Determination of the epimeric composition of ibuprofenyl-CoA.
Ibuprofen [racemic2-(4-isobutylphenyl)propionic acid] is a 2-arylpropionic acid nonsteroidal anti-inflammatory drug which undergoes unidirectional, R to S chiral inversion in vivo. It has been proposed that this chiral inversion phenomenon occurs via a coenzyme A (CoA) thioester intermediate. To characterize the formation and metabolism of this metabolic intermediate, ibuprofenyl-CoA, reference standards were needed and thus the CoA derivatives of (R)-, (S)-, and racemic ibuprofen were chemically synthesized. An HPLC assay employing a C18 reverse-phase column was developed to quantitate "total" ibuprofenyl CoA. Samples collected from this assay were then analyzed for ibuprofenyl-CoA epimeric composition by chiral chromatography employing a Chiral-AGP alpha 1-acid glycoprotein column. The applicability of these methods was demonstrated by assessing (R)- and (S)-ibuprofenyl-CoA hydrolysis and epimerization following incubation with rat liver homogenates. Rat liver homogenate catalyzed the complete and rapid epimerization of ibuprofenyl-CoA and the rate constants for (R)- and (S)-ibuprofenyl-CoA hydrolysis were equal. ATP and CoA were found to inhibit rat liver-catalyzed ibuprofenyl-CoA hydrolysis by 70-80% with no effect on epimerization. Additionally, it was demonstrated that traditional indirect ibuprofenyl-CoA assays which employ basic hydrolysis result in erroneous epimeric ratio determinations due to chemical epimerization.
Tracy
T S
TS
Department of Medicine, Indiana University School of Medicine, Wishard Memorial Hospital, Indianapolis 46202.
Hall
S D
SD
D000214:Acyl Coenzyme A / Q000032:analysis / Q000138:chemical synthesis / Q000737:chemistry*; D000255:Adenosine Triphosphate / Q000737:chemistry; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D003065:Coenzyme A / Q000737:chemistry; D006868:Hydrolysis; D015394:Molecular Structure; D051381:Rats; D012015:Reference Standards; D013237:Stereoisomerism
D000214:Acyl Coenzyme A; C070652:ibuprofenyl-coenzyme A; D000255:Adenosine Triphosphate; D003065:Coenzyme A