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Vol. 31, Issue 1, 133-139, January 2003

Validation of Serotonin (5-Hydroxtryptamine) as an in Vitro Substrate Probe for Human UDP-Glucuronosyltransferase (UGT) 1A6

Soundararajan Krishnaswamy, Su X. Duan, Lisa L. von Moltke, David J. Greenblatt, and Michael H. Court

Comparative and Molecular Pharmacogenetics Laboratory (S.K., S.X.D., M.H.C.) and Clinical Pharmacology Laboratory (L.L.v.M., D.J.G.), Department of Pharmacology and Experimental Therapeutics, Tufts University School of Medicine, Boston, Massachusetts

Investigation of human UDP-glucuronosyltransferase (UGT) isoforms has been limited by a lack of specific substrate probes. In this study serotonin was evaluated for use as a probe substrate for human UGT1A6 using recombinant human UGTs and tissue microsomes. Of the 10 commercially available recombinant UGT isoforms, only UGT1A6 catalyzed serotonin glucuronidation. Serotonin-UGT activity at 40 mM serotonin concentration varied more than 40-fold among human livers (n = 54), ranging from 0.77 to 32.9 nmol/min/mg of protein with a median activity of 7.1 nmol/min/mg of protein. Serotonin-UGT activity kinetics of representative human liver microsomes (n = 7) and pooled human kidney, intestinal and lung microsomes and recombinant human UGT1A6 typically followed one enzyme Michaelis-Menten kinetics. Serotonin glucuronidation activity in these human liver microsomes had widely varying Vmax values ranging from 0.62 to 51.3 nmol/min/mg of protein but very similar apparent Km values ranging from 5.2 to 8.8 mM. Pooled human kidney, intestine, and lung microsomes had Vmax values (mean ± standard error of the estimates) of 8.8 ± 0.4, 0.22 ± 0.00, and 0.03 ± 0.00 nmol/min/mg of protein (respectively) and apparent Km values of 6.5 ± 0.9, 12.4 ± 2.0, and 4.9 ± 3.3 mM (respectively). In comparison, recombinant UGT1A6 had a Vmax of 4.5 ± 0.1 nmol/min/mg of protein and an apparent Km of 5.0 ± 0.4 mM. A highly significant correlation was found between immunoreactive UGT1A6 protein content and serotonin-UGT activity measured at 4 mM serotonin concentration in human liver microsomes (Rs = 0.769; P < 0.001) (n = 52). In conclusion, these results indicate that serotonin is a highly selective in vitro probe substrate for human UGT1A6.

Copyright © 2003 by The American Society for Pharmacology and Experimental Therapeutics


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Copyright © 2003 by the American Society for Pharmacology and Experimental Therapeutics.