanic acid. These intermediates were not observed when peroxisome-deficient cells from Zellweger patients were incubated with the same compounds, pointing to the involvement of peroxisomes in the formation of these acylcarnitine intermediates. Similar experiments with fibroblasts deficient in carnitine palmitoyltransferase I, carnitine-acylcarnitine translocase or carnitine palmitoyltransferase II revealed that mitochondrial carnitine palmitoyltransferase I is not required for the oxidation of phytanic acid or pristanic acid, whereas both carnitine-acylcarnitine translocase and carnitine palmitoyltransferase II are necessary. These studies demonstrate that both phytanic acid and pristanic acid are initially oxidized in peroxisomes to 4,8-dimethylnonanoyl-CoA, which is converted to the corresponding acylcarnitine (presumably by peroxisomal carnitine octanoyltransferase), and exported to the mitochondrion. After transport across the mitochondrial membrane and transfer of the acylgroup to coenzyme A, further oxidation to 2,6-dimethylheptanoyl-CoA occurs.

Carnitine Acyltransferases↗
PubMed1997

Phytanic acid alpha-oxidation in peroxisomal disorders: studies in cultured human fibroblasts.

We studied the alpha-oxidation of phytanic acid in human fibroblasts of controls and patients affected with classical Refsum disease, rhizomelic chondrodysplasia punctata, generalized peroxisomal disorders and peroxisomal bifunctional protein deficiency. Cultured fibroblasts were incubated with phytanic acid, after which medium and cells were collected separately. 2-Hydroxyphytanic acid and pristanic acid were measured in the medium and cells by stable isotope dilution gas chromatography mass spectrometry. In controls, 2-hydroxyphytanic acid and pristanic acid could be detected in the medium after incubation with phytanic acid, proving that alpha-oxidation of phytanic acid via 2-hydroxyphytanoyl-CoA to pristanic acid was active and intermediates were excreted into the medium. In cells from patients with a defective alpha-oxidation (Refsum disease, rhizomelic chondrodysplasia punctata and generalized peroxisomal disorders) 2-hydroxyphytanic acid and pristanic acid were low or not detectable, showing that in these disorders the hydroxylation of phytanoyl-CoA to 2-hydroxyphytanoyl-CoA is deficient. In cells with a peroxisomal beta-oxidation defect, 2-hydroxyphytanic acid and pristanic acid were formed in amounts comparable to those in the controls.

Cells, Cultured↗
PubMed1996

Prenatal diagnosis of mitochondrial fatty acid oxidation defects.

Amniocytes isolated from two pregnancies at risk for fatty acid oxidation defects were incubated with stable isotopically labelled palmitate, in the presence of L-carnitine, to probe that pathway. The labelled acylcarnitines were then quantitated using tandem mass spectrometry. Amniocytes from a pregnancy at risk for medium-chain acyl-CoA dehydrogenase (MCAD) deficiency produced a characteristic acylcarnitine profile with increased levels of octanoylcarnitine and decanoylcarnitine, indicative of MCAD deficiency. DNA analysis confirmed that the fetus was homozygous for the MCAD A985G mutation. Acylcarnitine and DNA analysis of the infant's blood obtained post-partum confirmed MCAD deficiency. Amniocytes from a pregnancy at risk for an unspecified fat oxidation defect produced increased levels of long-chain acylcarnitines consistent with a deficiency in very-long-chain acyl-CoA dehydrogenase (VLCAD). Measurements of the enzymatic activity confirmed VLCAD deficiency in amniocytes. Acylcarnitine profiles of the infant's blood obtained post-partum in addition to enzyme activities measured in fibroblasts confirmed VLCAD deficiency. The successful prenatal diagnosis of VLCAD and MCAD deficiencies using in vitro probes of fatty acid oxidation in fibroblasts suggests that this approach can potentially recognize many mitochondrial fatty acid oxidation defects even if no prior diagnosis is determined in the family at risk.

Acyl-CoA Dehydrogenase↗
PubMed1996

Improved detection of the G1528C mutation in LCHAD deficiency.

