Effects of age and gender on in vitro properties of human liver microsomal monooxygenases Aging in humans is associated with marked declines in the disposition of numerous drugs and other xenobiotics that require hepatic biotransformation before elimination. Considerable pharmacokinetic evidence in humans, coupled with data on in vitro liver microsomal monooxygenase functions generated in inbred male rodent models, has implicated impaired liver phase I drug metabolism (i.e., diminished efficacy of microsomal monooxygenases) in reduced drug clearance in the elderly. This study (1) assessed the in vitro activities and amounts of liver microsomal monooxygenases as a function of donor age and gender in healthy humans and (2) provides the most extensive and comprehensive data to date demonstrating the absence of significant age- and gender-dependent differences in the activities and contents of human liver monooxygenases. (CLIN PHARMACOL THER 1990;48:365-74.)
Douglas L. Schmucker, PhD, Kenneth W. Woodhouse, MD, Rose K. Wang, MS, Hilary Wynne, MD, Oliver F. James, MD, Michael McManus, PhD, and Pierre Kremers, PhD San Francisco, Calif, Newcastle-on-Tyne, England, Bedford Park, South Australia, and Liege, Belgium
There is considerable clinical pharmacokinetic evidence that suggests that drug disposition is compromised in the elderly.12 Studies have implicated alterations in the distribution, reductions in hepatic phase I metabolism, and impairment of renal clearance of drugs in this subpopulation. Unfortunately, mueh of these data are difficult to interpret because of deficiencies in experimental design, small sample sizes, and the use of institutionalized subjects or patients with complicating diseases. Substantial evidence for (1) a decline in the in vitro efficiency of hepatic
From the Section of Cell Biology, Aging, and Molecular Medicine, Veterans Administration Medical Center, and the Department of Anatomy and the Liver Center, the University of California, San Francisco, the Departments of Geriatrics and Medicine, the Royal Victoria Infirmary and the University of Newcastle-onTyne, Newcastle-on-Tyne, England; the Department of Clinical Pharmacology, the Flinders University, Bedford Park, South Australia, and Laboratoire de Chimie medicate et de Toxicologie, Centre Hospitalier Universitaire de Liege. Supported in part by the Veterans Administration and National Institutes of Health grant AG07226. Received for publication April 24, 1990; accepted June 30, 1990. Reprint requests: Douglas L. Schmucker, PhD, 151E, Veterans Administration Medical Center, 4150 Clement St., San Francisco, CA 94121.
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microsomal monooxygenases, (2) a loss of hepatic smooth-surfaced endoplasmic reticulum, the primary hepatocellular locus of monooxygenases and (3) alterations in the physiochemical properties of microsomal membranes that influence the efficacy of constituent monooxygenases has been reported in senescent male rats.3-6 Furthermore, aging in this animal model is characterized by a "feminization" of the liver monooxygenase system such that old rats exhibit a marked loss of male-specific cytochrome P-450 (cytochrome P-450, testosterone 16a-hydroxylase) and a concomitant increase in female-specific isozyme (cytochrome P-450 5a-androstane-3a , 1713-disulfate-1513hydroxylase).7-9 Although there is some suggestion that gender influences liver phase I drug metabolism in humans,'' this is controversial,' it has not been observed in nonhuman primates' and the "feminization" of the male monooxygenase system appears to be restricted to rats.16 There have been few in vitro studies on the efficacy of human liver microsomal monooxygenases as a function of age, none of which have legitimately considered gender differences."-'9 The extrapolation of data derived in rodents to humans is replete with difficulties and, furthermore, male rats may be unsuitable for studies on hepatic drug metabolism during aging.' Still, the clinical literature is characterized by the as-
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sumption that hepatic microsomal monooxygenasedependent drug metabolism is significantly impaired in the elderly.'"2 However, recent studies suggest that agerelated declines in the disposition of drugs metabolized through the hepatic phase I pathway reflect, in part, concomitant reductions in liver volume and blood flow."'" To assess the effects of age and gender on hepatic phase I drug metabolism in humans, we subjected liver microsomal monooxygenases from male and female subjects of varied ages to in vitro analyses.
