Brief communicationDetermination of the hepatocellularity number for human, dog, rabbit, rat and mouse livers from protein concentration measurements
Introduction
The number of hepatocytes per gram of liver must be used as a scaling factor when in vivo hepatic clearance for xenobiotics is predicted from hepatocyte intrinsic clearance (Houston, 1994). The present work was undertaken in order to determine the hepatocellularity number for human, dog, rabbit, rat and mouse livers from the protein concentration of liver homogenates and hepatocyte suspensions. Previously, most efforts to determine hepatocellularity numbers have involved the rat liver, whereas surprisingly little data for other species have been presented. To date, accurately determined hepatocellularity numbers for other species than rats and humans have not been reported.
Determination of the rat hepatocellularity number based on DNA, protein or cytochrome P450 (CYP) content as well as morphometric methods has taken place, with somewhat different results (Carlile et al., 1997, Ekins et al., 1995). The DNA, CYP and protein content methods are all based on comparison between a whole liver and a hepatocyte suspension. For example, the total DNA amount per gram of liver is compared with the amount of DNA per 106 cells. One drawback of using the DNA content method is that some cells are binuclear (Seglen, 1973), resulting in a somewhat uncertain hepatocellularity number. The specific concentration of CYP is difficult to measure in liver homogenate, probably due to quenching and light scattering by the large amount of other proteins and particles present. Thus, comparing the CYP content in whole liver with a hepatocyte suspension might also give uncertain results.
The commonly employed standard hepatocellularity number used for scaling of in vitro hepatocyte data is 120 × 106 cells/g liver for human and rat hepatocytes (Bayliss et al., 1990, Bayliss et al., 1999). Detailed information on the determination of the dog hepatocellularity number has not been presented, although Bayliss et al., 1990, Bayliss et al., 1999 suggested 240 × 106 cells/g liver. However, since these authors did not specify which method they used or the degree of variability in the obtained data, it is difficult to determine the accuracy of this figure.
Interspecies comparisons of in vitro/in vivo correlations of new entities are often essential within the pharmaceutical industry during the process of dose scaling to man. Moreover, various animal models are often employed to determine the effect of new entities, and it is therefore important to compare the metabolism in these different animal species with that in man. For these reasons, hepatocellularity numbers for animals are needed when intrinsic clearances in animal hepatocytes are used to predict in vivo clearance.
The aim of the present study was to investigate further the hepatocellularity number of the dog liver and to compare it with rat and human values. In addition, the hepatocellularity numbers for the mouse and the rabbit were also studied. Hepatocytes represent almost the same protein concentration in the liver as in hepatocyte suspensions (Worboys et al., 1996), for which reason the hepatocellularity number was calculated from the ratio between the liver homogenate protein concentration and the protein concentration in the corresponding hepatocyte suspension. The advantages of using this method compared to the CYP and DNA content methods, are that the protein concentration is easily measured in both cell suspensions and in homogenate, and that binuclear cells do not affect the results. In the present study, the liver density for human, dog, rabbit and rat livers was also measured.
Section snippets
Chemicals
Forene® (isoflurane) for anesthesia came from Abbott Scandinavia AB (Kista, Sweden); Krebs–Henseleit buffer, Williams’ E medium, collagenase type XI (⩾1200 collagen digestion units/mg solid), trypan blue and EGTA were obtained from Sigma Chemical Co. (St. Louis, Mo., USA). All other chemicals were of analytical grade and obtained from common commercial suppliers.
Preparation of rat hepatocytes
Male Sprague–Dawley rats (8–9 weeks old; B&K Universal AB, Sollentuna, Sweden) were maintained under a 12 h light/dark cycle in steel
Hepatocellularity numbers for human, dog, rabbit, rat and mouse livers
There was no significant difference in the amount of protein per gram of liver between humans, dogs, rabbits, rats or mice (Table 1). However, the protein amount per million cells was significantly lower in suspensions with dog hepatocytes than in suspensions with hepatocytes from the other species (Table 1). For human, dog, rabbit, rat and mouse livers, the hepatocellular values obtained were 139 ± 25, 215 ± 45, 114 ± 20, 117 ± 30 and 135 ± 10 million cells per gram of liver, respectively (Table 2).
Liver tissue density
The
Discussion
In vitro intrinsic clearances for xenobiotics in hepatocytes are frequently employed to predict in vivo hepatic clearance (Houston, 1994). For these predictions, the number of hepatocytes per gram of liver is used as a scaling factor. However, although scaling factors are essential for scaling, surprisingly little information about the determinations of these hepatocellularity numbers has been reported in the literature. In the present study the hepatocellularity numbers of human, dog, rabbit,
Acknowledgements
The author wishes to thank Lotta Halvarsson at AstraZeneca R&D Södertälje for her help with the rats and rat hepatocyte preparations; Eva Floby, AstraZeneca R&D Södertälje, for much help with the hepatocytes; Dr. Anna Nordmark and Dr.Gunnel Tybring, formerly of the Division of Clinical Pharmacology and Technology at the Karolinska Institute, for providing human liver samples; Ann-Louise Hagbjörk and Dr. Ylva Terelius, AstraZeneca R&D Södertälje, for providing dog and rabbit liver samples. Dr.
References (12)
Relevance of in vitro kinetic parameters to in vivo metabolism of xenobiotics
Toxicol. Vitro
(1994)- et al.
Cryopreserved human hepatocytes: characterization of drug-metabolizing enzyme activities and applications in higher throughput screening assays for hepatotoxicity, metabolic stability and drug–drug interaction potential
Chemico-Biologic. Interact.
(1999) - et al.
Protein measurement with the Folin phenol reagent
J. Biol. Chem.
(1951) Preparation of rat liver cells. 3. Enzymatic requirements for tissue dispersion
Exp. Cell Res.
(1973)- et al.
The isolation of hormone-sensitive rat hepatocytes by a modified enzymatic technique
Arch. Biochem. Biophys.
(1974) - et al.
Prediction of intrinsic clearance of loxtidine from kinetic studies in rat, dog and human hepatocytes
Biochem. Soc. Transact.
(1990)
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