Long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) deficiency, an autosomal recessive disorder of fatty-acid oxidation, is clinically characterized by skeletal myopathy, Reye-like syndrome, or sudden unexplained infant death. A common mutation, G1528C, has recently been reported. To avoid nonspecific amplification from a "pseudogene" and potential complications, we have developed a nested PCR/PstI digestion method. Here, we report mutation studies in 11 additional unrelated patients with LCHAD deficiency. Genomic DNA fragments (117 bp) were amplified by the nested PCR, digested with PstI, and subjected to electrophoresis on 12% polyacrylamide gel. Four patients were found to be homozygous for the G1528C mutation; 7 patients were compound heterozygous, indicating significant genetic heterogeneity. The G1528C mutation has been found on at least one allele in all patients with isolated LCHAD deficiency, suggesting that it is an excellent marker for this disease. This DNA test combined with tandem mass-spectrometric in vitro probe analysis easily identifies affected individuals and carriers in families which are compound heterozygous for G1528C.

3-Hydroxyacyl CoA Dehydrogenases↗
PubMed1996

Very long chain acyl-CoA dehydrogenase deficiency: successful treatment of acute cardiomyopathy.

Very-long-chain acyl-CoA dehydrogenase (VLCAD) deficiency is a severe defect of mitochondrial fatty acid oxidation characterized by hypertrophic cardiomyopathy, pericardial effusion, steatosis, and hypoglycemia, often resulting in death by 4-5 months of age. The onset of cardiomyopathy and pericardial effusion is insidious and sudden, necessitating early diagnosis and intervention to prevent death. A family affected with this defect is described in which dietary therapy with medium-chain triglycerides (MCT) was associated with rapid reversal of these severe clinical symptoms. Diagnosis by acylcarnitine analysis in the neonatal period can provide the opportunity for early clinical intervention. Prenatal diagnosis from amniocytes by enzymology or in vitro analysis of the fat oxidation pathway with deuterated fatty acid precursors has also been successful and permits intervention at birth. Of 10 affected children, 7 untreated cases died within the first several months while the remaining 3 cases survived when treated with medium-chain triglycerides as the major source of dietary fat.

Acute Disease↗
PubMed1996

Cloning and characterization of human very-long-chain acyl-CoA dehydrogenase cDNA, chromosomal assignment of the gene and identification in four patients of nine different mutations within the VLCAD gene.

Very-long-chain acyl-CoA dehydrogenase (VLCAD) is one of four straight-chain acyl-CoA dehydrogenase (ACD) enzymes, which are all nuclear encoded mitochondrial flavoproteins catalyzing the initial step in fatty acid beta-oxidation. We have used the very fast, Rapid Amplification of cDNA Ends (RACE) based strategy to obtain the sequence of cDNAs encoding human VLCAD from placenta and fibroblasts. Alignment of the predicted amino acid sequence of human VLCAD with those of the other human ACD enzymes revealed extensive sequence homology. Moreover, human VLCAD and human acyl-CoA oxidase showed extensive sequence homology corroborating the notion that these genes are evolutionarily related. Southern blot analysis of genomic DNA from hybrid cell lines was used to localize the VLCAD gene to human chromosome 17p11.2-p11.13105. Using Northern and Western blot analysis to investigate the tissue specific distribution of VLCAD mRNA and protein in several human tissues we showed that VLCAD is most abundant in heart and skeletal muscle. This agrees well with the fact that cardiac and muscle symptoms are characteristic for patients with VLCAD deficiency. Northern blot analysis and sequencing of cloned PCR amplified VLCAD cDNA from four unrelated patients with VLCAD deficiency showed that VLCAD mRNA was undetectable in one patient and that the other three have mutations in both VLCAD alleles. Western blot analysis of patient fibroblasts showed that the identified mutations result in severely reduced amounts of VLCAD protein. None of the patients harbored identical mutations suggesting that the mutational heterogeneity in VLCAD deficiency is large.

