In vitro metabolism of acitretin by human liver microsomes: evidence of an acitretinoyl-coenzyme A thioester conjugate in the transesterification to etretinate.
The aromatic retinoid acitretin is the primary active metabolite of etretinate, and in this study we investigated the ethyl esterification of acitretin to etretinate using [(14)C]acitretin and human liver microsomes. Samples were analysed by TLC, HPLC, and LC-MS. Essential requirements for the transesterification reaction were identified and included viable microsomal protein, ATP, CoASH, and ethanol. Human liver microsomes catalysed formation of acitretinoyl-CoA at the rate of 0.08 +/- 0.02 nmol/min/mg (mean +/- SD, N = 10). Acitretinoyl-CoA was pivotal for the transesterification to etretinate and in the presence of methanol, ethanol, n-propanol, n-butanol, and hexanol, the corresponding esters, namely methyl-, ethyl (etretinate)-, propyl-, butyl-, and hexyl-acitretinate, were formed. On average, 1.7% of the acitretin present in the incubation was converted to etretinate in the presence of ethanol. In the absence of ethanol, transesterification did not proceed. Inhibition of the ester hydrolysis of etretinate by bis-p-nitrophenylphosphate (BNPP, 1 mM) prevented futile cycling of etretinate via acitretinoyl-CoA. An additional finding was that acitretin (15-30 microM) activated significantly human liver microsomal long-chain fatty acid-CoA ligase (E.C.6.2.1.3, LCL), resulting in enhanced formation of palmitoyl-CoA. This study demonstrated that in the presence of ethanol the ethyl esterification of acitretin to etretinate proceeds via formation of acitretinoyl-CoA. Predicting clearance of acitretin in vivo via this unique metabolic pathway will be a challenge, as the intracellular concentration of ethanol could never be predicted with any degree of accuracy in humans.
Knights
K M
KM
Department of Clinical Pharmacology, School of Medicine, Faculty of Health Sciences, Flinders University, 5042, Adelaide, Australia. kathie.knights@flinders.edu.au
Gasser
R
R
Klemisch
W
W
D000079:Acetaldehyde / Q000378:metabolism; D000085:Acetates / Q000378:metabolism; D017255:Acitretin / Q000378:metabolism*; D000214:Acyl Coenzyme A / Q000032:analysis; D003066:Coenzyme A Ligases / Q000378:metabolism; D004951:Esterification; D000431:Ethanol / Q000378:metabolism; D005050:Etretinate / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D008862:Microsomes, Liver / Q000378:metabolism*; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D000098859:Long-Chain-Fatty-Acid-CoA Ligase
D000085:Acetates; D000214:Acyl Coenzyme A; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; C412045:acitretinoyl-coenzyme A; D000431:Ethanol; D005050:Etretinate; C007650:ethyl acetate; D003066:Coenzyme A Ligases; C090799:FAA2 protein, S cerevisiae; D000098859:Long-Chain-Fatty-Acid-CoA Ligase; D000079:Acetaldehyde; D017255:Acitretin