Evaluation of a screening method by liquid chromatography-tandem mass spectrometry for estimating effect of drugs on the activation and β-oxidation of fatty acids in mitochondria.
OBJECTIVE
Fatty acid metabolism is controlled not only by the acyl-coenzyme A (CoA) synthetases but by some enzymes in the β-oxidation cycle. Medium-chain and long-chain acyl-CoA esters are key metabolites in fatty acid metabolism. We have developed an enzymatic assay method for determining chain shortening of the acyl-CoAs via β-oxidation from palmitic and octanoic acids in liver mitochondria. We have evaluated the assay method for detecting whether drugs influence the activation or the β-oxidation of fatty acids.


METHODS
Liver mitochondria were used for investigating the effect of drugs on fatty acid metabolism. The drugs selected were salicylic acid, diclofenac, valproic acid and paracetamol. Each acyl-CoA formed was analysed by liquid chromatography-tandem mass spectrometry.


RESULTS
After less than 5 min of incubation, the levels of acyl-CoAs reflected the acyl-CoA synthetase activity, whereas after 60-min incubation they reflected the activity of some enzymes in the β-oxidation cycle. Salicylic acid, diclofenac and valproic acid inhibited the medium-chain acyl-CoA synthetases, whereas valproic acid only exhibited a weak inhibitory activity toward the β-oxidation of the medium-chain fatty acids. In the case of long-chain fatty acid metabolism, salicylic acid and diclofenac inhibited both the activation and β-oxidation, whereas valproic acid was a weak inhibitor for only the β-oxidation activity. Paracetamol showed hardly any influence on the metabolism of medium-chain and long-chain fatty acids.


CONCLUSIONS
These findings suggest that salicylic acid, diclofenac, valproic acid and paracetamol exert a different influence on fatty acid metabolism depending on the length of the acyl chain. This assay allows sensitive and selective analysis for predicting the pathways by which drugs exert a greater influence over fatty acid metabolism.
Biochemical Toxicology Laboratory, Faculty of Pharmaceutical Sciences, Kobegakuin University, Kobe, Japan. kasuya@pharm.kobegakuin.ac.jp
Fumiyo
F
Kasuya
Ryota
R
Nishizawa
Teiichi
T
Masuyama
Maya
M
Kazumi
D000082:Acetaminophen / Q000494:pharmacology; D018712:Analgesics, Non-Narcotic / Q000494:pharmacology; D000818:Animals; D000890:Anti-Infective Agents / Q000494:pharmacology; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000032:analysis / Q000494:pharmacology; D002853:Chromatography, Liquid / Q000379:methods; D003066:Coenzyme A Ligases / Q000037:antagonists & inhibitors / Q000378:metabolism; D004008:Diclofenac / Q000378:metabolism / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005227:Fatty Acids / Q000037:antagonists & inhibitors / Q000378:metabolism* / Q000494:pharmacology; D050356:Lipid Metabolism / Q000187:drug effects*; D008297:Male; D051379:Mice; D008817:Mice, Mutant Strains; D008930:Mitochondria, Liver / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D010084:Oxidation-Reduction; D020156:Salicylic Acid / Q000494:pharmacology; D053719:Tandem Mass Spectrometry / Q000379:methods; D014635:Valproic Acid / Q000494:pharmacology
D018712:Analgesics, Non-Narcotic; D000890:Anti-Infective Agents; D000894:Anti-Inflammatory Agents, Non-Steroidal; D004791:Enzyme Inhibitors; D005227:Fatty Acids; D004008:Diclofenac; D000082:Acetaminophen; D014635:Valproic Acid; D003066:Coenzyme A Ligases; D020156:Salicylic Acid