METHODS Sources of human liver tissue. A total of 54 liver samples was obtained from several sources: (1) organ donors from the Liver Tissue Procurement and Distribution System (University of Minnesota, Minneapolis, Minn.) and the Flinders University Medical Center (Bedford Park, South Australia) and (2) wedge biopsy specimens from consenting patients undergoing laparotomy at the Royal Victoria Infirmary (Newcastleupon-Tyne, England). Light microscopic analysis of the liver tissue revealed normal hepatic morphologic findings in all samples examined. Only those donors with disorders not affecting hepatic phase I drug metabolism were included in the study. The distribution of surgical procedures for all biopsy donors was: cholecystectomy (76%), gastrectomy (14%), and exploratory laparotomy, vagotomy, and other (10%). Approximately 24% of the subjects were light to heavy smokers and none were assessed to use alcohol to excess. Six subjects (three female and three male subjects) were judged to be taking medications known to be inducers of liver monooxygenases (phenytoin, phenobarbital, spironolactone, and dichloralphenazone). Seven subjects (six female and one male subject) were identified as having received drugs that inhibit hepatic monooxygenases (e.g., cimetidine, oxprenolol, propranolol, and oral contraceptives). Segregation and separate analysis of data obtained from subjects receiving inducers or inhibitors of liver drug metabolism (i.e. , medications, cigarettes, and alcohol) did not yield results significantly different from those obtained from the remaining subject pool. The donors ranged in age from 9 to 89 years with a mean of 51.2 ± 20 years. The gender distribution was 20 male (48.5 ± 23 years) and 34 female subjects (52.8 -± 18 years). Preparation of liver microsomes. Wedge biopsy specimens or organ donor samples were immediately flash frozen in liquid nitrogen (N2) and stored at 80° C until used. Once thawed, the liver tissue was homogenized in 0.25 mol / L sucrose, 0.25 mol / L KPO,/ 0.15 mol / L KCI (pH 7.4), or 0.1 mol /L KPO4
CLIN PHARMACOL THER OCTOBER 1990
(pH 7.4), and microsomes were prepared by differential centrifugation.4''6 The 105,000 g pellet was washed and resuspended in the respective homogenization media with or without 20% glycerol. After the protein concentration was determined," the suspended microsomes were aliquoted, frozen, and kept at 80° C until used. A comparison of NADPH cytochrome P-450 reductase activity in freshly isolated and frozen/ stored ( 80° C) monkey and human liver microsomes yielded equivalent values -± 5% (Schmucker DL, Wang RK. Unpublished data. 1986-1988.). Analytic procedures and reagents. NADPH cytochrome P-450 reductase activity was measured by the method of Masters et al.' using cytochrome C as the substrate, and the rates are expressed as nanomoles of cytochrome C reduced per minute per milligram of microsomal protein or gram of liver. The microsomal content of immunoprecipitable reductase was determined by direct enzyme-linked immunosorbent assay (ELISA) with intact microsomes, biotinylated goat antihuman reductase polyclonal antibodies, and avidin-horseradish peroxidase." Rhesus monkey liver NADPH cytochrome P-450 reductase, purified to homogeneity, was used as a standard." The data were expressed as reciprocal titers (titer') and nanomoles of reductase per milligram of microsomal protein or per gram of liver. The concentration of total cytochrome P-450 was estimated by measuring the CO-binding Soret spectra and expressing the data as nanomoles of P-450 per milligram of microsomal protein or gram of liver." The amount of immunoprecipitable cytochrome P-450 was determined in a direct ELISA with biotinylated rabbit antirat liver cytochrome P-450 antiserum and the values were expressed as reciprocal titers. These polyclonal antibodies have not been fully characterized; thus no specific human orthologs have been identified. The relative concentrations of cytochromes P-4505 (P450IIIA2) and P-4508 (P-450I1C8) were measured in an ELISA with monoclonal antibodies directed against each isozyme." Cytochrome P-4509, a member of the P-45011C family, was also measured by ELISA with a monoclonal antibody that has not, as yet, been completely characterized. Statistical analysis. The data were grouped according to gender and subjected to linear regression, as well as Kendall and Spearman correlation analyses. Probability values 0.05; women: r = 0.02, p > 0.05).
