Analysis of genes for bilirubin UDP-glucuronosyltransferase in Gilbert's syndrome.

Gilbert's and Crigler-Najjar syndromes are characterised by unconjugated hyperbilirubinaemia due to complete and partial absence of bilirubin UDP-glucuronosyltransferase (UGT). Nucleotide sequences of the genes for bilirubin UGT were analysed in six patients with Gilbert's syndrome. All patients had a missense mutation caused by a single nucleotide substitution and the mutations were heterozygous. In addition, relatives of patients with Crigler-Najjar syndrome types I and II, and of those with Gilbert's syndrome were analysed. All ten relatives with mild hyperbilirubinaemia were heterozygotes with respect to each defective allele. These results suggest that Gilbert's syndrome is inherited as a dominant trait.

Angiotensin II directly induces follicle rupture and oocyte maturation in the rabbit.

To investigate the possible direct involvement of angiotensin II (Ang II) in ovulation and oocyte maturation, Ang II at 100 or 10 micrograms was administered at 2-h intervals in the in-vitro perfused rabbit ovaries. The addition of Ang II in the perfusate induced ovulation in vitro in the absence of gonadotropin, while ovulation did not occur in any contralateral control ovaries. However, the ovulatory efficiency in the Ang II-treated ovaries was significantly lower than in hCG-treated ovaries. Ang II significantly stimulated the meiotic maturation of ovulated ova and follicular oocytes. Concomitant addition of the specific receptor antagonist of Ang II, saralasin, 30 min before the onset of Ang II administration blocked Ang II-induced ovulation in a complete manner. Although saralasin did not inhibit completely hCG-induced ovulation and oocyte maturation, these results suggest that Ang II produced in the ovary may act locally in the process of ovulation.

Stimulatory role of cyclic adenosine monophosphate as a mediator of meiotic resumption in rabbit oocytes.

The present study was undertaken to assess the role of alterations of intraoocyte cAMP concentrations in the meiotic maturation of isolated and follicle-enclosed oocytes. In isolated oocyte culture, the intracellular cAMP content of denuded oocytes declined within 15 min of incubation, whereas the cAMP content of cumulus-enclosed oocytes did not change substantially for 1.5 h of incubation, and then declined abruptly. Commitment to meiotic maturation was preceded by reduced concentrations of intraoocyte cAMP. Forskolin inhibited the spontaneous maturation of cumulus-enclosed oocytes in a dose-dependent manner. However, this inhibition was attenuated as the duration of incubation increased. Forskolin significantly stimulated the intracellular cAMP content of denuded and cumulus-enclosed oocytes, but intraoocyte cAMP returned to pretreatment values within 4 h. The decline in intraoocyte cAMP was followed by the meiotic maturation of isolated oocytes. In in vitro perfused rabbit ovaries, exposure to forskolin at 10(-4) M, as well as to 50 IU human CG, accelerated the meiotic maturation of follicle-enclosed oocytes. The intraoocyte cAMP content increased significantly within 30 min and reached its maximum 2 h following exposure to forskolin. Thereafter, cAMP decreased abruptly and returned to pretreatment levels by 6 h. These alterations of intraoocyte cAMP contents following exposure to forskolin paralleled those observed in human CG-treated ovaries. The decline in cAMP content of follicle-enclosed oocytes was followed by their meiotic maturation. In conclusion, the sustained elevation of intraoocyte cAMP levels inhibits the initiation of meiotic maturation in isolated and follicle-enclosed oocytes. Within the follicle, resumption of meiosis is triggered via a transient increase in intraoocyte cAMP, but commitment to meiosis must await the decline of intraoocyte cAMP.

Protein kinase C mediates gonadotropin-releasing hormone agonist-induced meiotic maturation of follicle-enclosed rabbit oocytes.

