Free fatty acid determination by peroxidase catalysed luminol chemiluminescence.
A sensitive, specific, and partly automatic method for the analysis of free fatty acids is described. The assay involves activation of free fatty acids by acyl-CoA synthetase (EC 6.2.1.3) followed by oxidation of the thioesters by acyl-CoA oxidase. The H2O2 formed is determined in a reaction catalysed by horseradish peroxidase (EC 1.11.1.7) using luminol as electron donor. The assay has a linear range of 0.05 to 5 nmol of different free fatty acids (C10-C18) in the original sample. The efficiency of the method toward capric, lauric, myristic, palmitic, palmitoleic, stearic, oleic, and linoleic acid measured as recovery of light emission compared to that of H2O2 standards, was over 90%. AffiGel 501 was used to covalently bind the free thiol group in CoASH eliminating interference of this substance in the peroxidase-luminol reaction.
Research Centre, Karolinska Institute, Huddinge University Hospital, Sweden.
B M
BM
Näslund
K
K
Bernström
A
A
Lundin
P
P
Arner
D042962:Acyl-CoA Oxidase; D003066:Coenzyme A Ligases; D005230:Fatty Acids, Nonesterified / Q000032:analysis* / Q000592:standards; D006735:Horseradish Peroxidase; D008163:Luminescent Measurements; D008165:Luminol; D010088:Oxidoreductases; D012015:Reference Standards; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D000098859:Long-Chain-Fatty-Acid-CoA Ligase
D005230:Fatty Acids, Nonesterified; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D008165:Luminol; D010088:Oxidoreductases; D006735:Horseradish Peroxidase; D042962:Acyl-CoA Oxidase; D003066:Coenzyme A Ligases; C090799:FAA2 protein, S cerevisiae; D000098859:Long-Chain-Fatty-Acid-CoA Ligase