Stable isotope dilution analysis of isovalerylglycine in amniotic fluid and urine and its application for the prenatal diagnosis of isovaleric acidemia.

A stable isotope dilution method was developed to measure accurately small amounts of isovalerylglycine in amniotic fluid and urine for the prenatal diagnosis of isovaleric acidemia. [4,4,4-D3]Isovalerylglycine was synthesized and used as an internal standard. Samples were extracted, methylated, and analyzed by chemical ionization gas chromatography-mass spectrometry operated in the selected ion monitoring mode. This method is very sensitive (lower limit approximately 5 ng/ml), linear over three orders of magnitude above 10 ng/ml up to at least 10 micrograms/ml and reproducible. No isovalerylglycine was detected at all in amniotic fluids from eleven normal pregnant women with an exception of a single case which contained 6 ng/ml. Amniotic fluids from five pregnancies at risk were analyzed. Two of these samples had isovalerylglycine concentrations of 957 and 556 ng/ml. Three others contained 18, 18, and 17 ng/ml of isovalerylglycine. Postpartum diagnostic tests and/or in vitro assay of isovaleryl-CoA dehydrogenase of [1-14C] isovaleric acid oxidation using amniocytes confirmed that the first two fetuses were affected by isovaleric acidemia, whereas the latter three were unaffected. The method described in this report provides a highly accurate and reliable technique for the prenatal diagnosis of isovaleric acidemia.

Mechanism of action of short-chain, medium-chain, and long-chain acyl-CoA dehydrogenases. Direct evidence for carbanion formation as an intermediate step using enzyme-catalyzed C-2 proton/deuteron exchange in the absence of C-3 exchange.

The mechanisms of the initial interactions of three rat liver acyl-CoA dehydrogenases (short-chain, medium-chain, and long-chain acyl-CoA dehydrogenases) and their fatty acyl-CoA substrate were studied using enzyme-catalyzed deuterium exchange. The reaction products were identified and quantitated using mass spectroscopy and 1H-NMR. When fatty acyl-CoA substrates were incubated with catalytic amounts of acyl-CoA dehydrogenase in D2O in the absence of an electron acceptor, a rapid monodeuteration of the substrate occurred to replace one of the prochiral C-2 hydrogens, while no C-3 hydrogens were exchanged with deuterium. The C-2 monodeuteration proceeded to the extent of 80% of the total amount of substrate added at 90 min and almost to completion at 120 min. The pKa values and optimum pD values for the C-2 proton/deuteron exchange reactions were 6.0 and 7.5, respectively, for each of the three acyl-CoA dehydrogenases. The apparent turnover numbers were 3.0, 3.3, and 0.5 s-1 for short-chain, medium-chain, and long-chain acyl-CoA dehydrogenases, respectively. These results provide the first direct evidence for carbanion formation via abstraction of a C-2 hydrogen by a base in the enzyme, as the first step of the catalytic pathway of acyl-CoA dehydrogenation. When the acyl-CoA dehydrogenases were reacted with moderate excesses of acyl-CoA substrates in D2O in the absence of an electron acceptor, maximum bleaching of the FAD absorbance and the appearance of the long wavelength absorbance, attributed to a charge transfer complex, were observed. However, the dehydrogenation products, 2-enoyl-CoAs, were produced either not at all or in an amount which represented only a minor fraction of the amount of the enzyme added, while the substrates in the enzyme-substrate complexes rapidly turned over as indicated by the extensive monodeuteration which concomitantly occurred. Unlike previous hypothesis, these results indicate that the hydride ion transfer from C-3 of the substrate to the enzyme-FAD is not yet complete in the charge-transfer complex. The transfer of the hydride ion to alloxazine N-5 and the release of products are completed only in the presence of electron-transfer flavoprotein or another suitable electron acceptor.

Capillary gas chromatographic/mass spectrometric analysis of abnormal metabolites in hypoglycin-treated rat urine.