The present study was undertaken to determine the effects of a protein kinase C inhibitor, staurosporine, on gonadotropin-releasing hormone agonist (GnRHa)-induced oocyte maturation and follicular prostaglandin (PG) production, and the response to direct activators of protein kinase C using rabbit mature follicle culture. Treatment of mature follicles with GnRHa (buserelin and leuprolide acetate) neither stimulated nor inhibited cAMP accumulation in both the follicle and oocyte. Exposure to staurosporine at 10(-6) M 60 or 15 min before GnRHa (buserelin) administration reduced significantly the meiotic maturation of follicle-enclosed oocytes induced by GnRHa at 10(-7) M. However, staurosporine addition coincident with the agonist or thereafter did not inhibit meiotic maturation. Staurosporine suppressed GnRHa-induced meiotic maturation in a dose-dependent manner, whereas hCG-stimulated oocyte maturation was not inhibited. Similarly, staurosporine administered 60 min before exposure to GnRHa suppressed GnRHa-stimulated PG production by mature follicles. The active phorbol esters, 10(-6) M 12-0-tetra-decanoyl phorbol 13-acetate (TPA) and 10(-6) M 4 beta-phorbol 12,13-didecanoate (4 beta-PDD) stimulated meiotic maturation whereas the biological inactive isomer, 4 alpha-PDD, did not. The kinetics of germinal versicle breakdown of follicle-enclosed oocytes in the presence of active phorbol esters paralleled that of GnRHa-treated oocytes. Furthermore, the concomitant addition of staurosporine at 10(-6) M to the culture medium inhibited significantly (p less than 0.05) TPA-induced meiotic maturation. These data demonstrate that GnRHa stimulated both the meiotic maturation of follicle-enclosed oocytes and follicular PG formation via a mechanism other than the cAMP-mediated process.(ABSTRACT TRUNCATED AT 250 WORDS)

Direct effect of gonadotropin-releasing hormone agonists on the rabbit ovarian follicle.

OBJECTIVE: To determine if gonadotropin-releasing hormone agonist (GnRH-a)-induced oocyte maturation and degeneration can be attributed to the direct actions on the follicle. DESIGN: Mature rabbit follicle culture. INTERVENTIONS: The mature follicles were cultured with human chorionic gonadotropin (hCG) (100 ng/mL), buserelin acetate (10(-9) to 10(-6) M), leuprolide acetate (10(-9) to 10(-6) M), or buserelin acetate (10(-7) M) with a GnRH antagonist (10(-8) to 10(-6) M) for 14 hours. MAIN OUTCOME MEASURES: The percentage of oocytes achieving germinal vesicle breakdown, the oocyte degeneration rate, prostaglandins (PG) production by mature follicles, and the frequency of fertilization and embryonic development. RESULTS: Gonadotropin-releasing hormone agonist induced the meiotic maturation of follicle-enclosed oocytes in a dose-dependent manner while concomitantly increasing oocyte degeneration. The simultaneous addition of GnRH antagonist inhibited significantly GnRH-a-induced oocyte maturation and PG production by the mature follicles. Furthermore, a GnRH antagonist reversed the oocyte degeneration rate that had been increased by GnRH-a. The rates of normal fertilization and early embryonic development were significantly reduced in the oocytes matured by GnRH-a as compared with those matured by hCG. CONCLUSIONS: Gonadotropin-releasing hormone agonist acts directly on mature rabbit follicles to trigger the oocytes to undergo meiotic maturation, but oocytes matured in vitro by GnRH-a are not necessarily cytoplasmically mature.

Possible involvement of lipoxygenase products in human corpora lutea.

