Gm Liver Research Articles

Gender differences in plasma pharmacokinetics and hepatic metabolism of geissoschizine methyl ether from Uncaria hook in rats

Ethnopharmacological relevanceGeissoschizine methyl ether (GM), an indole alkaloid from Uncaria hook, is an active ingredient in the traditional Japanese Kampo medicine yokukansan, which is used to treat neurosis, insomnia, irritability, and night crying in children. Aim of the studyRecent our pharmacokinetic studies suggested that there may be gender differences in the plasma concentrations of GM in rats, but not in humans. However, the details of this difference remain unverified. The purpose of this study was to clarify the reasons for the gender differences in rats. Materials and methodsGM plasma pharmacokinetics was compared in male and female rats orally administered yokukansan (4 g/kg). To confirm the involvement of cytochrome P450 (CYP) in GM liver metabolism, GM was incubated with male and female rat liver S9 fraction in the absence or presence of 1-aminobenzotriazole (a nonspecific CYP inhibitor). CYP isoforms involved in GM metabolism were estimated using recombinant rat CYP isoforms and anti-rat CYP antibodies. ResultsThe maximum GM plasma concentrations were significantly higher in female than in male rats. When GM was incubated with rat liver S9 fractions, GM reduction was more striking in male S9 (69.3%) than that in female S9 (10.0%) and was completely blocked with nonspecific CYP inhibitor 1-aminobenzotriazole. Screening experiments using recombinant rat cytochrome P450 (CYP) isoforms showed that CYP1A1, CYP2C6, CYP2C11, CYP2D1, and CYP3A2 were involved in GM metabolism. Of these CYP isoforms, the use of anti-rat CYP antibodies indicated that male-dependent CYP2C11 and CYP3A2 were predominantly involved in the liver microsomal GM metabolism with gender differences. ConclusionsThese results suggest that the cause of gender differences in plasma GM pharmacokinetics in rats is most likely because of male-dependent CYP2C11 and CYP3A2, and provide also useful information to further evaluate the pharmacological and toxicological effects in future. This study is the first to demonstrate that the gender differences in plasma GM pharmacokinetics in rats are caused by the gender-dependent metabolism of GM.

Starvation Induced Alterations in Hepatic Lysine Metabolism in Different Families of Rainbow Trout (Oncorhynchus mykiss)

Lysine is the second limiting amino acid in fish meal based diets, second only to methionine. However, little is known about lysine metabolism in rainbow trout (RBT). Therefore, lysine catabolism by the lysine α-ketoglutarate reductase (LKR) pathway was studied. Additionally, since genetically improved strains could influence fish production, these studies were performed in 4 distinct families of RBT. Two full-sibling families, differing in feed efficiency, were selected from each of 2 strains (A and B) of RBT. Eight fish from each of the 4 families were allotted to individual tanks. Fish were fed until satiation for 5 weeks when four fish within each family were randomly selected for 2 weeks of starvation. After the starvation period, all fish were harvested. Hepatic in-vitro LKR activity and lysine oxidation were measured as was LKR mRNA. No effect of family within strain on LKR activity or lysine oxidation was detected. Strain A exhibited a 55% reduction (p < 0.01) in LKR transcripts compared to strain B pooled across both feeding levels. Within each family, LKR mRNA was decreased (p < 0.01) in starved vs. fed fish. On average, there was a 68% decrease in LKR transcripts for starved fish. LKR activity averaged 104 ± 33 and 150 ± 31 nmol/min*gm liver (p > 0.1) in fed and starved fish, respectively. Lysine oxidation averaged 1.2 ± 0.5 and 2.2 ± 0.4 nmol/min*gm liver (p > 0.1) in fed and starved fish, respectively. LKR transcripts were positively correlated to weight gain (p < 0.01). These data are consistent with multiple modes of LKR regulation in fish.

