Induction of drug-metabolizing enzymes in liver microsomes of mice and rats by softwood bedding.

Induction of three drug-metabolizing enzymes occurred in liver microsomes of mice and rats kept on softwood bedding of either red cedar, white pine, or ponderosa pine. This induction was reversed when animals were placed on hardwood bedding composed of a mixture of beech, birch, and maple. Differences in the capacity of various beddings to induce may partially explain divergent results of studies on drug-metabolizing enzymes. The presence of such inducing substances in the environment may influence the pharmacologic responsiveness of animals to a wide variety of drugs.

Disposition of morphine in man.

The disposition of morphine was investigated by means of radioimmunoassay after a single intravenous dose (10 milligrams per 70 kilograms) was administered to 10 adult normal male subjects who had not received other drugs for 2 weeks preceding the study. A multiphasic decline in serum concentrations of morphine occurred. Detectable blood concentrations of morphine, or of a metabolite, or of both persisted for 48 hours after a single intravenous dose.

Genetic control of drug levels in man: phenylbutazone.

Phenylbutazone was administered to seven pairs of identical (monozygotic) twins and to seven pairs of fraternal (dizygotic) twins. Individual half-lives ranged from 1.2 to 7.3 days. Subjects with identical genotypes (monozygotic twins) exhibited very similar phenylbutazone half-lives; significantly greater differences in half-life occurred in dizygotic twins. The previously established large variations among individuals in phenylbutazone metabolism appear to be genetically, rather than environmentally, determined.

Lactate dehydrogenase isozymes: further kinetic studies at high enzyme concentration.

In the report "Lactate dehydrogenase isozymes: Further kinetic studies at high enzyme concentration" by Thomas Wuntsch, Raymond F. Chen, and Elliot S. Vesell [169, 480 (1970)], 14.0 mM NAD in line 2 of the heading of Table 1 should read 14.0 , microM NAD.

Genetic control of drug levels in man: antipyrine.

Antipyrine was administered to identical or monozygotic twins and to fraternal or dizygotic twins. Individuals with identical genotypes (monozygotic twins) exhibited significantly less variability in antipyrine halflife than did genetically different individuals (dizygotic twins). Therefore variations in antipyrine metabolism appear to be determined genetically rather than environmentally. In the 36 twins tested, antipyrine half-lives varied between 5.1 and 16.7 hours. No significant correlation occurred between half-lives for phenylbutazone and antipyrine in the 28 twins who received both drugs.

Lactate dehydrogenase isozymes: further kinetic studies at high enzyme concentration.

By competition with lactate dehydrogenase (LDH) for nicotinamide adenine dinucleotide (NAD), commonly occurring intracellular proteins, such as glyceraldehyde-3-phosphate dehydrogenase, malate dehydrogenase, and albumin, can protect LDH-1 and LDH-5 from inhibition and ternary complex formation with NAD and pyruvate. The existence of intracellular proteins that compete with LDH for NAD renders unphysiological a model for estimating the extent of intracellular LDH inhibition based on incubations of only LDH, NAD, and pyruvate.

Lactate dehydrogenase isozymes: kinetic properties at high enzyme concentrations.

The kinetic properties of lactate dehydrogenase (LDH) isozymes have been determined at high enzyme concentrations. Spectrophotofluorometric assays revealed that the extent of substrate inhibition of LDH-1 and LDH-5 depends on enzyme concentration. At high enzyme concentrations, in the range of those that exist in most mammalian cells, no inhibition by pyruvate occurred. Pyruvate concentrations up to and including 20.0 millimoles per liter were used for each isozyme at 25 degrees and 40 degrees C at pH 7.0 and 7.4. These results suggest that substrate inhibition of LDH may not occur in vivo but only in vitro after appreciable dilution from physiologic enzyme concentrations. These experiments provide further evidence against the theory that substrate inhibition of LDH-1 in vivo accounts for the distribution of LDH isozymes within various tissues. They raise the possibility that, for other enzymes, kinetic properties determined at highly dilute concentrations in vitro may also be quite different from kinetic properties at the much higher concentrations that exist in vivo.

Liquid chromatographic assay of warfarin: similarity of warfarin half-lives in human subjects.

A high pressure liquid chromatographic assay was developed to measure warfarin concentrations in biological fluids. Twelve healthy, unrelated volunteers received a single oral dose of warfarin (0.75 mg per kilogram of body weight). The mean plasma warfarin half-life was 36.3 +/- 3.5 hours by liquid chromatography but 55.9 +/- 8.4 hours by a currently used fluorimetric assay that fails to separate warfarin from its metabolites. Interindividual variation was greater and each half-life longer by the fluorimetric than by the chromatographic procedure. Warfarin shows less interindividual variation than that observed for other drugs primarily metabolized by hepatic microsomal mixed function oxidases. Advantages of specificity, rapidity, sensitivity, accuracy, and simplicity recommend liquid chromatography in the development of other drug assays.