The present study was undertaken to determine the ability of cultured luteal cells from human corpora lutea to secrete progesterone (P4) and prostaglandins (PGs), and to assess the effects of the products of the lipoxygenase pathway on luteal P4 production. Luteal cells responded to human chorionic gonadotropin (hCG) with a significant increase (2- to 7-fold) in P4 production. Arachidonic acid significantly stimulated PGE2 synthesis by luteal cells in a dose-dependent manner. Both basal PGE2 production and the responsiveness to arachidonic acid were maintained for 8 days. In contrast, both PGF2 alpha and 6-keto-PGF1 alpha production abruptly declined as the culture proceeded. However, the addition of hCG did not further stimulate the accumulation of the 3 PGs assayed. In the subsequent experiment, 5-hydroxyeicosatetraenoic acid (5-HETE) and the reaction products of soybean lipoxidase of arachidonic acid (AA-LIP) were utilized for evaluating the involvement of the lipoxygenase pathway in luteolysis. The addition of 5-HETE dose-dependently inhibited P4 production by the cultured luteal cells. Although treatment with either arachidonic acid or lipoxidase alone had no effect on P4 production, AA-LIP significantly reduced P4 production in the presence or absence of hCG. These results suggest that the products of the lipoxygenase as well as of the cyclo-oxygenase pathway may be important in regulating the life span and function of human corpora lutea.

Novel high-performance liquid chromatography for determination of membrane phospholipid composition of rat hepatocytes.

We have developed a new, quick and efficient method of high-performance liquid chromatography (HPLC) for the isolation and quantitative determination of phospholipids in hepatocyte membranes. A silica gel column was used for the isolation and determination, and an isocratic mixture of acetonitrile, methanol and 85% phosphoric acid (130:5:1.7, v/v/v) was used as a mobile phase. Six kinds of phospholipids, i.e. phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidylcholine (PC), lysophosphatidylcholine (LPC) and sphinogomyelin (SPH), in this order, were completely isolated within 45 min. The phospholipid composition of sinusoidal membrane vesicles (SMV) and canalicular membrane vesicles (CMV) obtained from rat liver, as well as of human erythrocyte ghosts were determined by this HPLC method. The level of SPH in CMV was significantly higher than that in SMV, and the level of PC in CMV was significantly lower than that in SMV. These results were considered attributable to the low fluidity of CMV. The phospholipid composition of human erythrocyte membrane was different from that of rat SMV and CMV. The present technique is suitable for quantitative determination of phospholipids in cell membranes.

Gonadotropin-releasing hormone agonists induce meiotic maturation and degeneration of oocytes in the in vitro perfused rabbit ovary.

The present study was undertaken to assess the effects of gonadotropin-releasing hormone agonists (GnRH-a, buserelin and leuprolide acetate [LA]) on ovulation, oocyte maturation and degeneration, and steroid and prostaglandin production in the perfused rabbit ovary preparation. Ovulation did not occur in any of ovaries treated with buserelin or LA (10(2) to 10(4) ng/mL) in the absence of gonadotropin. Gonadotropin-releasing hormone agonists were associated with the resumption of meiosis in follicular oocytes in a dose-related manner. Furthermore, the addition of GnRH-a to the perfusate significantly increased the percentage of follicular oocytes that showed evidence of degeneration compared with contralateral untreated or human chorionic gonadotropin-treated controls. Prostaglandin E2 and prostaglandin F2 alpha production by the perfused rabbit ovaries were stimulated significantly by GnRH-a treatment. Exposure to GnRH-a failed to increase either progesterone or estradiol production by the perfused rabbit ovaries. These data demonstrate that GnRH-a act directly in the rabbit ovary to trigger meiotic maturation in oocytes within the follicles, concomitantly increasing oocyte degeneration.

[Treatment of leiomyomas with luteinizing hormone-releasing hormone agonist].

