UGTs

Figure 32-14. Conjugation of bilirubin with glucuronic acid. The glucuronate donor, UDP-glucuronic acid, is formed from UDP-glucose as depicted. The UDP-glucuronosyl-transferase is also called bilirubin-UGT.

Type I Crigler-Najjar syndrome is a rare autosomal recessive disorder. It is characterized by severe congenital jaundice (serum bilirubin usually exceeds 20 mg/dL) due to mutations in the gene encoding bilirubin-UGT activity in hepatic tissues. The disease is often fatal within the first 15 months of life. Children with this condition have been treated with phototherapy, resulting in some reduction in plasma bilirubin levels. Phenobarbital has no effect on the formation of bilirubin glucuronides in patients with type I Crigler-Najjar syndrome. A liver transplant may be curative. 

This rare inherited disorder also results from mutations in the gene encoding bilirubin-UGT, but some activity of the enzyme is retained and the condition has a more benign course than type I. Serum bilirubin concentrations usually do not exceed 20 mg/dL. Patients with this condition can respond to treatment with large doses of phenobarbital. 

Again, this is caused by mutations in the gene encoding bilirubin-UGT, but approximately 30% of the enzyme s activity is preserved and the condition is entirely harmless. 

The assessment of clearance is complicated by the numerous mechanisms by which compounds may be cleared from the body. These mechanisms include oxidative metabolism, most commonly by CYP enzymes, but also in some cases by other enzymes including but not limited to monoamine oxidases (MAO), flavin-containing monooxygenases (FMO), and aldehyde oxidase [45, 46], Non-oxidative metabolism such as conjugation or hydrolysis may be effected by enzymes such as glucuronyl transferases (UGT), glutathione transferases (GST), amidases, esterases, or ketone reductases, as well as other enzymes [47, 48], In addition to metabolic pathways, parent compound may be excreted directly via passive or active transport processes, most commonly into the urine or bile. 

In Slater s half electron transition state scheme the integral is replaced by a calculation of the orbital energy occupied by half an electron. In the generalised transition state method (GTS) [75] and its related method (UGTS) using unrestricted DFT, the integral is replaced by the expression shown in Equation (14). 

Although there are many enzymes involved in drug metabolism, the in vitro systems containing CYPs and UGTs are often used for metabolite synthesis due to the reasons discussed before. 

Screening is usually carried out with liver microsomes from humans, rats, mice, dogs and monkeys and liver S9 fraction from aroclor 1254-induced rats. The incubation is typically mn with a volume of 0.2-1. OmL in a microcentrifuge or a glass tube. Different incubation conditions are used for CYP and UGT reactions. The incubation mixture for formation of oxidative metabolites and/or GSH conjugates contains ... 

D-Saccharic acid 1,4-lactone (SAL 5-10 mM), a selective inhibitor of/3-glucuronidase [29], is often included in UGT reactions for improving yield. As /3-glucuronidases and UGTs have different optimal pH ranges, use of SAL in preparative-scale reactions may be avoided. 

Acetonitrile, methanol and DMSO had no apparent effect on umbelliferone glucuronidation in human hepatocytes at concentration up to 2% [32]. With HLMs or expressed UGTs, inhibitory effects of organic solvents on glucuronidation of 7-hydroxy-4-trifluoromethyl-coumarin (7-HFC) and estradiol generally followed the order acetonitrile > ethanol > methonal > DMSO [33], DMSO did not inhibit estradiol-3-glucuronidation activity at a concentration up... 

A typical CYP reaction length is 1-2 h, but CYP activity can survive longer at 37 °C. Figure 9.3 shows that product turnover of the CYP reactions occurred over a 6 h incubation (Li, unpublished results). UGT activity can last longer than 24 h [23], With accumulation of product, secondary reactions, such as further oxidation of product or hydrolysis of glucuronide, may become noticeable. Therefore, monitoring the reaction with HPLC-U V-MS is critical for identifying the best time to terminate the reaction. 

Dehal, S.S., Gagne, P.V., Crespi, C.L. and Parren, C.J. (2002) Effect of common organic solvent on human UGT enzyme activities. 11th North American ISSX Meeting, October 27-31, 2002, Orlando, Florida, USA, Abstract 370. 

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