Table 2

MDZ 1′-hydroxylation kinetics in Caco-2 cell cultures at the 1–2-hr incubation interval

1′-OH-MDZ formation rate75.9 pmol/min/g of cells
Average MDZ concentration
 Basolateral650  nm
 Apical2740  nm
Unbound MDZ concentration
 Basolateral66.3  nm
 Apical280 nm
Intrinsic formation clearance1.14 ml/min/g of cells

MDZ (4 μm) was added at time zero to the medium in the apical compartment of replicate Caco-2 cell cultures that had been treated with 0.25 μm1α,25-(OH)2-D3 for 2 weeks after confluence. 1α,25-(OH)2-D3 was not present in the MDZ incubation medium. Both apical and basolateral media were sampled 1 and 2 hr later (from separate cultures) for measurement of MDZ and 1′-OH-MDZ. The unbound fraction of MDZ in culture medium, measured by equilibrium dialysis, was found to be 10.2%. Calculations were performed as described in Experimental Procedures.