Twenty-five premenopausal women, 36-54 years of age, with uterine myomas were treated with 600-1,200 micrograms/day of luteinizing hormone-releasing hormone agonist (LHRHa) for 4 months. Eight patients reached menopause following the treatment with LHRHa (menopause group), while the resumption of menstruation occurred within 12 weeks after cessation of the therapy in 17 patients (menstruation group). Although the mean hemoglobin (Hb) concentration in the menopause group increased during treatment and was maintained within the normal range after cessation of the therapy, the Hb concentration in the menstruation group decreased after the resumption of menstruation. Both estradiol and CA125 in the menopause group were reduced during and after treatment. However, these parameters in the menstruation group increased concomitantly with the resumption of ovarian function. LH and FSH were suppressed during treatment, but these gonadotropins in the menopause group increased significantly to the levels of menopause. About a 50% reduction in uterine volume was observed in the menopause group. Three months after completing therapy, the restoration of uterine volume occurred in the menstruation group. Bone density findings in microdensitometry 12 weeks after cessation of the therapy did not differ significantly from those before the treatment. These results demonstrate that LHRHa therapy significantly reduces the uterine volume in patients with leiomyoma. It may be possible to treat selected patients with leiomyoma, including perimenopausal women and high surgical risk women with LHRHa, thus avoiding the need for surgery.

Effects of gonadotropin-releasing hormone agonists on meiotic maturation of follicle-enclosed oocytes in rabbits.

The effects of GnRH agonists on in vitro maturation of rabbit follicle-enclosed oocytes were studied. Rabbit preovulatory follicles were cultured with or without hCG (10(2) ng/ml), buserelin (10(2)-10(5) ng/ml), or leuprolide (10(2)-10(5) ng/ml) for 14 hours in vitro. GnRH agonists induced the resumption of meiosis in the follicle-enclosed oocytes in a dose-dependent manner. The percentage of oocytes achieving GVBD following treatment with 10(5) ng/ml buserelin (87.9 +/- 6.3%) or 10(5) ng/ml leuprolide (86.0 +/- 4.1%) did not differ significantly from hCG-treated control (87.3 +/- 3.8%). Mature oocytes initially were detected within 2 hours of GnRH agonist exposure. Concomitant addition of a GnRH antagonist at 10(4) ng/ml significantly blocked the stimulatory effect of GnRH agonist on oocyte maturation. GnRH agonists significantly stimulated both prostaglandin (PG) E2 (PGE2) and PGF2 alpha production by preovulatory follicles (p less than 0.01), but secreted prostanoid levels did not differ significantly among different concentrations of GnRH agonists. Meiotic maturation of follicle-enclosed oocytes following GnRH agonist exposure began 2 hours earlier than production of PGs. PG production stimulated by GnRH agonists was reduced significantly by indomethacin. However, oocyte maturity in the presence of GnRH agonist plus indomethacin did not differ significantly from that of GnRH agonist alone. GnRH agonistic analogues induce the resumption of meiosis in follicle-enclosed oocytes in rabbits by a mechanism other than PG stimulation.

Proportion of conjugated bilirubin in bile in relation to hepatic bilirubin UDP-glucuronyltransferase activity.

To elucidate the diagnostic relevance of biliary conjugated bilirubin, biliary bilirubin from normal volunteers (NV), patients with Gilbert's syndrome (GS) and Crigler-Najjar syndrome type II (C-N II), and from various rat strains was fractionated. Biliary bilirubin diglucuronide (BDG) was present at lower levels, and bilirubin monoglucuronide (BMG) and unconjugated bilirubin were present at higher levels in GS and C-N II compared with NV, which is consistent with decreased hepatic bilirubin UDP-glucuronyltransferase activity (BGTA). The level of biliary BDG was higher in Wistar-Kyoto rats and lower in heterozygous (Jj) Gunn rats than in SD and Wistar rats. The hepatic BGTA level in heterozygous (Jj) Gunn rats was decreased to 60% of that in Wistar rats, in accordance with decreased biliary BDG. On the other hand, BGTA in Wistar-Kyoto rats whose biliary BDG level was high, was not different from that of Wistar and SD rats. Thus, a correlation between BGTA and biliary bilirubin fractions may not exist on some occasions.

A case of Gilbert's syndrome combined with macroamylasemia.