Numerous abnormal metabolites were identified in large amounts in the urine of hypoglycin-treated rats using capillary gas chromatography/mass spectrometry-computer analysis. These metabolites are not detectable in significant amounts in normal rats' urine. Ten of them have not been previously associated with hypoglycin administration: these are several hydroxy compounds, including those from the valine and isoleucine pathways, 2-oxo-adipic acid, n-butyrylglycine and isovaleryl glucuronide. These results indicate that the pathways of isoleucine and valine metabolism are inhibited at their respective acyl-CoA dehydrogenation steps, as is the case for fatty acid, leucine and lysine metabolism, as previously shown. The mass spectra of the trimethylsilyl derivatives of cis, cis-4,7-decadiene-1,10-dioic, cis-4-decene-1,10-dioic, cis-4-octene-1,8-dioic acids, and (methylenecyclopropyl)acetylglycine, which were previously identified using nuclear magnetic resonance and oxidative cleavage or acid hydrolysis, are presented for the first time.

The identification and the excretion pattern of isovaleryl glucuronide in the urine of patients with isovaleric acidemia.

We identified isovaleryl glucuronide in the urine of patients with isovaleric acidemia by using gas chromatography-mass spectrometry (GC-MS) and by identifying the products of enzymatic hydrolysis. Conjugation of isovaleryl-CoA with glycine, by the action of glycine-N-acylase, is the main detoxification mechanism in isovaleric acidemia. The identification of isovaleryl glucuronide demonstrates a hitherto unknown, additional detoxification mechanism in patients with isovaleric acidemia. Quantitative analysis of 72 urine specimens from four patients with isovaleric acidemia shows that isovaleryl glucuronide is more likely to be excreted when the amount of urinary 3-hydroxyisovaleric acid excretion is high. This suggests that detoxification via glucuronide conjugation plays an important role when the glycine conjugation system is saturated.

Compilation of gas chromatographic retention indices of 163 metabolically important organic acids, and their use in detection of patients with organic acidurias.

Gas chromatographic retention indices have been compiled for 163 metabolically important compounds (mostly organic acids) in the form of methylene units, as trimethylsilyl derivatives, on 10% OV-1 and 10% OV-17 columns. Comprehensive references on metabolic diseases that can be diagnosed by detection of these metabolites are cross-indexed to facilitate the use of the methylene-unit list. The gas chromatographic method, which utilizes extraction of urine with ethyl acetate and trimethylsilylations, is described. Modified methods, one for neutral compounds and one for highly polar organic acids, both of which utilize appropriate ion exchange and lyophilization, are also described. Practical applications of these methods and the use of the methylene-unit in the diagnosis of eleven patients with various metabolic disorders are also shown.

Gas-chromatographic method of analysis for urinary organic acids. I. Retention indices of 155 metabolically important compounds.

Gas-chromatographic retention indices are given, in terms of methylene units, for 155 metabolically important compounds (mostly organic acids) as trimethylsilyl derivatives on 10% OV-1 and 10% OV-17 columns. Comprehensive references on metabolic diseases that can be diagnosed by detection of these metabolites are cross-indexed to facilitate the use of the methylene-unit list. With the data presented here, it is now possible to diagnose more than 25 well-defined organic acidurias by use of gas chromatography alone.

Gas-chromatographic method of analysis for urinary organic acids. II. Description of the procedure, and its application to diagnosis of patients with organic acidurias.

A gas-chromatographic method for urinary organic acid analysis is described, designed to be used routinely for the diagnosis of organic aciduria. It involves extraction of urine with ethyl acetate, dehydration of extract residues, and trimethylsilylation. Organic acids are identified by using an extensive list of retention indices published in the accompanying paper (this issue). Quantitative values are given for organic acids in urines from 50 ostensibly normal subjects. Typical chromatograms of urinary organic acids from patients with eight well-established organic acidurias are also shown.