Enzymatic assay for 3-hydroxyacyl-CoA and 2-trans-enoyl-CoA intermediates of beta-oxidation.
An enzymatic assay is presented for determining 3-hydroxyacyl-CoA and 2-trans-enoyl-CoA esters in tissue samples. The procedure includes extraction of acyl-CoA esters from frozen tissue samples with chloroform/methanol, stochiometric oxidation of the acyl esters to 3-keto-acyl-CoAs with 3-hydroxyacyl-CoA dehydrogenase in the presence or absence of enoyl-CoA hydratase, and an enzymatic cycling amplification of NADH produced for fluorometric determination. The procedure allows measurement of these intermediates of beta-oxidation at the picomole level. The method has been used successfully to measure the concentrations of 3-hydroxyacyl-CoA and 2-trans-enoyl-CoA esters in isolated rat hearts perfused with glucose or oleate or under anoxia.
Latipää
P M
PM
Department of Medical Biochemistry, University of Oulu, Finland.
Hassinen
I E
IE
Hiltunen
J K
JK
D015094:3-Hydroxyacyl CoA Dehydrogenases; D000214:Acyl Coenzyme A / Q000032:analysis*; D000818:Animals; D002384:Catalysis; D004746:Enoyl-CoA Hydratase; D044943:Fatty Acid Desaturases / Q000378:metabolism; D005227:Fatty Acids / Q000378:metabolism; D005260:Female; D006836:Hydro-Lyases; D008929:Mitochondria, Heart / Q000201:enzymology; D009243:NAD / Q000096:biosynthesis; D010084:Oxidation-Reduction; D051381:Rats; D011919:Rats, Inbred Strains; D013050:Spectrometry, Fluorescence
D000214:Acyl Coenzyme A; D005227:Fatty Acids; D009243:NAD; D015094:3-Hydroxyacyl CoA Dehydrogenases; D044943:Fatty Acid Desaturases; D006836:Hydro-Lyases; D004746:Enoyl-CoA Hydratase