Plasma membranes of the rat liver. Isolation and enzymatic characterization of a fraction rich in bile canaliculi.

A method is described for the rapid isolation of a plasma membrane fraction containing a high concentration of intact bile canaliculi from the rat liver. Isolated bile canaliculi retain most of the ultrastructural features exhibited in the intact liver cell. The final fraction contains 5'-nucleotidase activity at approximately the same concentration as that in previous preparations of plasma membranes. In the presence of 0.01 M Mg(++), 5'-nucleotidase exhibits a double pH optimum at pH values of 7.5 and 9.5. The activities of glucose-6-phosphatase and alkaline phosphatase are present in low amounts. Cytochrome P-450 is not detectable. Na(+)-K(+)-activation of ATPase is observed to the extent of 20-36% in about half of the assays. The availability of a method for preparation of intact bile canaliculi should prove useful for studying the biochemical events associated with the transport of bile constituents into canaliculi.

Follicle-stimulating hormone (FSH) and luteninizing hormone (LH) in the urine of prepubertal children.

Follicle-stimulating hormone (FSH) and luteinizing hormone (LH) have been measured by specific bioassays in pooled urine samples from prepubertal children, aged 2-6 yr, and from male adults. For children the mean urinary excretion of FSH was 2.2 U 2nd International Reference Preparation (2nd IRP) per liter and the mean urinary excretion of LH was 0.44 U 2nd IRP per liter. For adults the mean FSH excretion was 5.6 U 2nd IRP per liter and the mean LH excretion was 4.7 U 2nd IRP per liter. Our data show a 2.5-fold increase of FSH, a 10.7-fold increase of LH, and a consequent decrease in the FSH: LH ratio from 5 to 1 between childhood and adulthood. FSH and LH in urine from three patients with gonadal abnormalities have also been studied. The results from normal children, adults, and abnormal patients form a spectrum and reveal that sexual maturity is accompanied by a marked increase in the excretion of LH with relatively smaller increases in FSH.

Stimulators and inhibitors of hepatic porphyrin formation in human sera.

Human sera were found to contain factors that stimulate and factors that inhibit porphyrin formation by cultured avian liver cells. The capacity of sera to stimulate or inhibit porphyrin formation varied in different hormonal states and in the porphyrias. Sera from 31 post partum women, eight of whom were not lactating, inhibited porphyrin formation to a mean level 30% below the level in control cultures and also inhibited drug and steroid stimulation of porphyrin formation. In contrast, mean porphyrin formation compared to control cultures was increased between 9 and 21% by sera from 52 normal subjects, 16 women on oral contraceptives, and 11 pregnant women. It was increased 193% by sera from nine subjects with acute intermittent porphyria and 172% by sera from 13 subjects with porphyria cutanea tarda. Heated sera or ethanol extracts of sera from all groups of subjects further increased the mean porphyrin stimulation by sera and, for the post partum subjects, eliminated the inhibitory effect. Ethanol extracts of sera from 28 oral contraceptive-treated women caused significantly greater mean stimulation of porphyrin formation than did extracts of sera from 30 normal women. While sera from 17 out of 22 porphyric subjects contained both stimulatory and inhibitory factors, 5 out of 22 had no evidence of an inhibitory component. There appeared to be heterogeneity in the occurrence of the factors among porphyrics.The factor(s) in sera responsible for porphyrin stimulation were heat-stable and insensitive to trypsin; were present in the supernates after ethanol precipitation of plasma proteins; were extractable in ethyl acetate and nondialyzable; and they migrated with the albumincontaining fraction of serum during electrophoresis. The factor(s) responsible for porphyrin inhibition were heat labile, sensitive to trypsin, and resistant to neuraminidase; were present in the ethanol precipitates of sera and were nondialyzable; and they migrated with the gamma globulin fraction of serum during electrophoresis. Inhibition of porphyrin formation was not attributable to heme, free or bound as hemoglobin, hemopexin, or hemalbumin.

Studies of the pituitary-Leydig cell axis in young men with hypogonadotropic hypogonadism and hyposmia: comparison with normal men, prepuberal boys, and hypopituitary patients.

