3-Hydroxy-3-methylglutaryl-CoA lyase deficiency as detected by radiochemical assay in cell extracts by thin-layer chromatography, and identification of three new cases

Journal Article · Thu Feb 01 04:00:00 EST 1990 · Clinical Chemistry (Winston-Salem, North Carolina); (USA)

OSTI ID:6945626

Gibson, K M; Lee, C F; Kamali, V; Johnston, K; Beaudet, A L; Craigen, W J; Powell, B R; Schwartz, R; Tsai, M Y; Tuchman, M ^[1]

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1. Kimberly H. Courtwright Joseph W. Summers Metabolic Disease Center, Dallas, TX (USA)

In this rapid radiochemical assay for 3-hydroxy-3-methylglutaryl-coenzyme A lyase (I) activity in cell extracts, DL-3(glutaryl-3-14C)hydroxy-3-methylglutaryl-coenzyme A is used as substrate and the radiochemical product, (3-14C)acetoacetic acid, is converted to the more stable (3-14C)-3-hydroxybutyric acid in the presence of added NADH and 3-hydroxybutyrate dehydrogenase. Substrate and product are separated and quantified by thin-layer chromatography on cellulose (solvent system: butanol/water/formic acid, 77:13:10 by vol). All reagents for the assay are commercially available. No detailed column chromatography or spectrophotometry is required. Thus the assay is suited for any clinical laboratory. Using this procedure, we studied cultured fibroblasts or lymphocytes isolated from whole blood from five patients in whom the urinary organic acid profile was suggestive of deficiency of I. Three patients had less than or equal to 18% of control I activity in fibroblast or lymphocyte extracts. The other two had activity within the normal range. In one of the latter cases, urinary excretion of three of the characteristic acids disappeared with age, and 3-hydroxyisovaleric acid excretion was within normal limits. The other case presented with urinary excretion of moderate amounts of all four metabolites and the characteristic absence of urinary ketone bodies. Evidently, confirmatory enzyme studies should be undertaken, even when the profile of urinary organic acids appears definitive for this deficiency.

• OSTI ID: 6945626
• Journal Information: Clinical Chemistry (Winston-Salem, North Carolina); (USA), Journal Name: Clinical Chemistry (Winston-Salem, North Carolina); (USA) Vol. 36:2; ISSN 0009-9147; ISSN CLCHA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
AMINO ACIDS
ANIMAL CELLS
BIOLOGICAL MATERIALS
BLOOD
BLOOD CELLS
BODY FLUIDS
CARBON 14 COMPOUNDS
CARBOXYLIC ACIDS
CELL CULTURES
CHROMATOGRAPHY
CONNECTIVE TISSUE CELLS
ENZYME ACTIVITY
ENZYMES
FIBROBLASTS
ISOTOPE APPLICATIONS
LABELLED COMPOUNDS
LEUCINE
LEUKOCYTES
LYASES
LYMPHOCYTES
MATERIALS
ORGANIC ACIDS
ORGANIC COMPOUNDS
OXIDOREDUCTASES
RADIOASSAY
SEPARATION PROCESSES
SOMATIC CELLS
THIN-LAYER CHROMATOGRAPHY
TRACER TECHNIQUES