age and the kinetic profile (maximum rate of metabolism and elimination rate constant) of human liver 7ethoxycoumarin-O-deethylase.19 A recent in vivo study with the aminopyrine breath test concluded that human hepatic microsomal phase I monooxygenase function was well preserved throughout the lifespan." These observations are supported by data obtained in nonhuman primates. 15'2634 Collectively, these studies do not substantiate an age-related dysfunction in liver drug metabolism at the level of the monooxygenases. Still, many of these studies are characterized by inherent problems that render them virtually uninterpretable.' Our finding that hepatic microsomal protein concentration does not change significantly with increasing age is in agreement with other reports in humans's.' and in nonhuman primates.15'26 However, these data contradict those reported in inbred male rats that exhibit significant age-related declines in the yields of hepatic microsomal protein." The slight increase in microsomal protein observed in females may compensate for reduced liver volume such that total hepatic microsomal protein content remains unchanged with increasing age. The absence of a significant correlation between age and the specific activity of NADPH cytochrome P-450 reductase is in agreement with the only other human study and two nonhuman primate studies in which this particular in vitro parameter was measured. '532'34 Furthermore, the absence of sex-linked differences in P-450 reductase activity and, with the exception of
certain steroid-specific isozymes, several cytochrome P-450 activities in human liver microsomes reported by Kremers et al. has been confirmed. The marked interindividual variability observed in all of the parameters measured is consistent with that reported by others and most likely reflects the outbred nature of the human population. Interestingly, the degree of interindividual variability was substantial even within the pool of younger subjects and did not appear to be enhanced by increasing age. Previous studies in male rats demonstrated an agerelated accumulation of catalytically inactive (50%), immunoprecipitable, hepatic P-450 reductase." However, because the total liver reductase content in old rats was approximately twice that measured in young adult animals, the total liver reductase activity was equivalent in both age groups.' In this study the microsomal content of immunoprecipitable reductase in female, but not male, subjects exhibited a significant negative correlation with increasing age when the data were expressed per milligram of protein. The disappearance of this correlation when these data are expressed per gram of liver suggests that, volume for volume, human livers contain similar reductase concentrations regardless of donor age. However, the marked decline in human liver volume during aging may result in reduced liver complements of enzyme in the elderly.21,22 The response of cytochrome P-450 (CO-binding
,'
VOLUME 48 NUMBER 4
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Age (years) Fig. 6. A, The relative hepatic microsomal concentration of the cytochrome P-450 isozyme, P-4505, in male and female subjects between 9 and 89 years of age (males: r = 0.22, p > 0.05; females: r = 0.32, p > 0.05). B, Microsomal contents of cytochrome P-4508 measured as in A (males: r = 0.32, p > 0.05; females: r = 0.32, p > 0.05). C, Relative microsomal concentrations of cytochrome P-4509 (males: r = 0.31, p > 0.05; females: r = 0.31, p > 0.01 < 0.5). The data were obtained by ELISA with intact microsomes and biotinylated monoclonal antibodies directed against the respective cytochrome P-450 isozyme. Solid squares, Male subjects; open squares, female subjects.
VOLUME 48 NUMBER 4
spectra) to aging was similar to that observed for reductase activity and content (i.e. , apparent age and gender differences when the data are expressed per milligram of protein, which subsequently diminish when expressed per gram of liver). The small but significant decline in the relative amount of immunoprecipitable cytochrome P-450 in females, but not males, during aging corresponds to the patterns exhibited by P-450 reductase. The interpretation of these data is tempered because whether or not the antiserum to rat cytochrome P-450 immunoprecipitates all of the isozymes, their human orthologs or some combination of both has not been resolved. Nevertheless, we suspect that these age and gender differences may disappear when these data are expressed per gram of liver, but the absence of a suitable ELISA standard precluded such analyses in this study.
