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Biomedical subjects

D S Millington

Publications and source records attributed to D S Millington.

At least 19 recordsLinked to original sources
PubMed1992

Formation of a free acyl adenylate during the activation of 2-propylpentanoic acid. Valproyl-AMP: a novel cellular metabolite of valproic acid.

A study of the activation of valproic acid (2-n-propylpentanoic acid) by a soluble extract of rat liver mitochondria in the presence of ATP, CoASH, and MgCl2 revealed that, in addition to valproyl-CoA, an unknown UV-absorbing compound is formed which is the sole product when CoASH is omitted from the incubation mixture. The unknown compound, which was purified by high performance liquid chromatography, was identified as valproyl adenylate (valproyl-AMP) by mass spectrometry and by its enzymatic conversion to valproyl-CoA in the presence of CoASH. Valproyl-AMP exists, at least partially, in a free, not-enzyme-bound form. Its rate of formation is linear with time and increases 5-fold when the pH is decreased from 8 to 6.8. Valproyl-AMP was also identified when the metabolism of valproate was investigated with rat liver mitochondria and rat hepatocytes. Since the synthesis of valproyl-AMP is inhibited by octanoate, medium-chain acyl-CoA synthetase (EC. 6.2.1.2) may be responsible for its formation. This study establishes that during the activation of valproic acid to valproyl-CoA free valproyl-AMP is formed which is a novel cellular metabolite of valproic acid.

Acyl Coenzyme A↗
PubMed1992

Rapid optimization of enzyme substrates using defined substrate mixtures.

A strategy is described for the rapid optimization of kcat/Km for protease substrates. Selected positions of a given peptide substrate sequence are varied through synthesis with mixtures of amino acids. Incubation of the resulting peptide mixture with the enzyme of interest and analysis by high pressure liquid chromatography provides a direct measure of analogs with enhanced kcat/Km. High performance liquid chromatography/continuous flow fast atom bombardment mass spectrometry is used to assign structure to each peak in the chromatogram. As an example of the utility and efficiency of "substrate mapping" we describe optimization of the collagenase substrate Dnp-Pro-Leu-Gly-Leu-Trp-Ala-D-Arg-NH2 (where Dnp is dinitrophenyl) at the P'1 and P'2 positions. Six different mixtures were prepared for evaluation, representing the synthesis of 128 different synthetic substrates. "Substrate mapping" has led to Dnp-Pro-Leu-Gly-Cys(Me)-His-Ala-D-Arg-NH2, a substrate that possesses a 10-fold better kcat/Km than Dnp-Pro-Leu-Gly-Leu-Trp-Ala-D-Arg-NH2.

Amino Acid Sequence↗
PubMed1992

Glutaric aciduria type I: unusual biochemical presentation.

We describe a patient with glutaryl-coenzyme A dehydrogenase deficiency, demonstrated by a residual enzyme activity of only 1% in cultured fibroblasts. Although the clinical presentation was typical of glutaric aciduria type I, the urine concentrations of glutaric, glutaconic, and 3-hydroxyglutaric acids remained normal, even during episodes of clinical decompensation. An increased free glutarate level was demonstrated only in cerebrospinal fluid.

Carnitine↗
PubMed1992

Anoxia induces phospholipase A2 activation in rabbit renal proximal tubules.

Phospholipase A2 (PLA2) activation during anoxic cell injury was determined by use of a variety of approaches in rabbit proximal renal tubules. Arachidonic acid (AA) mass release increased from 4 +/- 1 (normoxia control) to 40 +/- 6 ng/mg protein after 20 min and 106 +/- 16 ng/mg protein after 40 min of anoxia. PLA2 activity was measured by estimating the amount of sn-2 fatty acid released from either 14C-labeled Escherichia coli membranes or [14C]phosphatidylethanolamine (PE) micelles incubated with membrane and cytosolic fractions obtained from normoxic or anoxic tubules. At pH 7.4 and 1 mM Ca, PLA2 activity increased in the 20-min anoxic membrane fractions from 8.1 +/- 2.3 (normoxic) to 15.2 +/- 2.1 pmol.min-1.mg protein-1 (anoxic). When the proximal tubules were homogenized in the absence of Ca, the anoxia-induced PLA2 activity was found to be soluble. Preincubation with pancreatic PLA2 antibody inhibited 50% of both basal and anoxia-stimulated PLA2 activity. Two protein bands (40- and 21-kDa species) immunoreactive to PLA2 antibody were detected in the membrane fraction. A sixfold increase in the immunoreactivity of the 40-kDa band was detected after 40 min of anoxia of proximal tubules. These results suggest that anoxia induces an intracellular PLA2 activity in kidney cells that could be immunologically related to pancreatic PLA2.

