UDPGA

For the reaction to proceed, glucuronate must be delivered by UDPGA, which is generated in the cytosol, not in the endoplasmic reticulum. It is synthesized in a two-step process ... 

Microsomes are widely used to study the metabolism of xenobiotics. Enzymes can be chararacterized on the basis of their requirement for cofactors (e.g., NADPH, UDPGA), and their response to inhibitors. Kinetic studies can be carried out, and kinetic constants determined. They are very useful in studies of comparative metabolism, where many species not available for in vivo experiment can be compared with widely investigated laboratory species such as rats, mice, feral pigeon, Japanese quail, and rainbow trout. 

Abbreviations NADPH, b-nicotinamide adenine dinucleotide phosphate reduced from 5 -phosphosulfate UDPGA, uridine diphosphate-glucuronic acid. 

To prepare glucuronide conjugates of products formed from initial oxidation (to yield a hydroxyl or carboxylic acid group), the oxidative metabolite should be prepared first and used as a substrate in the incubation. Alternatively, an incubation with parent drug, NADPH and UDPGA may produce the desired glucuronide. 

A typical procedure for setting up a preparative incubation is as follows the substrate solution with a minimal amount of organic solvent or solvent mixture is mixed with buffer (with necessary additives) in an Erlenmeyer flask. Microsomes or S9 is then added to the mixture, followed by addition of NADPH (or UDPGA) stock solution. The flask is incubated at 37 °C in a water bath with gentle shaking. The reaction kinetics in a large-volume incubation will be different from the 0.2-1.OmL incubation, so progress of the reaction should be monitored closely with a short HPLC-UV-MS method. 

FIGURE 7.1 Glucuronidation of a substrate containing an OH group utilizing UDPGA as a cofactor. 

Figure 12.2 The X-ray structure of human UGT2B7 (left) showing the UDPGA-binding site (left), and their molecular interaction fields (right) obtained using GRID force field [21], showing the large cavity and the hydrophilic regions (in blue).

Finally, the different atoms in each substrate are assigned a similarity score. Because of the computational mechanism, the score is proportional to the exposure of such substrate atoms toward the reactive heme or UDPGA (in CYP or in UGT, respectively) and represents the accessibility component. 

Tris-HCl (120 gL), CaCl2 (60 gL), BSA (60 gL), DTT (3 gL), water (87 /iL) and freshly thawed microsomal fraction (30 gL) were mixed well and divided into three reaction tubes one reaction and two control tubes (1 and 2). 40 uL of freshly prepared UDPGA was added to the reaction tube and control 1 and 40 yfL of water to the control 2. The tubes were pre-incubated for 2 min in a water bath shaker at 35 °C. Then, 20 yiL of freshly prepared solutions of substrate in 20 % DMSO was added to the reaction and control 2 tubes, as well as 20 /rL of water to the control 1 mbe. Aliquots of 50 /rL were withdrawn from aU tubes and were quenched in acidified ice-cold methanol (160 gL per each 50 gL aliquot). The quenched aliquots collected during the incubation were left on ice for at least 20 min. After centrifugation for 10 min at 12000 rpm, the supernatants were collected into glass mbes and the remaining proteinaceous pellets were washed twice with 50 /iL of water and reprecipitated with 150 /iL of ice-cold methanol. The combined supernatants were dried under N2 gas and reconstimted into 50 /iL of appropriate mobile phase. 

UDPGA (2 mL, 100 mM) and freshly thawed microsomal fraction (1.5 mL, 20 mg protein/mL) were added to a 25 mL glass tube containing tris-HCl solution(2 mL), 1 mL CaCl2 (1 mL), BSA (1 mL), DTT (50 microL) and H2O (1.45 mL). The mixture was mixed well, sealed and pre-incubated in a reciprocal shaker for 2 min at 35 °C. Freshly prepared 1 mL substrate solution was added to the reaction mixture, which was subsequently sealed with mbber cap, purged with N2 gas and left in the reciprocal shaker (130 rpm) for 6-8 h at 35 °C. 

Note The production of minor products, alcohol (9-glucuronides, in given examples was prevented by saturation with both UDPGA and substrate, which usually does not happen in vivo. 

UDPGA uridine diphosphate glucuronic acid glucuronic acid donor. 

In phenylbutazone (86a) and sulfinpyrazone (86b) the acidity of the hydrogen on the methylene carbon of the 1,3-dicarbonyl system facilitates nucleophilic attack at C-l of the glucuronic acid moiety of UDPGA (see Scheme 7). The glucuronides formed (87a, 87b)... 

PDC pyruvate decarboxylase (R)-PAC (R)-phenylacetylcarbinol KDN 2-keto-3-deoxy-D-glycero-D-galacto-nonopyranulosonic add UDPGA /J-D-uridine diphosphoglucuronic add dTDP deoxythymine diphosphate ... 

Glucuronides were produced continuously in an enzyme membrane reactor from aglycones and /3-D-uridine diphosphoglucuronic acid (UDPGA) in the presence of a guinea-pig liver preparation of /3-glucuronidase for between 24 and 118 h (Pfaar, 1999). 

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