The absence of significant differences in the microsomal concentrations of several cytochrome P-450 isozymes (i.e. , P-4505, P-4508, and P-4509) suggests that monooxygenase substrate specificities for these particular heme proteins do not change appreciably during aging in either sex. Subsequent examination of isozyme/ total cytochrome P-450 ratios failed to reveal any apparent inductive or inhibitory response and substantiates this contention. McManus et al.37 reported microsomal NADPH cytochrome P-450 reductase/ cytochrome P-450 concentration ratios of 7: 1 in human livers. The twofold higher values obtained in this study may relect procedural differences. For example, we express reductase content as micrograms of enzyme per milligram of microsomal protein, with Macaca mulatta reductase used as a standard, whereas McManus et al. reported values as picomoles per milligram of microsomal protein using the more appropriate human enzyme as a standard. In this study only cytochrome P-450 (nanomoles per milligram of protein) was measured versus both the cytochromes P-450 and P-420 (picomoles per milligram of protein) measured by McManus et al. Regardless, our observations confirm the absence of marked age- and gender-dependent differences in this parameter. Recent evidence in humans strongly suggests that reduced liver volume, coupled with diminished liver blood flow, is primarily responsible for reduced hepatic drug clearance in the elderly.21,22.31 The absence of significant age- or gender-related losses in monooxygenase activities or microsomal concentrations in nonhuman primates or humans lends credence to this suggestion .15,17,19,26,32,34 On the other hand, the absence of agerelated declines in reductase activity and content per gram of liver and the significant declines in hepatic
Age, sex, and human liver monooxygenases
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volume may complement each other and result in diminished total liver enzyme content and activity. This, of course, would be reflected in reduced hepatic drug clearance rates in vivo, as have been reported in the elderly.2.3' The data generated during this study demonstrate that (1) neither age nor gender significantly influences human liver microsomal monooxygenases and (2) reduced hepatic drug clearance in the elderly most likely reflects concomitant declines in organ volume and blood flow.
References Schmucker DL. Age-related changes in drug disposition. Pharmacol Rev 1979;30:445-56. Schmucker DL. Age-related changes in drug disposition: an update. Pharmacol Rev 1985;37:133-48. Kato R, Vassenelli P, Frontina G, Chiesara E. Variation in the activity of liver microsomal drug-metabolizing enzymes in rats in relation to age. Biochem Pharmacol 1964;13:1037-51. Schmucker DL, Wang RK. Age-related changes in liver drug-metabolizing enzymes. Exp Gerontol 1980; 15:321-9. Schmucker DL, Mooney JS, Jones AL. Stereological analysis of hepatic fine structure in the Fischer 344 rat: influence of sublobular location and animal age. J Cell Biol 1978;78:319-37. Schmucker DL, Vessey DA, Wang RK, James J, Maloney A. Age-dependent alterations in the physicochemical properties of rat liver microsomes. Mech Ageing Dev 1984;27:207-17. Kamataki T, Maeda K, Shimada M, Kitani K, Nagai T, Kato R. Age-related alteration in the activities of drugmetabolizing enzymes and contents of sex-specific forms of cytochrome P-450 in liver microsomes from male and female rats. J Pharamcol Exp Ther 1985;233:222-8. Fujita S, Kitagawa H, Chiba M, Suzuki T, Ohta M, Kitani K. Age and sex association differences in the relative abundance of multiple species of cytochrome P-450 in rat liver microsomes: a separation by HPLC of hepatic microsomal cytochrome P-450 species. Biochem Pharmacol 1985;34:1861-2. Conney AH. Induction of microsomal cytochrome P-450 enzymes: the first Bernard B. Brodie lecture. Life Sci 1986;39:2493-518. Tuenissen MW, Grivastava AK, Breimer 00. Influence of sex and oral contraceptives on antipyrine metabolite formation. CLIN PHARMACOL THER 1987;32:240-6. Roberts RK, Desmond PV, Wilkinson GR, Schenker S. Disposition of chlordiazepoxide: sex differences and effects of oral contraceptives. CLIN PHARMACOL THER 1979;25:826-31. Allen MD, Greenblatt DJ, Harmatz JS, Shader RI. Desmethyldiazepam kinetics in the elderly after oral prazepam. CLIN PHARMACOL THER 1980;28:196-202. Greenblatt DJ, Allen MD, Harmatz JS, Shader RI.
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374 Schmucker et al. Diazepam disposition determinants. CLIN
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THER 1980;27:301-12.
