C R Roe

Tandem mass spectrometry: A new method for acylcarnitine profiling with potential for neonatal screening for inborn errors of metabolism

ConclusionA method for analysis of acylcarnitines in blood at physiological concentrations has been developed. Preliminary results from umbilical cord blood and neonatal blood spotted onto Guthrie cards are encouraging. This method will detect up to at least eight inherited metabolic disorders which exhibit diagnostic acylcarnitine profiles, including medium-chain acyl-CoA dehydrogenase deficiency. The speed and simplicity of the method permit automation with existing technology, and could enable routine neonatal screening to be carried out in an efficient and cost-effective manner.

Application of fast atom bombardment with tandem mass spectrometry and liquid chromatography/mass spectrometry to the analysis of acylcarnitines in human urine, blood, and tissue

Using a precursor-ion scan function on a triple quadrupole mass spectrometer, acylcarnitines were detected in the target matrices at or below concentrations of 1 nmol per gram by fast atom bombardment mass spectrometry. Acylcarnitine profiles from patients with known metabolic disorders were consistent with previously acquired data. Putative acylcarnitine signals were confirmed in one case by administration of stable isotope-labeled carnitine, which equilibrated rapidly with the endogenous pool. The addition of a continuous flow system enabled rapid sequential analysis without operator intervention, indicating the potential for automation of the analytical procedure. Incorporation of a micro-LC column enabled on-line liquid chromatographic/mass spectrometric analysis of selected patient samples. Large-scale screening and quantitative analysis of urine or blood for diagnostic acylcarnitines are now practicable.

2-Methylbutyryl-coenzyme A dehydrogenase deficiency: a new inborn error of L-isoleucine metabolism.

An 4-mo-old male was found to have an isolated increase in 2-methylbutyrylglycine (2-MBG) and 2-methylbutyrylcarnitine (2-MBC) in physiologic fluids. In vitro oxidation studies in cultured fibroblasts using 13C- and 14C-labeled branched chain amino acids indicated an isolated block in 2-methylbutyryl-CoA dehydrogenase (2-MBCDase). Western blotting revealed absence of 2-MBCDase protein in fibroblast extracts; DNA sequencing identified a single 778 C>T substitution in the 2-MBCDase coding region (778 C>T), substituting phenylalanine for leucine at amino acid 222 (L222F) and absence of enzyme activity for the 2-MBCDase protein expressed in Escherichia coli. Prenatal diagnosis in a subsequent pregnancy suggested an affected female fetus, supporting an autosomal recessive mode of inheritance. These data confirm the first documented case of isolated 2-MBCDase deficiency in humans.

L-carnitine therapy in isovaleric acidemia.

Isovaleric acidemia, resulting from isovaleryl-coenzyme A dehydrogenase deficiency, is associated with marked reduction of free carnitine in both plasma and urine. Fast atom bombardment-mass spectrometry, hydrolysis, and gas chromatography/mass spectrometry have unequivocally identified the existence of isovalerylcarnitine, a new metabolite specific for this disorder. Administration of equimolar amounts of glycine or L-carnitine separately with leucine demonstrated that isovaleryl-coenzyme A is removed by supplemental L-carnitine in the form of isovalerylcarnitine as effectively as it is by glycine, in the form of isovalerylglycine. When L-carnitine is given alone, excretion of isovalerylglycine decreases in preference to enhanced excretion of isovalerylcarnitine and hippurate. Treatment with L-carnitine alone has proven effective in preventing further hospitalizations in a patient with this genetic disorder.

2,4-Dienoyl-coenzyme A reductase deficiency: a possible new disorder of fatty acid oxidation.

Several inherited disorders of fatty acid beta-oxidation have been described that relate mainly to saturated precursors. This study is the first report of an enzyme defect related only to unsaturated fatty acid oxidation and provides the first in vivo evidence that fat oxidation in humans proceeds by the reductase-dependent pathway. The patient was a black female, presenting in the neonatal period with persistent hypotonia. Biochemical studies revealed hyperlysinemia, hypocarnitinemia, normal organic acid profile, and an unusual acylcarnitine species in both urine and blood. The new metabolite was positively identified by mass spectrometry as 2-trans,4-cis-decadienoylcarnitine, derived from incomplete oxidation of linoleic acid. In spite of dietary therapy, the patient died of respiratory acidosis at four months of age. Samples of liver and muscle from the autopsy were assayed for 2,4-dienoyl-coenzyme A reductase activity. Using the substrate 2-trans,4-cis-decadienoylcoenzyme A, the reductase activity was 40% of the control value in liver and only 17% of that found in normal muscle. It is suggested that unsaturated substrates should be used for in vitro testing to cover the full range of potential beta-oxidation defects and that acylcarnitine species identification be used for in vivo detection of this disorder.

Identification of 3-methylglutarylcarnitine. A new diagnostic metabolite of 3-hydroxy-3-methylglutaryl-coenzyme A lyase deficiency.

Deficiency of 3-hydroxy-3-methylglutaryl-coenzyme A (CoA) lyase affects the metabolism of leucine as well as ketogenesis. This disorder is one of an increasing list of inborn errors of metabolism that presents clinically like Reyes Syndrome or nonketotic hypoglycemia. Four patients with proven 3-hydroxy-3-methylglutaryl-CoA lyase deficiency were shown to excrete a new diagnostically specific metabolite. The technique of fast atom bombardment and tandem mass spectrometry revealed that only 3-methylglutaryl-CoA is a substrate for acylcarnitine formation. Neither 3-methylglutaconyl-CoA nor 3-hydroxy-3-methylglutaryl-CoA are excreted as acylcarnitines. The excretion of 3-methylglutarylcarnitine may explain, in part, the apparent secondary carnitine deficiency in this disorder. Carnitine supplementation with moderate dietary restrictions may be a useful treatment strategy for this disorder.

Intravenous L-Carnitine and Acetyl-L-Carnitine in Medium-Chain Acyl-Coenzyme A Dehydrogenase Deficiency and Isovaleric Acidemia

ABSTRACT: 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 β-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.

Analysis of abnormal urinary metabolites in the newborn period in medium-chain acyl-CoA dehydrogenase deficiency

SummaryIn order to determine which are useful early diagnostic markers for medium-chain acyl-CoA dehydrogenase (MCAD) deficiency, we have analysed urine from an asymptomatic neonate. Profiling of urinary organic acids followed by peak confirmation by electron impact mass spectrometry revealed a high suberate/adipate ratio (>1.0) and the presence of n-hexanoylglycine (HG). Acylcarnitine analysis by fast atom bombardment mass spectrometry (FAB-MS) was inconclusive, but FAB-MS/MS (tandem mass spectrometry) revealed diagnostic amounts of octanoylcarnitine and hexanoylcarnitine. Quantitative analysis of acylglycines by stable istotope dilution and chemical ionization mass spectrometry revealed a 30-fold increase in HG and increased suberylglycine, but no increase in 3-phenylpropionylglycine.