Microsomal protein concentrations

Tran, T.H., Von Moltke, L.L., Venkatakrishnan, K., Granda, B.W., Gibbs, M.A., Obach, R.S., Harmatz, J.S. and Greenblatt, D.J. (2002) Microsomal protein concentration modifies the apparent inhibitory potency of CYP3A inhibitors. Drug Metabolism and Disposition, 30 (12), 1441-1445. 

Figure 4. Effect of carp liver microsomal protein concentration on its mfo activity (NADPH 3 mg/mL, pH 7.4 incubated for 15 min at 25°C)...

Figure 5. Effect of incubation time on the carp liver microsomal mfo activity (NADPH 2 mg/mL, pH 7.4 incubated at 25° C. Microsomal protein concentration...

Several properties of hepatic microsomal AHH activity were compared in control and DBA-pretreated male little skates as shown in Table I. Following treatment there was an approximately 35-fold increase in specific enzyme activity, as quantitated by fluorescence of the phenolic metabolites formed (3, 21). The pH optimum, which was fairly broad, and the concentration of benzo(a)-pyrene (0.06 mM) that had to be added to the incubation mixture to achieve maximum enzyme activity were the same for both control and induced skate hepatic microsomes. The shorter periods observed for linearity of product formation with microsomes from the induced skates is thought to be related to the much higher AHH activity present, and may be due to substrate depletion or the formation of products which are inhibitory (i.e., compete with the MFO system as they are substrates themselves). A similar explanation may be relevant for the loss of linear product formation at lower microsomal protein concentrations in the induced animals. 

Low microsomal protein concentrations of 0.1 mg/mL or below reduce effects of microsomal binding. 

Zhao and Lou [164] studied the metabolism of omeprazole to its two major metabolites, hydroxyomeprazole and omeprazole sulfone, in rat liver microsomes by a reversed-phase HPLC assay. The formation of metabolites of omeprazole depended on incubation time, substrate concentration, microsomal protein concentration, and was found to be optimal at pH 7.4. The Pmax and Km of omeprazole hydroxylation in the rat liver microsomal preparation were 2033 nmol /(min mg protein), and 46.8 ymol/l, respectively. The effects of seven drugs on omeprazole metabolism were tested. Mephenytoin, five benzodiazepines and pava-verine caused inhibition of omeprazole metabolism. 

Figure 3 Effect of protein concentration on the inhibition of CYP2C8 (paclitaxel 6a-hydroxylation) by montelukast in human liver microsomes. The inhibitory effect of montelukast (CYP2C8 inhibitor) on the conversion of paclitaxel to 6a-hydroxypaclitaxel declined almost 20-fold when the microsomal protein concentration increased 20-fold due to nonspecific protein binding.

Preincubation time with inhibitor Microsomal protein concentration ... 

Figure 12 Effect of microsomal protein concentration on the mechanism-based inhibition of CYP1A2 by furafylline (A) and CYP2A6 by 8-methoxypsoralen (B). The typical IC50 experiment was conducted, except that microsomal protein concentrations of 0.1 and 1 mg/mL were utilized, a 15-minute preincubation period was used, and, to prevent over metabolism, coumarin (CYP2A6 substrate) was incubated at 50 pM. Phenacetin (60 pM) was used to measure CYP1A2 activity.

For CYP inhibition assays, organic solvent concentrations need to be kept low, usually with DMSO at no more than 0.2%, so once again insoluble compounds will be unsuitable for such assays. When microsomes are used, microsome protein concentration can drastically affect results. At high concentrations extensive binding to phospholipids and rapid metabolism may occur, leading to an underestimate of inhibitory potential.Because of this, microsome protein concentrations need to be kept low. Substrate concentrations can also affect results, and for maximum relevance they should be kept close to concentrations that they might reach in vivo. 

Gertz M, Kilford PJ, Houston B, Galetin A. 2008. Drng hpophilic-ity and microsomal protein concentration as determinants in the prediction of the fraction nnbound in microsomals incubations. Drug Metab Dispos 36 535-542. 

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