Pituitary and gonadal function was studied in seven chromatin-negative men, ages 15-27 yr, with retarded sexual and somatic development, skeletal anomalies, and hyposmia. These hyposmic patients were compared with normal men, prepuberal boys and hypogonadal patients with hypopituitarism. The urinary follicle-stimulating hormone (FSH) and luteinizing hormone (LH) levels of hyposmic subjects were the same as those of normal boys and hypopituitary patients but significantly lower than those of normal men. Clomiphene citrate did not cause an increase in plasma FSH and LH levels in either hypogonadal group as it does in normal men. In contrast to hypopituitary patients, thyroid and adrenocortical function and release of growth hormone in the hyposmic subjects were normal. The plasma testosterone levels were equally low in prepuberal, hypopituitary, and hyposmic patients but were increased to a greater extent by human chorionic gonadotropin (HCG) treatment in prepuberal and hypopituitary subjects than in the hyposmic patients. Prolonged treatment with HCG has failed to return plasma testosterone levels to normal in two hyposmic patients. These observations suggest that there are defects of both pituitary and Leydig cell function in men with the syndrome of hypogonadism, skeletal anomalies, and hyposmia. They have impaired secretion of FSH and LH and a Leydig cell insensitivity to gonadotropin.

Induction of tamoxifen-4-hydroxylation by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), beta-naphthoflavone (beta NF), and phenobarbital (PB) in avian liver: identification of P450 TCDDAA as catalyst of 4-hydroxylation induced by TCDD and beta NF.

Tamoxifen has been found to be metabolized by liver primarily into three metabolites, tamoxifen-N-oxide, formed by the flavin-containing monooxygenase, and N-desmethyl- and 4-hydroxytamoxifen, formed by cytochrome P450. The N-demethylation was demonstrated to be catalyzed by P4503A in rat and human liver; however, the P450s catalyzing the 4-hydroxylation have not been identified. Although 4-hydroxytamoxifen exhibits more potent estrogen agonist/antagonist activity than tamoxifen, the relative contributions of the parent drug and its 4-hydroxy metabolite(s) to the activity of tamoxifen in vivo have not been established. We report here that the rate of tamoxifen 4-hydroxylation is higher in livers of adult chicken and chick embryos than in livers of mammalian species. Tamoxifen 4-hydroxylation was increased by treatment of chick embryos with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), beta-naphthoflavone (beta NF), and to a lesser extent by phenobarbital (PB). The major effect of PB treatment was an increase in tamoxifen N-demethylation. Tamoxifen 4-hydroxylase activity of reconstituted purified chicken P450s was highest for TCDDAA, a P450 active in arachidonate epoxygenation and estradiol 2-hydroxylation, and one of the two major P450s induced by TCDD and beta NF in chick embryo liver. The second P450, TCDDAHH, which is active in aryl hydrocarbon hydroxylase and 7-ethoxyresorufin deethylase was inactive in tamoxifen 4-hydroxylation. Anti-TCDDAA IgG immunoinhibited tamoxifen 4-hydroxylation in microsomes from beta NF-treated embryos by over 80%, but was ineffective against this reaction in the controls. The immunochemical findings together with the reconstitution data identify TCDDAA as the P450 responsible for TCDD/beta NF-induced tamoxifen 4-hydroxylation in chick liver. In PB-treated livers, a P450 fraction containing CYP2H1/H2, the major PB-induced P450s, had the highest tamoxifen 4-hydroxylase and N-demethylase activities, a finding compatible with one or both of those P450s being responsible for the PB-induced tamoxifen 4-hydroxylation and N-demethylation. The findings reported here raise the possibility that exposure of women undergoing tamoxifen therapy to agents that induce human CYP1A2 or CYPB1/2 analogues may produce increased levels of 4-hydroxytamoxifen and that this may affect the therapeutic potency of tamoxifen.

Aryl hydrocarbon hydroxylase activity and microsomal cytochrome content of human fetal tissues.

Aryl hydrocarbon hydroxylase (AHH) and microsomal cytochromes were measured in tissues of human features aborted by prostaglandins. Cytochrome P-450 concentrations and AHH activity were about 4 times higher in adrenal glands than in liver. AHH was present in testes, ovaries, and vagina and uterus at levels equal to or greater than those in the liver. In lung, kidney, and intestines it was present at levels lower than those in the liver. Mean hepatic AHH and hepatic and adrenal cytochromes P-450 and b5 were not significantly different in prostaglandin and hysterotomy abortuses; mean adrenal AHH was significantly lower in prostaglandin abortuses, but the ranges in both groups were overlapping. Neither fetal sex nor maternal cigarette smoking affected hepatic or adrenal AHH activity or microsomal cytochrome concentrations or difference spectra. Hepatic and adrenal AHH activities were concentrated in microsomes and were correlated with microsomal P-450 content. These findings are constant with P-450 mediation of AHH in human fetal tissues. The data demonstrate the utility of prostaglandin abortuses for studies of fetal tissue mixed-function oxidase activity and point to the endocrine glands and target tissues in addition to the liver as potential sites for activating chemical carcinogens and cytotoxins in the human fetus.