ICremers P, Beaune P, Cresteil T, et al. Cytochrome
P-450 monooxygenase activities in human and rat liver microsomes. Eur J Biochem 1981;118:599-606. Maloney AG, Schmucker DL, Vessey DA, Wang RK. The effects of aging on the hepatic microsomal mixedfunction oxidase system of male and female monkeys. Hepatology 1986;6:282-7. Kitani K. Hepatic drug metabolism in the elderly. Hepatology 1986;6:316-9. James OFW, Rawlins MD, Woodhouse K. Lack of aging effect on human microsomal monooxygenase enzyme activities and on inactivation pathways for reactive metabolic intermediates. In: Kitani K, ed. Liver and aging1982: liver and drugs. Amsterdam: Elsevier Biomedical Press, 1982:395-408. Woodhouse KW, Mutch E, Williams FM, Rawlins MD, James OFW. The effect of age on pathways of drug metabolism in human liver. Age Ageing 1984;13:32834. Wynne HA, Mutch E, James OFW, Wright P, Rawlins MD, Woodhouse KW. The effect of age upon the affinity of microsomal monooxygenase enzymes for substrate in human liver. Age Ageing 1988;17:401-5. Schmucker DL. Is the rat a suitable model for aging research? In: Woodhouse KW, Yelland C, James OFW, eds. The Liver: Metabolism and Aging. Rijswijk, The Netherlands: EURAGE, 1989:3-18. Marchesini G, Bua V, Brunori A, et al. Galactose elimination capacity and liver volume in aging man. Hepatology 1988;8:1079-83. Wynne HA, Cope LH, Mutch E, Rawlins MD, Woodhouse KW, James OFW. The effect of age upon liver volume and apparent liver blood flow in healthy man. Hepatology 1989;9:297-301. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951;193:265-75. Masters BS, Williams C, Kamin H. The preparation and properties of TPNH cytochrome C reductase from pig liver. Methods Enzymol 1965;10:565-73. McManus ME, de la M Hall P, Stupans I, et al. Immunohistochemical localization and quantitation of NADPH cytochrome P-450 reductase in human liver. Mol Pharmacol 1987;32:189-94.
Schmucker DL, Wang RK. Effects of aging on the properties of rhesus monkey liver microsomal NADPH cytochrome C (P-450) reductase. Drug Metab Dispos 1987; 15:225-32. Omura T, Sato R. The carbon monoxide binding pigment of liver microsomes. J Biol Chem 1964;239:2370-8. Beaune P, Kremers P, Letawe-Goujon F, Gielen JE. Monoclonal antibodies against human liver cytochrome P-450. Biochem Pharmacol 1985;34:3547-52. Cheng KC, Gelboin HV, Song BJ, Park SS, Friedman FK. Detection and purification of cytochromes P-450 in animal tissues with monoclonal antibodies. J Biol Chem 1984;259:12,279-84. Distlerath LM, Guengerich FP. Characterization of a human liver cytochrome P-450 involved in the oxidation of debrisoquin and other drugs by using antibodies raised to the analogous rat enzyme. Proceedings of the National Academy of Sciences (USA) 1984;81:7348-52. Vestal RE. Aging and determinants of hepatic drug clearance. Hepatology 1989;9:331-4. Brodie MJ, Boobis AR, Bulpuitt CJ, et al. Influence of liver disease and environmental factors on hepatic monooxygenase activity in vitro. Eur J Clin Pharmacol 1981;20:39-40. Arora S, Kassarjian Z, Krasinski SD, Croffey B, Kaplan MM, Russell RM. Effect of age on tests of intestinal and hepatic function in healthy humans. Gastroenterology 1989;96:1560-5 . Sutter M, Wood G, Williamson L, Strong R, Pickham K, Richardson A. Comparison of the hepatic mixedfunction oxidase systems of young, adult and old nonhuman primates (Macaca nemistrina). Biochem Pharmacol 1985;34:2983-7. Schmucker DL, Wang RK. Age-dependent alterations in rat liver microsomal NADPH cytochrome C (P-450) reductase: a qualitative and quantitative analysis. Mech Ageing Dev 1983;21:137-56. Schmucker DL, Wang RK, Wong KP. Age-dependent alterations in rat liver microsomal NADPH cytochrome C (P-450) reductase. In: Kitani K, ed. Liver and aging1982: liver and drugs. Amsterdam: Elsevier Biomedical Press, 1982:75-97. McManus ME, Huggett A, Burgess W, Robson R, Birkett DJ. Immunochemical and catalytical characterization of the human liver NADPH cytochrome P-450 reductase. Clin Exp Pharmacol Physiol 1989;16:121-34.