A 30-year-old Japanese male, who had no remarkable family history, visited our hospital with a complaint of abdominal pain, and unconjugated hyperbilirubinemia and hyperamylasemia were observed. He showed negative hemolysis tests, positive nicotinic acid test, low hepatic bilirubin UDP-glucuronyltransferase activity, decreased bilirubin diglucuronide and increased bilirubin monoglucuronide in bile, and a decrease in serum bilirubin after phenobarbital administration. He also showed high serum amylase level, low urine amylase level, and low amylase-creatinine clearance ratio. Gel filtration of serum with Sephadex G-200 revealed the existence of macroamylase. Countercurrent immunoelectrophoresis proved binding of serum amylase to lambda type IgA. From these results, the case was diagnosed as Gilbert's syndrome combined with macroamylasemia.

Serum binding and biliary excretion of bilirubin after bilirubin loading in Nagase analbuminemic rats and heterozygous (Jj) Gunn rats.

To elucidate the effects of albumin on the handling of serum bilirubin, hepatic metabolism and biliary excretion of bilirubin were examined during intravenous bilirubin infusion in Sprague-Dawley (SD) rats, Gunn (heterozygous, Jj) rats, and Nagase analbuminemic rats (NARs). Serum bilirubin was primarily bound to a protein fraction with a molecular weight of about 600 x 10(3) or more in NARs. About 39.2% +/- 12.5% of the serum bilirubin during infusion of bilirubin was bound to the same fraction in Gunn rats. Bilirubin was substantially taken up into the liver and excreted into the bile in NARs, suggesting the role of a high molecular protein, probably a lipoprotein, in its blood transport and the hepatic uptake process. In NARs, biliary bilirubin secretion reached the peak between 20 and 40 minutes after the initiation of bilirubin loading and decreased thereafter, whereas it continued to increase in SD rats and in NARs to which albumin was administered 20 minutes after the start of bilirubin loading. Biliary bilirubin fractions before bilirubin loading were similar in SD rats and NARs, whereas an increase in bilirubin monoglucuronide (BMG) and a decrease in bilirubin diglucuronide (BDG) were observed in Gunn rats. After the initiation of bilirubin loading, a decrease in biliary BDG and an increase in BMG and unconjugated bilirubin were observed in all groups of rats.

Determination of individual serum bile acids in chronic liver diseases: fasting levels and results of oral chenodeoxycholic acid tolerance test.

Fifteen bile acids in serum of 5 normal subjects and 21 patients with chronic liver diseases were fractionated by high performance liquid chromatography. Fasting total bile acids (TBA), glycocholic acid, taurocholic acid, glycochenodeoxycholic acid (GCDCA), and taurochenodeoxycholic acid (TCDCA) were significantly increased in patients with liver cirrhosis as compared with normal subjects. The cholic acid (CA) level and the ratio of the sum of free and conjugated CA to the sum of free and conjugated chenodeoxycholic acid (CDCA) were significantly elevated in patients with compensated as compared with decompensated liver cirrhosis, and were useful for differentiation of the two conditions. Serum bile acid levels were determined after oral administration of 500 mg of CDCA in the 5 normal subjects and 11 patients with liver disease. The TBA level reached a peak 90 min after CDCA administration in patients with chronic hepatitis and after 120 min in those with liver cirrhosis. The increase in the TBA level was significantly greater in patients with liver disease than in normal subjects. CDCA, GCDCA, and TCDCA showed changes similar to those in TBA. In patients with decompensated liver cirrhosis, the reduction in the TBA and CDCA levels after the peaks was slow, and GCDCA and TCDCA levels continued to increase until 180 min after the administration of CDCA. The TBA and CDCA levels 180 min after CDCA administration were significantly different among normal subjects, patients with chronic hepatitis, those with compensated liver cirrhosis, and those with decompensated liver cirrhosis, suggesting the usefulness of CDCA administration in differentiation of these conditions.

An autopsy case of histiocytic medullary reticulosis presenting with marked hepatosplenomegaly for 13 years before the onset.