Reduced warfarin binding of albumin variants.

Binding studies of albumins A/A and A/Me from human plasma and isolated albumin revealed small, but statistically significant, reductions in warfarin binding of albumin A/Me compared to albumin A/A. Such differences in binding in vitro could result in altered pharmacological responses to warfarin administered to individuals possessing albumin A/Me. To determine if clinically significant differences in warfarin distribution exist, observations should be made in vivo on patients of different albumin phenotype who are receiving warfarin therapeutically.

Genetic control of chloroform toxicity in mice.

Mouse strain differences suggest intermediate or multifactorial gentic control of chloroform-induced renal toxicity and death. The chloroform dose lethal to 50 percent of animals was four times higher in C57BL/6J males than in DBA/2J males. Twice as much chloroform accumulated in the kidneys of the sensitive as the resistant strain. First generation offspring were midway between parental strains for both parameters.

Hepatic drug metabolism in rats: impairment in a dirty environment.

Reduction of aniline hydroxylase activity, ethylmorphine N-dementhylase activity, and cytochrome P-450 content occurred in hepatic microsomes of rats kept under dirty conditions, defined as accumulation for 1 week of urine and feces in pans under the wire mesh cages. In comparison with rats that had urine and feces removed twice daily from such pans, rats kept over Kimpak bedding or over Litter Green, changed twice daily, also showed reduced drug-metabolizing activity in hepatic microsomes, but to a lesser degree than the dirty rats. Placement of a filter top on cages for 1 week also decreased drug-metabolizing activity. These experiments suggest that the relative cleanliness of an animal's environment can influence hepatic microsomal drug metabolism.

Monogenic control of variations in antipyrine metabolite formation. New polymorphism of hepatic drug oxidation.

To investigate mechanisms that control large variations among normal uninduced subjects in the elimination of the model compound antipyrine (AP) and other drugs, AP was administered to 144 subjects (83 unrelated adults and 61 members of 13 families). Thereafter, at regular intervals for 72 h, the urine of each subject was collected and concentrations of AP and its three main metabolites measured. From these urinary concentrations, rate constants for formation of each AP metabolite were calculated. Trimodal curves were observed when values for each AP rate constant were plotted in 83 unrelated subjects; probit plots of these values showed inflections at the two antimodes of each trimodal distribution. All members of our 13 families were assigned one of three phenotypes determined by where their AP metabolite rate constant placed them in the trimodal distributions derived from the 83 unrelated subjects. In each family, pedigree analysis to identify the mode of transmission of these three phenotypes was consistent with their monogenic control. These results provide evidence for a new polymorphism of drug oxidation in man.

Genetic control of dicumarol levels in man.

The mean half-life of dicumarol in the plasma of seven sets of identical and seven sets of fraternal twins after a single oral dose of 4 mg/kg was 43.6+/-SD 17.9 hr. Half-lives ranged from 7 to 74 hr in these 28 normal adults not receiving other drugs for 2 wk preceding dicumarol administration. Large differences among unrelated individuals in dicumarol half-life disappeared almost completely in identical twins, but persisted to some extent in most sets of fraternal twins. These results indicate that marked differences among subjects in dicumarol half-life are under genetic rather than environmental control. Reproducibility of values for dicumarol half-life was demonstrated. A direct relationship between the dose and the half-life of dicumarol occurred in unrelated volunteers administered progressively larger doses at 10-day intervals. Dose dependence of the half-life of a drug results in increased variability of half-life and hence in greater risks of toxicity on long-term therapy. Risks of toxicity on the one hand and of failure to anticoagulate adequately on the other can be reduced by determining dicumarol half-life before starting long-term therapy. Half-lives for dicumarol and phenylbutzone tended to be correlated in the 28 twins, but no correlation occurred between dicumarol and antipyrine half-lives.

Genetic control of the phenobarbital-induced shortening of plasma antipyrine half-lives in man.