Studies in porphyria. VI. Biosynthesis of porphyrins in mammalian skin and in the skin of porphyric patients.

Porphyrin biosynthesis in mammalian skin and in skin obtained from patients with selected types of porphyria has been studied. Cutaneous porphyrinogenesis required the precursor delta-aminolevulinic acid (ALA) which, when added to murine, rat, and human skin in vitro, was rapidly converted to porphyrins. Total porphyrin content was quantitated by fluorescence assay, and spectral studies indicated that more than 80% of the porphyrin produced was protoporphyrin. The majority of skin porphyrinogenesis occurrred in epidermis or in epidermal derivatives such as hair roots. Known inducers of hepatic delta-aminolevulinic acid synthetase (ALAS), the rate-limiting enzyme for heme biosynthesis, were not inducers when added to skin in vitro. Skin from patients with acute intermittent porphyria demonstrated a 43% decrease in cutaneous porphyrin production as compared to unaffected normals. This is consistent with the known deficiency of uroporphyrinogen synthetase that has been previously demonstrated in the liver and red blood cells of these patients. Porphyrinogenesis in skin of patients with porphyria cutanea tarda was not different from controls. These studies demonstrate that skin has the enzymatic capacity to synthesize porphyrins from added ALA and that cutaneous porphyrinogenesis from ALA is deficient in patients with acute intermittent porphyria.

Danazol, a new steroidal inducer of delta-aminolevulinic acid synthetase.

Danazol (a derivative of 17 alpha-ethinyl testosterone) has recently been reported to cause clinical exacerbations of acute intermittent porphyria. In the present study, we demonstrate that danazol is an inducer of hepatic delta-aminolevulinic acid synthetase and that it increases hepatocyte porphyrin production using a chick embryo model. The findings provide an explanation for the clinical observations. Although danazol is a porphyrinogenic agent in the chick embryo and humans it has been reported to be ineffective as a porphyrinogen in rats. The present findings, therefore, also confirm the superiority of the chick embryo as a model system for the screening of drugs and chemicals which may have the potential to cause clinical exacerbations of porphyria.

Changes in drug metabolism in children with thyroid disorders.

The rate of disappearance of antipyrine from the plasma is a useful indicator for the in vivo capacity of mixed function oxidation. The half-life of antipyrine was measured before and after treatment in three hypothyroid and three hyperthyroid children, aged three months to 14 years, in order to examine the effect on drug metabolism of thyroid disorders in children. The half-life of antipyrine decreased in all three hypothyroid subjects and increased in all three hyperthyroid subjects after treatment. The mean half-life decreased from 34.5 h to 8.6 h after treatment of the hypothyroid subjects and increased from 6.1 to 10.1 h after treatment of the hyperthyroid subjects. The mean metabolic clearance rate of antipyrine increased from 11.7 to 25 ml/h in the hyothyroid patients while in the hyperthyroid children there was a decrease from 43 to 25 ml/h. The apparent volume of distribution did not change significantly in the treatment, thus changes in the half-life of antipyrine were most likely attributable to alterations in the metabolic clearance rate of antipyrine.

Antipyrin clearance in homozygous beta-thalassemia.

Serum half-lives for antipyrine were normal or shorter than normal in 19 subjects between 7 and 23 yr of age with beta-thalassemia major. The mean antipyrine serum half life (+/-SE) for the group as a whole was 8.5 +/- 0.6 hr. The mean antipyrine half-lives (t1/2) for the younger subjects were within the range reported for normal children, while the mean t1/2 for the older males approached the values reported for normal adult males. The mean t1/2 for the older females was shorter than has been reported for normal adult females. The mean apparent volume of distribution for antipyrine (+/-SE) in the subjects with thalassemia was 0.69 +/- 0.01 L/kg. Thus, total body water appears to be increased in thalassemia. The mean metabolic clearance rate for antipyrine (+/-SE) in the group as a whole (1.07 +/- 0.08 ml/min/kg) is substantially higher than the metabolic clearance rates for antipyrine reported in normal adults. Thus, the relatively short t1/2s of antipyrine in subjects with thalassemia are attributable to rapid rates of clearance of the drug. The data indicate that antipyrine clearance is unimpaired in patients with thalassemia despite evidence of liver damage and iron overload. Our study supports the proposition that hepatic microsomal hemoprotein synthesis is not adversely affected in homozygous beta-thalassemia.

Effect of glucocorticoids and ACTH on antipyrine clearance in children.