Adenosine Triphosphate↗
PubMed1992

Automated analysis for free and short-chain acylcarnitine in plasma with a centrifugal analyzer.

We describe a fully automated, spectrophotometric assay of free and total carnitine in plasma ultrafiltrates. The method, suitable for routine application in most hospital laboratories, incorporates the hydrolysis of acylcarnitines to free carnitine within the program of a Cobas Fara II centrifugal analyzer. The hydrolysis is monitored and calibrated with standard solutions containing octanoylcarnitine. Results correlated well with those from a reference isotope-dilution mass spectrometric assay. The ability to analyze a batch of samples for both free and total carnitine within 90 min enables analysis of > or = 100 samples per day. Used in conjunction with acylcarnitine species identification by mass spectrometry, the Cobas assay facilitates the diagnosis of carnitine-deficiency syndromes and specific metabolic disorders.

Acetylcarnitine↗
PubMed1991

Biomedical applications of high-performance liquid chromatography-mass spectrometry with continuous-flow fast atom bombardment.

This report describes the application of high-performance liquid chromatography combined with continuous-flow fast atom bombardment mass spectrometry to analytical problems in the biomedical laboratory. Applications include the compound-specific detection of diagnostic acylcarnitines in human urine, the separation and analysis of acyl-coenzyme A thioesters, and qualitative studies on complex mixtures of modified peptides (dansyl and dinitrophenyl derivatives). For each of these applications standard analytical columns (3.9 mm I.D.) and 1 ml/min flow-rates were employed with post-column stream splitting (1:100) before mass spectrometry. Various mobile phase compositions and solvent gradients were employed. The addition of 1-5% glycerol to the mobile phase was shown to have little effect on the chromatography. For all compounds studied (acylcarnitines, acyl-coenzyme A thioesters, and derivatized peptides) molecular weight information was obtained and sufficient sensitivity was achieved to allow unambiguous identification of trace components in complex mixtures.

Acyl Coenzyme A↗
PubMed1990

Combined high-performance liquid chromatographic-continuous-flow fast atom bombardment mass spectrometric analysis of acylcoenzyme A compounds.

A high-performance liquid chromatographic method for the analysis of coenzyme A thioesters which employs continuous-flow fast atom bombardment mass spectrometric detection is presented. The chromatographic system utilizes gradient elution with reversed-phase conditions using ammonium acetate-acetonitrile from both standard analytical (3.9 mm I.D.) and microbore (1 mm I.D.) columns. Applications to coenzyme A thioesters of various acyl group chain length (C2-C18) and functionality (-COOH, -OH, -C = C-) are described. The system is also applied to an in vitro enzyme reaction (crotonase) to directly follow the disappearance of substrate and appearance of product. The mass spectrometry of coenzyme A thioesters, their chromatographic behavior, system stability, and sensitivity of detection are discussed.

Acyl Coenzyme A↗
PubMed1990

Quantitative assay of free and total carnitine using tandem mass spectrometry.

A new, specific method for isotope dilution assay of total and free carnitine in urine has been developed. The method utilizes fast atom bombardment ionization with tandem mass spectrometry and requires minimal sample preparation. It compared well with radioenzymatic assay in terms of specificity, precision and accuracy, but was much more convenient in terms of analysis time and sample throughput. The new method is also applicable to the determination of free and short-chain total carnitine in plasma.

Carnitine↗
PubMed1990

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

In 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 (greater than 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 isotope dilution and chemical ionization mass spectrometry revealed a 30-fold increase in HG and increased suberylglycine, but no increase in 3-phenylpropionylglycine.

Acyl-CoA Dehydrogenase↗
PubMed1990

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.

Fatty Acid Desaturases↗
PubMed1990

Carnitine homeostasis in the organic acidurias.

The detection of acylcarnitines by FAB-MS in human physiological fluids has added another valuable diagnostic tool for the recognition of specific metabolic diseases. The newly developed technique of FAB-MS/MS, which embodies the principles of tandem mass spectrometry, affords a quantum leap in specificity, enabling the detection and quantification of acylcarnitines in concentrations well within the physiological ranges in urine, blood and tissue. The therapeutic use of L-carnitine appears to offer protection against the harmful consequences of catabolism. A reduction in the number of clinical episodes requiring hospitalization has been observed in most cases. When such occurrences have necessitated intravenous fluids, the addition of L-carnitine to the IV infusate has led to dramatic reduction in recovery times. It has also been shown that there is no discernable toxicity associated with carnitine therapy, even under high-dose IV conditions.

Carnitine↗