Hepatocytes measure

Hewitt NJ, Utesch D. Cryopreserved rat, dog and monkey hepatocytes measurement of dmg metabolizing enzymes in suspensions and eultures. Hum Exp Toxieol 2004 23 307-316. 

Primary Cultures of Human Hepatocytes Measurement of CYP3A4 induction in primary human hepatocyte cultures involves three steps isolation of hepatocytes from autopsy- or biopsy-derived human donor livers treatment with a test compound for three days following a 2-day recuperation culture period and measurement of the expression or activity of CYP3A4 (Fig. 17.1). 

Vaillant, C., Monod, G., and Volataire, Y. et al. (1989). Measurement and induction of cytochrome P450 and monooxygenases in a primary culture of rainbow trout hepatocytes. Comptes Redus de L Academic des Sciences 308, 83-88. 

By applying an extension of the clearance concept 30, 31), in vitro metabolism was used to predict in vivo toxin elimination. Hepatocytes were incubated with 0.5 to 10 pg unlabeled PbTx-3 containing 0.1 pg radiolabeled toxin as tracer. Disappearance of parent compound and the appearance of metabolites were measured by HPLC equipped with a Radiomatic isotope detector. (1.6 nmol/min/g liver)... 

Mirsalis JC, Tyson CK, Steinmetz KL, et al. 1989. Measurement of unscheduled DNA synthesis and S-phase synthesis in rodent hepatocytes following in vivo treatment Testing of 24 compounds. Environ Mol Mutagen 14 155-164. 

As in the case in the analysis of food samples, the introduction of relatively inexpensive MS detectors for GC has had a substantial impact on the determination of methylxanthines by GC. For example, in 1990, Benchekroun published a paper in which a GC-MS method for the quantitation of tri-, di-, and monmethylxanthines and uric acid from hepatocyte incubation media was described.55 The method described allows for the measurement of the concentration of 14 methylxanthines and methyluric acid metabolites of methylxanthines. In other studies, GC-MS has also been used. Two examples from the recent literature are studies by Simek and Lartigue-Mattei, respectively.58 57 In the first case, GC-MS using an ion trap detector was used to provide confirmatory data to support a microbore HPLC technique. TMS derivatives of the compounds of interest were formed and separated on a 25 m DB-% column directly coupled to the ion trap detector. In the second example, allopurinol, oxypurinol, hypoxanthine, and xanthine were assayed simultaneously using GC-MS. 

As described above, it will be normal to assume that the dose interval is 24 hours, i.e., once-a-day dosing. Absorption can be estimated with good confidence from absorption in the rat (see Section 6.1). Clearance is the sum of the predicted hepatic, renal, biliary and extrahepatic clearance. Hepatic clearance can be derived from in vitro studies with the appropriate human system, using either microsomes or hepatocytes. We prefer to use an approach based on that described by Houston and Carlile [83], Renal clearance can be predicted allometrically (see section 6.8.1). The other two potential methods of clearance are difficult to predict. To minimize the risks, animal studies can be used to select compounds that show little or no potential for clearance by these routes. As volume can be predicted from that measured in the dog, after correction for human and dog plasma protein binding (see Section 6.2), it is possible to make predictions for all of the important parameters necessary. 

Bradley MO. 1985. Measurement of DNA single-strand breaks by alkaline elution in rat hepatocytes. In Ashby J, de Serres FJ, et al., eds. Progress in mutation research. Vol. 5. Evaluation of short- term tests for carcinogens. Amsterdam, The Netherlands Elsevier Science Publishers, 353-357. 

Donateo, M.T., Gomez-Lechon, M.J., and Castell, J.V. 1993. A microassay for measuring cytochrome P450IA1 and P450IIB1 activities in intact human and rat hepatocytes cultured on 96-well plates. Anal. Biochem. 213 29. 

Chatton JY, Liu H, Stucki JW 1995 Simultaneous measurements of Ca2+ in the intracellular stores and the cytosol of hepatocytes during hormone-induced Ca2+ oscillations. FEBS Lett 368 165-168... 

Bradley, M.O., Dysart, G., Fitzsimmons, K., Harback, P., Lewin, J. and Wolf, G. (1982). Measurement hy filter elution of DNA single- and double-strand breaks in rat hepatocytes Effects of nitrosamines and y-irradiation. Cancer Res. 42 2569-2597. 

Several kinetic parameters can be measured on different experimental systems to account for the interaction of a compound with CYPs. For example when studying the metabolic stability of a compound, it could be measured in a recombinant CYP system, in human liver microsomes, in hepatocytes and so on. Each system increases in biological complexity. Although in the recombinant CYP system only the cytochrome under consideration is studied, in the case of the human liver microsomes, there is a pool of enzyme present that includes several CYPs, and finally in the hepatocyte cell system, metabolizing enzymes play an important role in the metabolic compound stability. In addition, transport systems are also present that could involve recirculation or other transport phenomena. The more complex the experimental system, the more difficult it is to extract information on the protein/ligand interaction, albeit it is closer to the in vivo real situation and therefore to the mechanism that is actually working in the body. 

Although both LDL and HDL are primarily cholesterol particles, most of the cholesterol measured in the blood is assodated with LDL. The normal role of LDL is to deliver cholesterol to tissues for biosynthesis. When a cell is repairing membrane or dividing, the cholesterol is required for membrane synthesis. Bile acids and salts are made from cholesterol in the liver, and many other tissues require some cholesterol for steroid synthesis. As shown in Figure 1-15-6, about 80% of LDL are picked up by hepatocytes, the remainder by peripheral tissues. ApoB-100 is the only apoprotein on LDL, and endocytosis of LDL is mediated by apoB-100 receptors (LDL receptors) clustered in areas of cell membranes lined with the protdn clathrin. 

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