Acyl-CoA Dehydrogenase, Long-Chain↗
PubMed1995

Evidence for intermediate channeling in mitochondrial beta-oxidation.

The accumulation of beta-oxidation intermediates was studied by incubating normal and beta-oxidation enzyme-deficient human fibroblasts with [2H4]linoleate and L-carnitine and analyzing the resultant acylcarnitines by tandem mass spectrometry. Labeled decenoyl-, octanoyl-, hexanoyl-, and butyrylcarnitines were the only intermediates observed with normal cells. Intermediates of longer chain length, corresponding to substrates for the beta-oxidation enzymes associated with the inner mitochondrial membrane, were not observed unless a cell line was deficient in one of these enzymes, such as very-long-chain acyl-CoA dehydrogenase, long-chain 3-hydroxyacyl-CoA dehydrogenase, or electron transfer flavoprotein dehydrogenase. Matrix enzyme deficiencies, such as medium- and short-chain acyl-CoA dehydrogenases, were characterized by elevated concentrations of intermediates corresponding to their respective substrates (octanoyl- and decenoylcarnitines in medium-chain acyl-CoA dehydrogenase deficiency and butyrylcarnitine in short-chain acyl-CoA dehydrogenase deficiency). These observations agree with the notion of intermediate channeling due to the organization of beta-oxidation enzymes in complexes. The only exception is the incomplete channeling from thiolase to acyl-CoA dehydrogenase in the matrix. This situation may be a consequence of only one 3-ketoacyl-CoA thiolase being unable to interact with the several acyl-CoA dehydrogenases in the matrix.

Acyl-CoA Dehydrogenase↗
PubMed1995

Familial infantile apnea and immature beta oxidation.

Infants with inborn errors of fatty acid metabolism may present with apnea, periodic breathing, and sudden infant death syndrome (SIDS). Recognition of these disorders and initiation of appropriate therapy may prevent SIDS. Metabolic pathways develop during gestation and post-natally. We report three siblings with apnea and periodic breathing, as well as biochemical defects consistent with a non-specific abnormality of beta oxidation. One infant died a witnessed sudden infant death. The two survivors were treated with L-carnitine supplementation resulting in rapid resolution of both respiratory and metabolic abnormalities. Enzyme activity for short, medium, and long chain acyl coenzyme A dehydrogenases was normal in these two infants. Although a unique enzymatic deficiency was not identified, our experience with this family supports the need for routine biochemical evaluation of infants with "near miss" SIDS, also called acute life-threatening events (ALTE), as well as those who have died of SIDS.

Apnea↗
PubMed1995

Investigation of beta-oxidation intermediates in normal and MCAD-deficient human fibroblasts using tandem mass spectrometry.

Mitochondrial fatty acid beta-oxidation was studied by incubating stable isotope-labeled fatty acid probes with human fibroblasts in the presence of L-carnitine. The acylcarnitine intermediates produced were analyzed by tandem mass spectrometry. Oxidation by normal fibroblasts produced specific acylcarnitine intermediates corresponding to acyl-CoA dehydrogenase substrates mainly of 10 or less carbons. These probes demonstrated that the pathway, involving all beta-oxidative steps, could be examined. Oxidation of the same precursors by cells with medium chain acyl-CoA dehydrogenase (EC 1.3.99.2) (MCAD) deficiency, which is caused by different DNA mutations, produced acylcarnitine profiles which appear to be specific to this enzyme defect, regardless of the DNA mutation. Increased amounts of octanoyl-, decanoyl-, or decenoylcarnitine were detected. The ratios of octanoylcarnitine to decanoyl- or decenoylcarnitine appear specific for MCAD deficiency. Even though the concentration of labeled decenoylcarnitine (C10:1) was elevated in incubations of MCAD-deficient cells with labeled linoleate or with a fatty acid mixture which included palmitate, oleate, and linoleate, the predominant intermediate was octanoylcarnitines. These results suggest that MCAD-deficient cells readily convert decanoyl-CoA into octanoyl-CoA. This in vitro system could be utilized to study fatty acid oxidation disorders and to study the origins of metabolic intermediates associated with them.