In situ neural isolation of the entire canine upper gut: Effects on fasting and fed motility patterns

Background. Multiorgan upper gut transplantation is becoming clinically feasible; however, the effects of multivisceral transplantations on gastrointestinal motility are unknown. Our aim was to determine the neural and hormonal mechanisms controlling motility patterns after complete extrinsic denervation of the upper gut as a model of multivisceral upper gut autotransplantation. Methods. Seven dogs successfully underwent in situ neural isolation of the stomach, entire small intestine, proximal colon, liver, and pancreas by transecting all connections (distal esophagus, midcolon, all nerves, lymphatics) to this multivisceral complex except the celiac artery, superior mesenteric artery, and the suprahepatic and infrahepatic vena cava; these vessels were meticulously stripped of adventitia under optical magnification. Blood flow was not disrupted to prevent confounding effects of ischemia-reperfusion injury. After 1- to 2-week recovery, myoelectric and manometric recordings of stomach and myoelectric recordings of small bowel were obtained from conscious animals. Results. During fasting the characteristic cycling migrating motor complex (MMC) was observed in the stomach and small intestine. The gastric component of the MMC was absent in one of the seven dogs. Regular cycling of the MMC during fasting, however, was intermittently disrupted and replaced by a noncyclic pattern of intermittent contractions in two of seven dogs 43% of the recording time. A small meal (50 gm liver) did not abolish the MMC as occurs in normal dogs; in contrast, a large meal (500 gm liver) did abolish the MMC. Conclusions. Extrinsic innervation to the upper gut modulates but is not requisite for interdigestive and postprandial motility of the stomach. Because relatively normal global motility patterns are preserved, multivisceral upper gut transplantation should be a viable option in selected patients.

Hemodynamic Mechanisms of Emerging Portal Hypertension Caused by Schistosomiasis in the Hamster

A hamster model of schistosomiasis has provided the first opportunity to sequentially examine the early phases of the development of portal hypertension in a natural model of chronic liver disease. Groups of hamsters were infected with 50 cercariae of Schistosoma mansoni and underwent hemodynamic evaluation at intervals of 5, 8, 12 and 20 wk after infection. A progressive rise in intrahepatic resistance (from 4.0 +/- 0.4 to 8.4 +/- 1.0 mm Hg min.ml-1.gm liver weight [p less than 0.01]) appeared to play a major role in the initial stages of evolving portal hypertension. A gradual decline in portal blood flow (from 2.1 +/- 0.3 to 1.3 +/- 0.1 ml.min-1.gm-1 liver weight [p less than 0.01]) was only partially compensated for by an increase in hepatic arterial flow. Accordingly, by week 20, total hepatic blood flow decreased 23%. Liver weight that increased markedly between 5 and 12 wk after infection, as a result of the acute accumulation of obstructing granulomas, stabilized between wk 12 and 20, while a gradual but progressive rise in hepatic collagen content was seen. Portal pressure increased 75% during the study period. Chronic examination of this natural model should help define the pathogenesis of the complications of portal hypertension and contribute to the basis for effective intervention in this disease process.

Hemodynamic and metabolic responses to leukotriene C4 in isolated perfused rat liver.

Responses of isolated perfused rat liver to leukotriene C4 were studied in order to assess the mechanisms involved in leukotriene-mediated liver injury. Infusion of leukotriene C4 (11 and 44 pmoles per min per gm liver weight) into the portal vein resulted in a rise in portal pressure, a decrease in oxygen consumption, an increase in hepatic glucose and lactate efflux and lactate/pyruvate ratio in the perfusate and a small decrease in bile flow. Isoproterenol (1 microM) counteracted the effects of leukotriene C4 on respiration and portal pressure, whereas bile flow and glucose efflux were reversibly stimulated. The same changes were observed upon withdrawal of leukotriene C4. The release of glucose was correlated with the increase in oxygen consumption upon both isoproterenol addition and withdrawal of leukotriene C4. These results are indicative of leukotriene C4-induced microcirculatory redistribution of perfusate flow. Since, in the presence of nitroprusside (50 microM), both the effects of leukotriene C4 and their reversal by isoproterenol were diminished, a vascular site of action can be assumed. Accordingly, the accompanying metabolic responses can be explained by gradual changes in oxygen supply to parts of the liver. Reversibility of the leukotriene C4 effects and lack of short-term impairment of viability of the isolated liver suggest that leukotriene-mediated liver injury is a long-term effect related to events subsequent to microcirculatory changes.

Protein kinase C agonists inhibit bile secretion independently of effects on the microcirculation in the isolated perfused rat liver.