Douglas L. Schmucker, PhD, Kenneth W. Woodhouse, MD, Rose K. Wang, MS, Hilary Wynne, MD, Oliver F. James, MD, Michael McManus, PhD, and Pierre Kremers, PhD San Francisco, Calif, Newcastle-on-Tyne, England, Bedford Park, South Australia, and Liege, Belgium
There is considerable clinical pharmacokinetic evidence that suggests that drug disposition is compromised in the elderly.12 Studies have implicated alterations in the distribution, reductions in hepatic phase I metabolism, and impairment of renal clearance of drugs in this subpopulation. Unfortunately, mueh of these data are difficult to interpret because of deficiencies in experimental design, small sample sizes, and the use of institutionalized subjects or patients with complicating diseases. Substantial evidence for (1) a decline in the in vitro efficiency of hepatic
From the Section of Cell Biology, Aging, and Molecular Medicine, Veterans Administration Medical Center, and the Department of Anatomy and the Liver Center, the University of California, San Francisco, the Departments of Geriatrics and Medicine, the Royal Victoria Infirmary and the University of Newcastle-onTyne, Newcastle-on-Tyne, England; the Department of Clinical Pharmacology, the Flinders University, Bedford Park, South Australia, and Laboratoire de Chimie medicate et de Toxicologie, Centre Hospitalier Universitaire de Liege. Supported in part by the Veterans Administration and National Institutes of Health grant AG07226. Received for publication April 24, 1990; accepted June 30, 1990. Reprint requests: Douglas L. Schmucker, PhD, 151E, Veterans Administration Medical Center, 4150 Clement St., San Francisco, CA 94121.
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microsomal monooxygenases, (2) a loss of hepatic smooth-surfaced endoplasmic reticulum, the primary hepatocellular locus of monooxygenases and (3) alterations in the physiochemical properties of microsomal membranes that influence the efficacy of constituent monooxygenases has been reported in senescent male rats.3-6 Furthermore, aging in this animal model is characterized by a "feminization" of the liver monooxygenase system such that old rats exhibit a marked loss of male-specific cytochrome P-450 (cytochrome P-450, testosterone 16a-hydroxylase) and a concomitant increase in female-specific isozyme (cytochrome P-450 5a-androstane-3a , 1713-disulfate-1513hydroxylase).7-9 Although there is some suggestion that gender influences liver phase I drug metabolism in humans,'' this is controversial,' it has not been observed in nonhuman primates' and the "feminization" of the male monooxygenase system appears to be restricted to rats.16 There have been few in vitro studies on the efficacy of human liver microsomal monooxygenases as a function of age, none of which have legitimately considered gender differences."-'9 The extrapolation of data derived in rodents to humans is replete with difficulties and, furthermore, male rats may be unsuitable for studies on hepatic drug metabolism during aging.' Still, the clinical literature is characterized by the as-
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sumption that hepatic microsomal monooxygenasedependent drug metabolism is significantly impaired in the elderly.'"2 However, recent studies suggest that agerelated declines in the disposition of drugs metabolized through the hepatic phase I pathway reflect, in part, concomitant reductions in liver volume and blood flow."'" To assess the effects of age and gender on hepatic phase I drug metabolism in humans, we subjected liver microsomal monooxygenases from male and female subjects of varied ages to in vitro analyses.
METHODS Sources of human liver tissue. A total of 54 liver samples was obtained from several sources: (1) organ donors from the Liver Tissue Procurement and Distribution System (University of Minnesota, Minneapolis, Minn.) and the Flinders University Medical Center (Bedford Park, South Australia) and (2) wedge biopsy specimens from consenting patients undergoing laparotomy at the Royal Victoria Infirmary (Newcastleupon-Tyne, England). Light microscopic analysis of the liver tissue revealed normal hepatic morphologic findings in all samples examined. Only those donors with disorders not affecting hepatic phase I drug metabolism were included in the study. The distribution of surgical procedures for all biopsy donors was: cholecystectomy (76%), gastrectomy (14%), and exploratory laparotomy, vagotomy, and other (10%). Approximately 24% of the subjects were light to heavy smokers and none were assessed to use alcohol to excess. Six subjects (three female and three male subjects) were judged to be taking medications known to be inducers of liver monooxygenases (phenytoin, phenobarbital, spironolactone, and dichloralphenazone). Seven subjects (six female and one male subject) were identified as having received drugs that inhibit hepatic monooxygenases (e.g., cimetidine, oxprenolol, propranolol, and oral contraceptives). Segregation and separate analysis of data obtained from subjects receiving inducers or inhibitors of liver drug metabolism (i.e. , medications, cigarettes, and alcohol) did not yield results significantly different from those obtained from the remaining subject pool. The donors ranged in age from 9 to 89 years with a mean of 51.2 ± 20 years. The gender distribution was 20 male (48.5 ± 23 years) and 34 female subjects (52.8 -± 18 years). Preparation of liver microsomes. Wedge biopsy specimens or organ donor samples were immediately flash frozen in liquid nitrogen (N2) and stored at 80° C until used. Once thawed, the liver tissue was homogenized in 0.25 mol / L sucrose, 0.25 mol / L KPO,/ 0.15 mol / L KCI (pH 7.4), or 0.1 mol /L KPO4
