Covariation of Human Microsomal Protein Per Gram of Liver with Age: Absence of Influence of Operator and Sample Storage May Justify Interlaboratory Data Pooling

Abstract

The use of in vitro-in vivo extrapolation (IVIVE) of metabolic data in the prediction of population clearance has become an important tool in both discovery and preclinical phases of the drug development process (Rostami-Hodjegan and Tucker, 2007). Scaling of metabolic rate constants derived using microsomal protein (MSP) isolated from human livers or those from recombinantly expressed cytochrome P450 enzymes requires knowledge of the amount of microsomal protein per gram of liver (MPPGL) among other scaling factors (Barter et al., 2007). The most commonly used value of MPPGL in human IVIVE is 45 mg g Ϫ1 (Obach, 1997;Soars et al., 2002;Andersson et al., 2004;Uchaipichat et al., 2006) reported in a review by Houston (1994). However, this value is not obtained from human livers; instead it is a combination of data generated using rat tissue from a number of sources (Joly et al., 1975;Lin et al., 1978; Baarnhielm et al., 1986;Chiba et al., 1990). Several values of MPPGL determined using human tissue have been reported in the literature, and a detailed review of these studies has been the focus of a recent consensus article on the most appropriate values of MPPGL for use in IVIVE alongside other scaling factors such as human hepatocellularity (Barter et al., 2007). Collation of values of MPPGL from five studies (114 observations; age range 11-80 years, median 48 years; 47 females) has indicated a weak but statistically significant inverse relationship between MPPGL and donor age (Barter et al., 2007). Barter et al. (2007) assumed that different experimental procedures and technical staff carrying out the exercise ("operators") would not introduce bias into their analysis. However, these effects have not been assessed systematically. Assessing the operator effect also has implications for within-laboratory pooling of data. The preparation and analysis of replicate samples, which are required to differentiate between methodological and true biological variability in values of MPPGL, is time-consuming (2 days/liver). To increase output, processing of samples may be performed by more than one individual (operator); however, this will require an indication of consistency between estimates of MPPGL by these operators.In addition, determination of values of MPPGL from fresh tissue from large numbers of donors is hampered by the infrequent supply of human tissue and logistical problems associated with immediate analysis of the samples when they arrive at the laboratory. In theory, the use of tissue stored at Ϫ80°C solves this problem. However, the use of frozen samples assumes maintenance of protein structure and function through the freeze-thaw process. Thus, to ensure adequate compatibility between the results obtained within and between differ-Z.E.B. was supported by Simcyp Ltd., (Sheffield, UK) and European Union Framework 6 (BIOSIM).Article, publication date, and citation information can be found at http://dmd.aspetjournals.org. doi:10.1124/dmd.108.021311. ABBREVIATIONS:IVIVE, in vitro-in vivo extrapolation; MSP, micr...

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