Acyl Coenzyme A↗
PubMed1995

Rapid diagnosis of maple syrup urine disease in blood spots from newborns by tandem mass spectrometry.

We report a new method for the diagnosis of maple syrup urine disease (MSUD) from dried blood spots on newborn screening cards based on tandem mass spectrometry (MS-MS). The mean +/- SD concentration of Leu plus Ile in normal newborns was 151 +/- 47 mumol/L (n = 1096); for Val, 131 +/- 58 mumol/L (n = 791). SDs were lower when the concentrations of these amino acids were expressed relative to that of Phe. The mean ratio for Leu + Ile to Phe was 2.5 +/- 0.49; for Val to Phe, 2.18 +/- 0.51. These results compare well with values previously reported in the literature. With these criteria, samples from a collection categorized by a bacterial inhibition assay as normal or falsely positive for MSUD were normal by MS-MS [(Leu + Ile): Phe < 5.0]. Samples from confirmed MSUD patients were categorized as abnormal [(Leu+Ile): Phe > 9.0] by MS-MS.

False Positive Reactions↗
PubMed1994

Medium-chain acyl-coenzyme A dehydrogenase deficiency: clinical course in 120 affected children.

Medium-chain acyl-coenzyme A dehydrogenase deficiency is an autosomal recessive disorder of beta-oxidation of fatty acids manifested by episodic hypoglycemia, encephalopathy, apnea, and sudden death. Medical data were obtained on 120 patients with medium-chain acyl-coenzyme A dehydrogenase deficiency referred to Duke University Medical Center for biochemical testing. There were 55 male and 65 female subjects ranging from birth to 19 years of age; 118 subjects were white. Twenty-three children (19%) died before the diagnosis was made. Follow-up data were available in the 97 surviving patients for an average of 2.6 years after diagnosis. Psychodevelopmental data were collected on 73 patients older than 2 years of age. Unexpected morbidity included developmental and behavioral disability, chronic muscle weakness, failure to thrive, and cerebral palsy. We conclude that unidentified patients with this disorder have a significant risk of sudden death in early childhood and that survivors have a significant risk of developmental disability and chronic somatic illness.

Acyl-CoA Dehydrogenase↗
PubMed1994

Intravenous L-carnitine and acetyl-L-carnitine in medium-chain acyl-coenzyme A dehydrogenase deficiency and isovaleric acidemia.

The purpose of this study was to determine whether treatment with L-carnitine or acetyl-L-carnitine enhances the turnover of lipid or branched-chain amino acid oxidation in patients with inborn errors of metabolism. Increasing i.v. doses of L-carnitine and acetyl-L-carnitine were given to one patient with medium-chain acyl-CoA dehydrogenase deficiency and to another with isovaleric acidemia. Both patients were in stable condition and receiving oral L-carnitine supplements. The excretion of carnitine and disease-specific metabolites was measured. The incorporation of L-carnitine in the intracellular pool was demonstrated using stable isotopes and mass spectrometry. Increasing doses of either i.v. L-carnitine or acetyl-L-carnitine did not stimulate the excretion of octanoylcarnitine in the patient with medium-chain acyl-CoA dehydrogenase deficiency, nor did it raise the plasma levels of either cis-4-decenoate or octanoylcarnitine. Similarly, increasing doses of either i.v. L-carnitine or acetyl-L-carnitine did not enhance the excretion of isovalerylcarnitine in a patient with isovaleric acidemia. The excretion of isovalerylglycine actually decreased. We conclude that there was no evidence of enhanced fatty acid beta-oxidation or enhanced branched-chain amino acid oxidation in vivo by the administration of high doses of L-carnitine or acetyl-L-carnitine in these two patients. Because only one individual with each disorder was studied, the data are only indicative and may not necessarily be representative of all individuals with these disorders. Definite settlement of this issue will require further studies in additional subjects.

Acetylcarnitine↗