The role of hormones in the regulation of bile secretion is not known; however vasoactive agents, which act via the phosphoinositide signal transduction pathway, may mediate changes in bile flow by altering the hepatic microvasculature. We therefore examined the effects of phorbol esters and diacylglycerol, agonists of the protein kinase C branch of the phosphoinositide cascade, on perfusion pressure and bile flow in a single-pass, hemoglobin-free, isolated perfused rat liver system with constant perfusate flow. The active phorbol ester, 12,13-phorbol dibutyrate, produced a dose-dependent (maximal effect at 10(-6) M), sustained and reversible decrease in bile flow from 1.09 +/- 0.18 to 0.61 +/- 0.09 microliters per min per gm liver (37.2 +/- 5.9%) while simultaneously increasing perfusion pressure from 12.3 +/- 0.7 to 21.5 +/- 2.5 cm H2O (74.0 +/- 4.3%). Both effects were inhibited by the synthetic protein kinase C antagonist H-7. 1,2-Dioctanoyl-sn-glycerol, a diacylglycerol, produced changes in bile flow and perfusion pressure that were similar to, but more marked than, those caused by 12,13-phorbol dibutyrate, whereas the inactive phorbol ester 4 alpha-phorbol didecanoate and the vehicle dimethyl sulfoxide had no effects on either parameter. 12,13-Phorbol dibutyrate infusion resulted in reversible decreases in oxygen consumption (23.3%) and a reversible vascular redistribution of trypan blue dye but did not alter hepatic venous effluent concentrations of K+.(ABSTRACT TRUNCATED AT 250 WORDS)

Hepatic dopamine sulfotransferases in untreated rats and in rats subjected to endocrine or hypertension-related treatments.

Here we describe the dopamine sulfotransferase activity of rat liver cytosol. With cytosol, 3'-phosphoadenosine-5'-phosphosulfate and dopamine Km values were 17.2 +/- 4.1 and 22.4 +/- 3.5 microM. Females possessed 23 to 37% of dopamine sulfotransferase levels, per gm liver, in males. DEAE-Sephadex A-50 chromatography resolved dopamine sulfotransferase activity to dopamine sulfotransferase I and dopamine sulfotransferase II. Dopamine sulfotransferase II comprised 79 +/- 10 or 61 +/- 18% of dopamine sulfotransferase in males or females in routine assays. 4-Methoxytyramine gave 609 or 179% of mean dopamine sulfotransferase activity with dopamine sulfotransferase I or II. Dopamine and 3-methoxytyramine were comparable substrates. Epinephrine was less effective. Mn++, Cd++, Zn++, Na+ and K+ inhibited dopamine sulfotransferase II. Mg++ activated it. Dopamine sulfotransferase II from males was purified 184 +/- 64-fold. Its Km values for 3'-phosphoadenosine-5'-phosphosulfate and dopamine were 12.7 +/- 1.5 and 47.5 +/- 6.7 microM, respectively. Its dopamine sulfotransferase mechanism was sequential. The molecular weight of dopamine sulfotransferase II was 49,100 +/- 4,000 by Sephadex G-100 chromatography. Dopamine sulfotransferase II preferred phenol to catecholamines. Dopamine and 3,4-dihydroxybenzylamine were its best catecholamine substrates. Adrenalectomy or castration of males led to 35 or 45% mean decreases of dopamine sulfotransferase levels, indicating adrenal and gonadal participation in control of dopamine sulfotransferase production. Testosterone had no effect in either sex, whereas estradiol led to 40% mean decreases of dopamine sulfotransferase levels in males. This suggested a role for ovaries in dopamine sulfotransferase production, supported by 55 to 102% increased dopamine sulfotransferase levels after ovariectomy. Okamoto-hypertensive males or males given hypertensogenic doses of cortisol exhibited 37 or 48% mean increases of dopamine sulfotransferase levels per gm liver. Antihypertensive spironolactone or hydralazine led to 30% mean decreases of dopamine sulfotransferase levels. Altered dopamine sulfotransferase levels after all experimental manipulations were due mostly to changed dopamine sulfotransferase II content. Dopamine sulfotransferase II is compared to other reported enzymes that sulfate catecholamines.

Diabetes-induced cholestasis in the rat: possible role of hyperglycemia and hypoinsulinemia.

