Human liver S9 fractions stored at -70°C maintain high phase I and phase II enzymatic activities over multiple freeze/thaw cycles and for at least 10 years
Expanded interest in the biotransformation of xenobiotics by phase II enzymes and other various cytosolic enzymes has led to an increase in the use of S9 fractions in metabolic stability, clearance, and phenotyping studies. To ensure the integrity of an in vitro test system, it is important to understand the stability of metabolic enzymes during long-term storage and throughout multiple freeze/thaw cycles. Data regarding the stability of human liver microsomes have been previously published by multiple authors (Pearce et al, 1996, Yamazaki et al, 1996). However, metabolic enzyme stability for S9 stored for long periods and/or subjected to multiple freeze/thaw cycles has not been thoroughly investigated.
The objectives of this study were to evaluate the effects of long-term storage at -70°C or below and multiple freeze/thaw cycles on the enzymatic activities in human liver S9 fractions. Nine lots of pooled (n = 16 to 200)Human liver S9 samples were prepared over a ten year period and stored at -70°C or below for up to 10 years. These S9 fractions were analyzed for their ability to catalyze reactions for various phase I and phase II enzymes including cytochrome P450 (CYP), UDP-glucuronosyltransferase (UGT), glutathione S-transferase (GST), sulfotransferase (SULT), aldehyde oxidase (AO), and N-acetyltransferase (NAT). Additionally, a single lot of pooled (n = 200)Human liver S9 samples were subjected to up to 10 freeze/thaw cycles and further evaluated for potential loss of enzymatic activities with increasing freeze/thaw cycles.