TABLE 1

Effect of assay conditions on estradiol glucuronidation

Two separate microsomal preparations (phosphate or sucrose), each composed of a mixture of six human livers, were incubated with 100 μM estradiol using varying assay buffers (A, 100 mM potassium phosphate, pH 7.1, 1 mM MgCl2, 5 mM saccharic acid 1,4-lactone; B, 100 mM Tris/malate, pH 7.4, 10 mM MgCl2, 10 mM saccharic acid 1,4-lactone) and activation conditions (see Materials and Methods). Each value is the mean of duplicate incubations. The fold activation was calculated as estradiol glucuronidaton at the maximal level of activation divided by estradiol glucuronidation obtained with untreated microsomes. Maximal activation was obtained with 50 μg/mg alamethecin and 5 s of sonication, done four times, in all cases. However, the concentration of Brij 58 required varied with different assay conditions.


Microsomal Preparation

Assay Buffer

Activation

Estradiol-3-glucuronidation

Estradiol-17-glucuronidation
Fold Activation
Activity
Fold Activation
Activity
pmol/min/mg pmol/min/mg
Phosphate A Alamethecin 3 870 3 150
Phosphate B Alamethecin 2.5 1230 2.2 240
Sucrose A Alamethecin 3 540 2.2 150
Sucrose B Alamethecin 2.8 740 2 180
Phosphate A Sonication 2.5 480 2.8 60
Phosphate B Sonication 1.8 710 2.2 120
Sucrose A Sonication 1.4 230 1.5 40
Sucrose B Sonication 1.8 280 2 70
Phosphate A Brij 58 2.6 770 3.8 120
Phosphate B Brij 58 1.6 780 2.5 160
Sucrose A Brij 58 2.3 390 3 80
Sucrose
B
Brij 58
2.5
370
2.9
80