The mean half-life of antipyrine in the plasma of four sets of identical and four sets of fraternal twins after a single oral dose of 16 mg/kg of antipyrine was 12.7 +/-(SD) 3.3 hr. After 2 wk on sodium phenobarbital (2 mg/kg daily) the half-life of antipyrine in the plasma of these twins was reduced to 8.0 +/-(SD) 1.5 hr. Shortening of the plasma antipyrine half-life occurred in all but one of these 16 normal, adult volunteers, but there was considerable variation in the extent of reduction which ranged from 0 to 69%. Phenobarbital administration decreased individual variations in antipyrine metabolism as indicated by the smaller standard deviation of the plasma antipyrine half-lives after phenobarbital than observed initially and by the narrowed range of variation in plasma antipyrine half-lives from 2.8-fold initially to 1.8-fold after phenobarbital. These results suggest that some inducing agents may be used to minimize individual variations in drug metabolism where such variations create therapeutic problems by exposing patients who slowly metabolize certain drugs to toxicity and other patients who rapidly metabolize some drugs to undertreatment. During the course of phenobarbital administration blood levels were determined. Phenobarbital blood levels correlated neither with the final values for plasma antipyrine half-lives nor with the per cent reduction in plasma antipyrine half-life produced by phenobarbital treatment. There was a direct relationship between initial antipyrine half-lives and the per cent shortening of antipyrine half-life produced by phenobarbital administration: the shorter the initial antipyrine half-life, the less the reduction caused by phenobarbital treatment. Larger intrapair variances in fraternal than in identical twins indicate genetic, rather than environmental, control of phenobarbital-induced alterations in plasma antipyrine half-life.

Genetic and environmental factors that regulate cytosolic epoxide hydrolase activity in normal human lymphocytes.

To determine whether genetic mechanisms control large variations in cytosolic epoxide hydrolase (cEH) activity of unstimulated lymphocytes from normal human subjects, cEH activity was measured in (a) 6 sets of monozygotic (MZ) twins and 6 sets of dizygotic (DZ) twins; (b) 100 unrelated male subjects; and (c) 6 families. The twin study revealed predominantly genetic control (H2(1) = 0.95). Variability was markedly less within MZ (intrapair variance = 0.25) than DZ twins (intrapair variance = 6.33). In 100 unrelated male subjects the extent of interindividual variation was 11-fold. Unimodal distribution of values among 99 subjects encompassed a sixfold range. One outlier with very high activity clearly stood apart. Using the whole distribution curve we phenotyped members of six families. In the outlier's family, analysis of three generations suggested autosomal dominant transmission of high cEH activity. Analysis of the other 5 families and of 12 sets of twins, all from the large unimodal distribution, was consistent with either monogenic or polygenic control of variations within this mode. Several temporal host factors, including fever, the menstrual cycle, a 24-h fast, and diurnal variations, were investigated. Fever and fasting elevated cEH activity. Diurnal variations produced no observable alteration. During the menstrual cycle irregular fluctuations occurred.

Polymorphism of theophylline metabolism in man.

To determine whether genetic mechanisms control large interindividual variations in theophylline elimination in normal uninduced human subjects, and, if so, to test the possibility that these genetic factors are transmitted as a simple Mendelian trait, theophylline was administered to 79 unrelated adults, six sets of monozygotic twins, six sets of dizygotic twins, and six two-generation families. Thereafter, in urine collected from each subject at regular intervals for 48 h, concentrations of theophylline and its three principal metabolites were measured and rate constants of formation of these metabolites calculated. The twin study, designed to determine the relative contributions of genetic and environmental factors to large interindividual variation in theophylline elimination, revealed predominantly genetic control. Values for this genetic component, designated heritability (H1(2)), of interindividual variation in rate constants of metabolite formation were 0.61, 0.84, and 0.95 for 3-methylxanthine, 1-methyluric acid, and 1,3-dimethyluric acid, respectively. H1(2) for the overall theophylline elimination rate constant (kel) was lower (0.34). In the 79 unrelated adults, each distribution curve for rate constants of formation of each theophylline metabolite appeared to be trimodal. By contrast, the distribution curve for the overall theophylline elimination rate constant appeared to be either unimodal or bimodal. The extent of interindividual variation was fourfold for theophylline kel and 6-8-fold for the three principal metabolites. High correlations among the three rate constants in individual subjects suggested their regulation by a single shared factor. In six families carefully selected to be under near basal environmental conditions so that hepatic theophylline metabolism of each family member would be neither markedly induced nor inhibited, phenotypes for theophylline metabolite rate constants were assigned. This assignment of phenotype was made by the position of each family member's rate constant on the three distribution curves that were generated from the 79 unrelated subjects. In each family, pedigree analysis of the three phenotypes for each rate constant was consistent with their control by two alleles at a single genetic locus and with autosomal codominant transmission. Frequencies of the two alleles at each genetic locus controlling rate constants of formation of theophylline metabolites were similar (p = 0.49, 0.53, and 0.52). In the three families studied with antipyrine (AP) as well as with theophylline, AP k(el) correlated (r approximately 0.7) with each rate constant of theophylline metabolite formation, as well as with theophylline k(el). While these results are compatible with a common regulatory element in the AP and theophylline polymorphisms, other evidence suggests more than a single genetic polymorphism. This additional evidence includes different gene frequencies for the AP (p approximately 0.1) and theophylline (p approximately 0.5) polymorphisms, different genotype assignments in several families for some theophylline metabolites, different distribution curves for theophylline k(el) from those for the three theophylline metabolites in 79 unrelated subjects, and finally low correlations between AP metabolite rate constants and theophylline metabolite rate constants in the three families receiving both drugs.