A 32-year-old male was hospitalized with high fever, pancytopenia and hepatosplenomegaly. No atypical cells were found in the peripheral blood. Bone marrow aspiration resulted in dry taps. Superficial lymph node swelling was not observed. He had been treated twice for high fever, hepatosplenomegaly and leukopenia that were very similar to the present illness, 13 and 3 years before the onset and hepatosplenomegaly had been noted by the patient for 13 years. The patient died after a rapid course of 20 days. Histiocytic medullary reticulosis (HMR) was diagnosed at the autopsy, which revealed atypical histiocytic infiltration showing erythrophagocytosis in the liver, spleen, left adrenal, and mesenterial and pulmonary hilar lymph nodes. This patient had shown the same clinical signs and hepatosplenomegaly 13 years before the onset of HMR, which suggest a possible latent stage and acute exacerbation of HMR.

Serum bilirubin fractionation using multilayer film method in hepatobiliary diseases in comparison with high performance liquid chromatography.

The Ektachem multilayer film method (Ektachem) and high performance liquid chromatography (HPLC) were employed to fractionate and evaluate serum bilirubin species in 45 serum samples. The false-positive or false-high levels of bilirubin close-bonded with albumin (i.e. the delta bilirubin fraction (B delta] was obtained by Ektachem in sera of cases with normal bilirubin concentration and cases with unconjugated hyperbilirubinemia when compared with the results of HPLC. In the sera of cases with conjugated hyperbilirubinemia, Ektachem gave comparable levels of total bilirubin (TB), and unconjugated bilirubin (Bu) to those of HPLC, but underestimated conjugated bilirubin (Bc) and slightly overestimated B delta. To investigate the clinical significance of B delta in 113 cases of various hepatobiliary diseases with conjugated hyperbilirubinemia, the ratios of B delta to TB (B delta/TB) and to directly-reacting bilirubin fractions (B delta/(Bc + B delta] and that of Bc to B delta (Bc/B delta) were calculated based on the results of Ektachem and compared with each other during the course of jaundice. The mean B delta/TB was below 40% in various hepatobiliary diseases but became as high as approximately 60% in the convalescence stage. The mean B delta/(Bc + B delta) was below 50% in acute hepatitis (the serum bilirubin-elevating stage) and obstructive jaundice, and it increased to above 80% in the recovery stage. In decompensated liver cirrhosis and intrahepatic cholestasis the mean B delta/(Bc + B delta) was about 60%, indicating continuous backflow of Bc from liver cells. The changes in B delta/(Bc + B delta) were much greater than in B delta/TB.(ABSTRACT TRUNCATED AT 250 WORDS)

Induction of rat liver bilirubin-conjugating enzymes and glutathione S-transferase by rifampicin.

After oral administration of rifampicin and 25-desacetylrifampicin, which is a major metabolite of rifampicin in man but not in rat, to male Wister rats for 7 days, hepatic microsomal cytochrome P450, cytochrome b5, and activities of aniline hydroxylase, aminopyrine demethylase, bilirubin-conjugating enzymes and supernatant glutathione S-transferase were measured. Rifampicin induced bilirubin UDP-glucuronyltransferase, bilirubin UDP-glucosyltransferase, bilirubin UDP-xylosyltransferase and glutathione S-transferase activities, but did not induce mixed function oxidase activities. No inductive effect of desacetylrifampicin on any enzymes was observed. Serum bilirubin increased till the third day, and decreased after 7 days of rifampicin treatment. Plasma clearances of indocyanine green and sulfobromophthalein showed a marked delay after 1 day and 7 days of rifampicin treatment. Induction of bilirubin-conjugating enzymes and glutathione S-transferase by rifampicin in rats was different from that in humans, in which selective induction of mixed function oxidase is reported to occur. This species difference does not seem to be derived from the species difference of rifampicin metabolism, because no effect of desacetylrifampicin was observed. These results suggested that in rats rifampicin directly inhibits the hepatic excretion of bilirubin, whereas it enhances bilirubin conjugation due to enzyme induction.