A coupled assay detecting defects in fibroblast isoleucine degradation distal to enoyl-CoA hydratase: application to 3-oxothiolase deficiency

Clin Chim Acta. 1992 Jan 31;205(1-2):127-35. doi: 10.1016/s0009-8981(05)80007-5.

Abstract

We developed a coupled NaH14CO3 fixation assay to detect 3-oxothiolase deficiency in extracts of cultured human fibroblasts. Cell extracts were incubated with tiglyl-CoA, NAD, CoASH, ATP and NaH14CO3. The enzymatic activities of tiglyl-CoA (enoyl-CoA) hydratase, 2-methyl-3-hydroxybutyryl-CoA dehydrogenase and 2-methylacetoacetyl-CoA thiolase (3-oxothiolase) were coupled to produce propionyl-CoA. Propionyl-CoA produced in the assay was estimated by fixation of NaH14CO3 into [14C]methylmalonyl-CoA employing endogenous propionyl-CoA carboxylase. The control activity was 32 +/- 23 pmol/min per mg protein (+/- 1 S.D., range 7-94; 28 cell lines). Five known cases of 3-oxothiolase deficiency had a mean activity of 2% of the control; a sixth case of 3-oxothiolase deficiency was significantly higher at 27% of the mean control value. Coupled assay activity was also low (3% of control) in the cells from a patient with propionyl-CoA carboxylase deficiency.

MeSH terms

  • Acetyl-CoA C-Acetyltransferase / deficiency*
  • Acyl Coenzyme A / metabolism
  • Adenosine Triphosphate / pharmacology
  • Bicarbonates / metabolism
  • Bicarbonates / pharmacology
  • Carbon Radioisotopes
  • Cells, Cultured
  • Enoyl-CoA Hydratase / metabolism*
  • Fibroblasts / enzymology*
  • Humans
  • Isoleucine / metabolism*
  • Magnesium Chloride / pharmacology
  • NAD / metabolism
  • Sodium / metabolism
  • Sodium / pharmacology
  • Sodium Bicarbonate

Substances

  • Acyl Coenzyme A
  • Bicarbonates
  • Carbon Radioisotopes
  • Magnesium Chloride
  • Isoleucine
  • NAD
  • methylmalonyl-coenzyme A
  • propionyl-coenzyme A
  • tiglyl-coenzyme A
  • Adenosine Triphosphate
  • Sodium Bicarbonate
  • Sodium
  • Acetyl-CoA C-Acetyltransferase
  • Enoyl-CoA Hydratase