CLIN PHARMACOL THER OCTOBER 1990
(pH 7.4), and microsomes were prepared by differential centrifugation.4''6 The 105,000 g pellet was washed and resuspended in the respective homogenization media with or without 20% glycerol. After the protein concentration was determined," the suspended microsomes were aliquoted, frozen, and kept at 80° C until used. A comparison of NADPH cytochrome P-450 reductase activity in freshly isolated and frozen/ stored ( 80° C) monkey and human liver microsomes yielded equivalent values -± 5% (Schmucker DL, Wang RK. Unpublished data. 1986-1988.). Analytic procedures and reagents. NADPH cytochrome P-450 reductase activity was measured by the method of Masters et al.' using cytochrome C as the substrate, and the rates are expressed as nanomoles of cytochrome C reduced per minute per milligram of microsomal protein or gram of liver. The microsomal content of immunoprecipitable reductase was determined by direct enzyme-linked immunosorbent assay (ELISA) with intact microsomes, biotinylated goat antihuman reductase polyclonal antibodies, and avidin-horseradish peroxidase." Rhesus monkey liver NADPH cytochrome P-450 reductase, purified to homogeneity, was used as a standard." The data were expressed as reciprocal titers (titer') and nanomoles of reductase per milligram of microsomal protein or per gram of liver. The concentration of total cytochrome P-450 was estimated by measuring the CO-binding Soret spectra and expressing the data as nanomoles of P-450 per milligram of microsomal protein or gram of liver." The amount of immunoprecipitable cytochrome P-450 was determined in a direct ELISA with biotinylated rabbit antirat liver cytochrome P-450 antiserum and the values were expressed as reciprocal titers. These polyclonal antibodies have not been fully characterized; thus no specific human orthologs have been identified. The relative concentrations of cytochromes P-4505 (P450IIIA2) and P-4508 (P-450I1C8) were measured in an ELISA with monoclonal antibodies directed against each isozyme." Cytochrome P-4509, a member of the P-45011C family, was also measured by ELISA with a monoclonal antibody that has not, as yet, been completely characterized. Statistical analysis. The data were grouped according to gender and subjected to linear regression, as well as Kendall and Spearman correlation analyses. Probability values 0.05; women: r = 0.02, p > 0.05).
age and the kinetic profile (maximum rate of metabolism and elimination rate constant) of human liver 7ethoxycoumarin-O-deethylase.19 A recent in vivo study with the aminopyrine breath test concluded that human hepatic microsomal phase I monooxygenase function was well preserved throughout the lifespan." These observations are supported by data obtained in nonhuman primates. 15'2634 Collectively, these studies do not substantiate an age-related dysfunction in liver drug metabolism at the level of the monooxygenases. Still, many of these studies are characterized by inherent problems that render them virtually uninterpretable.' Our finding that hepatic microsomal protein concentration does not change significantly with increasing age is in agreement with other reports in humans's.' and in nonhuman primates.15'26 However, these data contradict those reported in inbred male rats that exhibit significant age-related declines in the yields of hepatic microsomal protein." The slight increase in microsomal protein observed in females may compensate for reduced liver volume such that total hepatic microsomal protein content remains unchanged with increasing age. The absence of a significant correlation between age and the specific activity of NADPH cytochrome P-450 reductase is in agreement with the only other human study and two nonhuman primate studies in which this particular in vitro parameter was measured. '532'34 Furthermore, the absence of sex-linked differences in P-450 reductase activity and, with the exception of
