Validation of serotonin (5-hydroxtryptamine) as an in vitro substrate probe for human UDP-glucuronosyltransferase...

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 V(max) values ranging from 0.62 to 51.3 nmol/min/mg of protein but very similar apparent K(m) values ranging from 5.2 to 8.8 mM. Pooled human kidney, intestine, and lung microsomes had V(max) 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 K(m) values of 6.5 +/- 0.9, 12.4 +/- 2.0, and 4.9 +/- 3.3 mM (respectively). In comparison, recombinant UGT1A6 had a V(max) of 4.5 +/- 0.1 nmol/min/mg of protein and an apparent K(m) 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 (R(s) = 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.