Administration of dexamethasone and infusion of adrenocorticotropic hormone (ACTH) to children for periods up to 4 days did not alter the half-life, apparent volume of distribution, or metabolic clearance rate of antipyrine, a drug principally metabolized by the mixed-function oxidase system of the liver. We conclude that the short-term administration of glucocorticoids and ACTH with ensuing stimulation of endogenous glucocorticoid production is unlikely to produce clinically significant changes in the rate of drug metabolism.

Effects of growth hormone on antipyrine kinetics in children.

The kinetics of antipyrine, a drug used as a clinical indicator of hepatic drug-metabolizing enzyme activity, were examined in children after short- (5 days to 1 wk) or long-term (6 wk to 1 yr) treatment with human growth hormone (hGH). After short-term treatment the mean volume of distribution of antipyrine (aVd) (also a measure of total body water) increased from 0.49 to 0.58 l/kg (p less than 0.005). The mean aVd after long-term treatment did not differ from the mean pretreatment value, but it rose in three of the eight subjects examined. Neither the mean serum half-life (t 1/2) nor the metabolic clearance rate of antipyrine for the group as a whole was altered after short- or long-term treatment with hGH. However, t 1/2 rose to 135% to 151% of control value in three of nine children and decreased to 63% of control value in one after short-term treatment, while after long-term treatment it rose to 128% to 176% of control value in four of eight children. The results indicate that hGH can increase total body water and should be used cautiously in children with impaired cardiac, renal, or hepatic function. The data further suggest that hGH may alter antipyrine t 1/2 in some children. The variable nature of the changes precludes any uniform prediction about growth hormone effects on drug metabolism, but it may be necessary in some children to modify the dosage of other drugs administered with hGH.

NAD(P)H: quinone oxidoreductase (DT-diaphorase) in chick embryo liver. Comparison to activity in rat and guinea pig liver and differences in co-induction with 7-ethoxyresorufin deethylase by 2,3,7,8-tetrachlorodibenzo-p-dioxin.

NAD(P)H:quinone oxidoreductase (EC 1.6.99.2; DT-diaphorase) was present in the liver of 18- and 19-day-old chick embryos as assayed both by reduction of resorufin and by the more traditional assay, reduction of 2,6-dichlorophenolindophenol (DCPIP). Both reductions had the classic characteristics of DT-diaphorase: they were equally supported by NADPH and NADH and almost entirely inhibited by dicumarol. Chick embryo liver DT-diaphorase was entirely cytosolic. It was undetectable in the microsomal and mitochondrial fractions. Chick embryo liver cytosol and mitochondrial fractions contained an enzyme oxidizer of resorufin but not of DCPIP. The Km for NADPH for resorufin reductase was an order of magnitude higher in chick embryo than in rat or guinea pig cytosol (1 mM vs 0.1 mM). Resorufin reductase activity was higher for chick embryo than for rat or guinea pig cytosols: Vmax (nmol resorufin reduced per mg cytosolic protein per min +/- SEM) 355 +/- 28 for chick embryo, 159 +/- 10 for guinea pig and 68 +/- 28 for rat. The Vmax for DCPIP reduction was also twice as high in chick embryo as rat liver cytosol. In the chick embryo, 7 days after treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) at 6.4 micrograms/kg egg (1 nmol/egg) mortality was increased 2.4-fold, hepatic DT-diaphorase 1.3-fold, and 7-ethoxyresorufin deethylase (7-EROD) 72-fold over control levels. At 32 micrograms/kg, mortality was increased 4.2-fold, DT-diaphorase 2.3-fold and 7-EROD 100-fold. In the guinea pig, 5 days after treatment with TCDD at 10 micrograms/kg, TCDD toxicity was also evident (loss of body weight and thymus weight); there was no change in DT-diaphorase as measured by resorufin reduction, confirming by a different assay the observation of Beatty and Neal (Biochem Pharmacol 27: 505-510, 1978) that TCDD does not induce DT-diaphorase in guinea pig liver, and 7-EROD was increased 8-fold. In contrast, in the rat, 7 days after exposure to TCDD at 10 micrograms/kg, there was no evidence of toxicity, DT-diaphorase was increased close to 7-fold and 7-EROD, 100-fold. The results demonstrate that avian liver contains DT-diaphorase and show that the extent to which DT-diaphorase is part of the pleiotypic response of the liver to an Ah (aryl hydrocarbon) receptor ligand is species dependent. They also suggest that DT-diaphorase induction and TCDD toxicity may be inversely related. The possibility that DT-diaphorase protects against TCDD toxicity and participates in species differences in sensitivity to TCDD toxicity warrants further investigation.