Genetic control of interindividual variations in the inducibility of aryl hydrocarbon hydroxylase in cultured human lymphocytes.

Interindividual and intraindividual variations in aryl hydrocarbon hydroxylase (AHH) induction by 3-methylcholanthrene were studied in cultured lymphocytes from normal adult volunteers. Using eight pairs of monozygotic and eight pairs of dizygotic twins, we examined to what extent these variations are controlled by heritable factors and whether AHH inducibility correlations in an individual with the plasma half-lives of three drugs. Substantial overestimation of the induction ratio (fold inducibility) may occur if the nonlinearity of the assay standard curve is not considered. Fold inducibility remains relatively constant for an individual, but large intraindividual variations occur in absolute "control" and "induced" AHH activities. Fetal calf serum may contain inducers of AHH activity that vary with the particular lot of serum, thereby rendering the apparent induction ratio an imprecise indicator of genetic susceptibility to induction by 3-methylcholanthrene. The index of heritability for AHH fold inducibility in twins studied with different lots of fetal calf serum (0.80) or with a single lot of fetal calf serum (0.77) suggests nonetheless that genetic rather than environmental factors are mainly responsible for interindividual variations in AHH inducibility by 3-methylcholanthrene in human lymphocytes. In these twins a significant but poor correlation (r=-0.551; 0.03 less than p less than 0.05) occurs between AHH inducibility in culture and the plasma antipyring half-life, but not between AHH inducibility and phenylbutazone or bishdroxycoumarin half-lives.

Variables affecting nicotine metabolism.

Nicotine metabolism is exceedingly sensitive to perturbation by numerous host factors. To reduce the large variations and discrepancies in the literature pertaining to nicotine metabolism, investigators in future studies need to recognize and better control these host factors. Recent advances in the understanding of nicotine metabolism have suggested new approaches to elucidating underlying mechanisms of certain toxic effects associated with cigarette smoking.

On the significance of host factors that affect drug disposition.

Reports on drug disposition in man often overemphasize statistical analyses or correlation coefficients at the expense of clear statements on the biological meaning of an experiment. Moreover, the results of many such studies are presented as though they were immutable. In reality, the levels of statistical significance often depend on, and are intimately related to, numerous host factors. Results can fall anywhere within a broad spectrum of values determined by the host factors of the subjects selected for investigation. The investigator's objectives determine whether subjects are selected for uniformity or heterogeneity of these host factors. Since only a small number of subjects can be studied, it is unlikely that all critical host factors will be represented. Therefore, studies repeated elsewhere may not duplicate subject host factors with sufficient precision to allow a similar outcome. Thus, a full description of critical host factors is required for all subjects who serve in studies on drug disposition. This requirement is particularly important because many subjects suffering from diseases exhibit simultaneous changes in critical host factors, thereby modifying the dosage requirements for drug therapy. The number of host factors recognized as capable of influencing drug disposition has risen due to recent discoveries in this active field.

Cimetidine-induced reduction in gastrointestinal absorption of antipyrine and rate constants for formation of its metabolites.

In 15 normal men, cimetidine taken orally in a dose of 300 mg twice a day for 3 days reduced to similar extents the rate constants for formation (ki) of the three principal metabolites of antipyrine (AP): 29.9% +/- 8.5% (mean +/- SD) for 4-hydroxyantipyrine (4-OH-AP); 28.3% +/- 6.3% for 3-hydroxymethylantipyrine (3-OHM-AP); and 22.4% +/- 5.6% for N-demethylantipyrine (NDM-AP). AP clearance declined 24.3%; AP salivary t 1/2 rose 33%; and corrected AP apparent volume of distribution was unchanged. In one apparently normal subject, however, kis for formation of 3-OHM-AP and NDM-AP rose after cimetidine even though AP clearance declined 19.7%. This surprising result, which suggests that cimetidine can exert an inductive effect on the hepatic mixed-function oxidases of some subjects, was checked by restudying the individual. Very similar values occurred on repetition. The average increase in kis for NDM-AP and 3-OHM-AP was 172.2% and 34.0%. These unusual results in this subject indicate that at least two distinguishable forms of cytochrome P-450 participate in AP metabolism in man. Cimetidine appeared to reduce the amount of AP absorbed from the gut in 10 of our 15 normal subjects.