Uridine Diphosphate Glucuronosyltransferase UDPGT

Metabolic implications. Glucuronidation is quantitatively the most important conjugation reaction of xenobiotics mediated by uridine diphosphate-glucuronosyl-transferase (UDPGT) (Clarke and Burchell, 1994). It is generally considered a low-affinity and high-capacity reaction. More than 95% of the drugs in the market are metabolized by cytochrome P450s, UDPGT, and sulfotransferases.

Reaction type. Glucuronidation (Fig. 8.3):

(a) O-glucuronidation:

R-OH R-O-glucuronic acid (ether glucuronidation)

R-COOH -v R-COO-glucuronic acid (acyl (or ester) glucuronidation)

(b) N, S-glucuronidation:

Substrates. Glucuronidation can occur on nucleophilic moieties of molecules such as alcohol, acid (O-glucuronidation), amine (N-glucuronidation), and thiol (S-glucuronidation).

Enzyme structure. Oligomers of between 1 and 4 subunits with a molecular weight of between 50 and 60 kDa.

Cofactor. Uridine-5'-diphospho-a-D-glucuronic acid (UDPGA).

Tissue distribution. Liver, lung, kidney, stomach, intestine, skin, spleen, thymus, heart, and brain Most tissues have some glucuronidation activity.

Subcellular location. Mainly endoplasmic reticulum and some in the nuclear membrane.

Isozymes. More than 15 UDPGTs are known in humans, and they can be categorized into two major subfamilies, UDPGT 1 and UDPGT2. The UDPGT protein sequences exhibit greater than 60% similarity within the subfamily.

Species differences. Glucuronidation occurs in most mammalian species with the exception of the cat and related felines and the Gunn rat.

Glucuronosyltransferase

OH OH

Figure 8.3. Substitution reaction of a nucleophilic substrate (R-OH, R-COOH) on the C, carbon atom of uridine diphosphate-glucuronic acid (UDPGA) by uridine diphosphate-glucuronosyltransferase (UDPGT).

OH OH

Figure 8.3. Substitution reaction of a nucleophilic substrate (R-OH, R-COOH) on the C, carbon atom of uridine diphosphate-glucuronic acid (UDPGA) by uridine diphosphate-glucuronosyltransferase (UDPGT).

INDUCIBILITY. Inducible by phenobarbital, 3-methylcholanthrene (MC), or preg-nenolone-1 6a -carbonitrile (PCN) in rats. In humans, the induction of various UDPGT activities by phenobarbital, phenytoin, and oral contraceptives has been observed.

Polymorphism. Approximately 2-5% of the population have a defective UDPGT1 gene complex causing hyperbilirubinemia (Gilbert's syndrome) (Burchell et al., 1995).

In vitro experimental conditions. There are important in vitro experimental considerations, which can affect the degree of activities and substrate specificity of UDPGTs as a result of the latency of enzyme activity and chemical instability of acyl glucuronides.

8.3.1.1. Latency and Membrane Disruption by Detergents

The active site of the UDPGT lies on the lumenal side of the endoplasmic reticulum (ER), which restricts the access of substrates and UDPGA from cytosol. It has been suggested that UDPGA transport from cytosol onto the lumenal side of the ER across the ER membranes may be the rate-determining step for glucuroni-dation in the intact microsomes. Owing to this membrane barrier, UDPGTs are latent enzymes and their activities in freshly isolated microsomes cannot be fully revealed without disrupting the membranes to a certain degree. For instance, more than 95% of UDPGT activity can be latent in liver microsomes prepared in 0.25 M sucrose with 5 mM HEPES, pH 7.4. Disruption of microsomal membranes with detergents such as Lubrol Px can remove UDPGT latency and increase enzyme activity by up to 10- to 20-fold under the optimal conditions. Often, the ratio of protein and detergent (0.01-0.5 mg detergent/mg microsomal protein) has to be tested empirically for optimal activation of UDPGT, and preincubation of micro-somes with detergent(s) is required before an experiment (Burchell and Coughtrie, 1989; Mulder, 1992).

8.3.1.2. Chemical Instability of Acyl Glucuronides

In buffer or biological matrices at neutral or slightly alkaline pH, acyl glucuron-ides of many drugs with carboxylic acid moiety can undergo hydrolysis converting back to the parent drugs (futile cycling) and/or rearrangement (intramolecular rearrangement and intermolecular transacylation), whereas ether glucuronides are relatively stable. When acyl glucuronidation is anticipated, it is important to treat biological samples with acetic acid or HCl upon collection from animals, adjusting the pH to below 5, in order to minimize hydrolysis or rearrangement so that the amount of acyl glucuronides can be measured accurately (Kaspersen and Van Boeckel, 1987; Musson et al, 1985; Watt et al, 1991).

8.3.1.3. Acyl Migration

The rearrangement or isomerization reaction of acyl glucuronides involves the nonenzymatic migration of the drug moiety from the biosynthetic C1 position of the glucuronic acid ring to the neighboring C2, C3, or C4 positions (Fig. 8.4). In particular, intramolecular rearrangement (isomerization via acyl migration) of acyl glucuronide of drugs in plasma or albumin solutions can potentially lead to covalent binding of the drug moiety to proteins (intermolecular transacylation). These protein adducts with the (rearranged) acyl glucuronides of drugs have been proposed as possible causes of the in vivo toxicity seen in drugs with acid moieties (Hayball, 1995; Smith et al., 1986).

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