certain steroid-specific isozymes, several cytochrome P-450 activities in human liver microsomes reported by Kremers et al. has been confirmed. The marked interindividual variability observed in all of the parameters measured is consistent with that reported by others and most likely reflects the outbred nature of the human population. Interestingly, the degree of interindividual variability was substantial even within the pool of younger subjects and did not appear to be enhanced by increasing age. Previous studies in male rats demonstrated an agerelated accumulation of catalytically inactive (50%), immunoprecipitable, hepatic P-450 reductase." However, because the total liver reductase content in old rats was approximately twice that measured in young adult animals, the total liver reductase activity was equivalent in both age groups.' In this study the microsomal content of immunoprecipitable reductase in female, but not male, subjects exhibited a significant negative correlation with increasing age when the data were expressed per milligram of protein. The disappearance of this correlation when these data are expressed per gram of liver suggests that, volume for volume, human livers contain similar reductase concentrations regardless of donor age. However, the marked decline in human liver volume during aging may result in reduced liver complements of enzyme in the elderly.21,22 The response of cytochrome P-450 (CO-binding
,'
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Age (years) Fig. 6. A, The relative hepatic microsomal concentration of the cytochrome P-450 isozyme, P-4505, in male and female subjects between 9 and 89 years of age (males: r = 0.22, p > 0.05; females: r = 0.32, p > 0.05). B, Microsomal contents of cytochrome P-4508 measured as in A (males: r = 0.32, p > 0.05; females: r = 0.32, p > 0.05). C, Relative microsomal concentrations of cytochrome P-4509 (males: r = 0.31, p > 0.05; females: r = 0.31, p > 0.01 < 0.5). The data were obtained by ELISA with intact microsomes and biotinylated monoclonal antibodies directed against the respective cytochrome P-450 isozyme. Solid squares, Male subjects; open squares, female subjects.
VOLUME 48 NUMBER 4
spectra) to aging was similar to that observed for reductase activity and content (i.e. , apparent age and gender differences when the data are expressed per milligram of protein, which subsequently diminish when expressed per gram of liver). The small but significant decline in the relative amount of immunoprecipitable cytochrome P-450 in females, but not males, during aging corresponds to the patterns exhibited by P-450 reductase. The interpretation of these data is tempered because whether or not the antiserum to rat cytochrome P-450 immunoprecipitates all of the isozymes, their human orthologs or some combination of both has not been resolved. Nevertheless, we suspect that these age and gender differences may disappear when these data are expressed per gram of liver, but the absence of a suitable ELISA standard precluded such analyses in this study.
The absence of significant differences in the microsomal concentrations of several cytochrome P-450 isozymes (i.e. , P-4505, P-4508, and P-4509) suggests that monooxygenase substrate specificities for these particular heme proteins do not change appreciably during aging in either sex. Subsequent examination of isozyme/ total cytochrome P-450 ratios failed to reveal any apparent inductive or inhibitory response and substantiates this contention. McManus et al.37 reported microsomal NADPH cytochrome P-450 reductase/ cytochrome P-450 concentration ratios of 7: 1 in human livers. The twofold higher values obtained in this study may relect procedural differences. For example, we express reductase content as micrograms of enzyme per milligram of microsomal protein, with Macaca mulatta reductase used as a standard, whereas McManus et al. reported values as picomoles per milligram of microsomal protein using the more appropriate human enzyme as a standard. In this study only cytochrome P-450 (nanomoles per milligram of protein) was measured versus both the cytochromes P-450 and P-420 (picomoles per milligram of protein) measured by McManus et al. Regardless, our observations confirm the absence of marked age- and gender-dependent differences in this parameter. Recent evidence in humans strongly suggests that reduced liver volume, coupled with diminished liver blood flow, is primarily responsible for reduced hepatic drug clearance in the elderly.21,22.31 The absence of significant age- or gender-related losses in monooxygenase activities or microsomal concentrations in nonhuman primates or humans lends credence to this suggestion .15,17,19,26,32,34 On the other hand, the absence of agerelated declines in reductase activity and content per gram of liver and the significant declines in hepatic
Age, sex, and human liver monooxygenases
373
volume may complement each other and result in diminished total liver enzyme content and activity. This, of course, would be reflected in reduced hepatic drug clearance rates in vivo, as have been reported in the elderly.2.3' The data generated during this study demonstrate that (1) neither age nor gender significantly influences human liver microsomal monooxygenases and (2) reduced hepatic drug clearance in the elderly most likely reflects concomitant declines in organ volume and blood flow.
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