In one of our earlier studies, an impaired biliary function in diabetes was suggested. We studied bile formation in rats with streptozotocin-induced diabetes (60 mg per kg body weight). Diabetic rats showed hyperglycemia and hypoinsulinemia, but no significant changes in hematocrit, plasma protein concentration or plasma osmolality. Bile flow was significantly (p less than 0.05) reduced (-23%) as compared with control animals, despite a higher (p less than 0.05) bile acid secretion rate (+56%). The biliary responses to three choleretic compounds (taurocholate, ursodeoxycholate and insulin), acting in a very different way upon bile formation, were not impaired in diabetes. The study of the relationship between bile acid output and bile flow, after infusion of taurocholate at different doses (0.25 to 1.5 mumoles per min per 100 gm body weight) showed that diabetes-induced cholestasis in the rat is mainly related to a decreased bile acid-independent fraction of the bile flow. We tested the possible role of hyperglycemia and hypoinsulinemia as cholestatic factors in diabetes. Glucose infusion [300 mM, 150 microliter per min (Group G)] induced a significant (p less than 0.05) reduction in bile flow (-0.33 microliter per min per gm liver) as compared to the basal period. After acute pancreatectomy (P) or mannoheptulose treatment [0.14 mmole per 100 gm body weight (Group M)], similar cholestatic effects were observed (-0.29 and -0.27 microliter per min per gm liver, respectively). However, plasma glucose and insulin concentrations were higher (p less than 0.01) in Group G than in P or M.(ABSTRACT TRUNCATED AT 250 WORDS)

Early disturbance of calcium translocation across the plasma membrane in toxic liver injury.

An increased influx and/or a decreased extrusion of calcium across the plasma membrane resulting in an increase in cytosolic-free calcium could play an important role in the initiation of irreversible cell injury. Therefore, the translocation of calcium across the plasma membrane was probed in the perfused rat liver using multiple indicator dilution methodology. The sucrose space corresponding to the extracellular space amounted to 0.35 +/- 0.13 ml per gm liver, and the water space corresponding to the extra- and intracellular spaces was 0.97 +/- 0.08 ml per gm. The calcium space was always slightly larger (0.42 +/- 0.10 ml per gm) than the sucrose space. The calcium space further increased during perfusion with the calcium ionophore A 23187, whereas the sucrose space remained unchanged. Two hours after administration to intact rats of acetaminophen (2 gm per kg) and carbon tetrachloride (2 ml per kg), respectively, the calcium space had increased markedly relative to the sucrose space and relative to the water space, indicating an increased accessibility of the cells to extracellular calcium. Similarly, reperfusion of livers after 90 min of ischemia was associated with an increase in calcium space relative to the sucrose and water spaces. These studies indicate that, in three models of acute liver injury, the net influx of calcium across the plasma membrane is increased early in the evolution of the injury before irreversible damage occurs.

Hepatic metabolism during acute ethanol administration: a phosphorus-31 nuclear magnetic resonance study on the perfused rat liver under normoxic or hypoxic conditions.

The effect of ethanol metabolism on the energetic parameters and intracellular pH of the isolated perfused rat liver from fed rats was studied by phosphorus-31 nuclear magnetic resonance spectroscopy. This technique allowed us to analyze nondestructively and in real time the role of low oxygen tension on the possible injurious effect of ethanol on the liver cells. A quantitative analysis of nuclear magnetic resonance data recorded on a perfused rat liver within a 30 mm diameter probe has been performed at 80.9 MHz. Under normoxic and normothermic conditions, the levels of phosphorylated metabolites detected by nuclear magnetic resonance were 2.8, 0.3 and 2 mumoles per gm liver wet weight for ATP, ADP and inorganic orthophosphate, respectively. The cytosolic pH was 7.25 +/- 0.05. During a period of 4 min of hypoxia induced by reducing the perfusion flow rate to 25% of its initial value (i.e., from 12 ml to 3 ml per min per 100 gm body weight), the level of ATP dropped to 2.2 mumoles per gm liver wet weight. Concomitantly, ADP and inorganic orthophosphate increased to 0.6 and 3.3 mumoles per gm liver wet weight. Cytosolic pH fell to 7.02 +/- 0.05. Perfusion of the liver with a Krebs medium containing 70 mM (0.4%) ethanol induced a sharp decrease in intracellular inorganic orthophosphate to reach 1.3 mumole per gm liver wet weight and after a lag time of 4 to 6 min, a decrease in ATP level (2.15 mumoles per gm liver wet weight). A large increase in phosphomonoesters (mainly sn-glycerol 3-phosphate) up to 6 mumoles per gm liver wet weight was also observed.(ABSTRACT TRUNCATED AT 250 WORDS)

THEOPHYLLINE METABOLISM DURING POSTNATAL DEVELOPMENT

Few data exist regarding theophylline (T) biotransformation in early postnatal life. In this study T metabolism was characterized in the rat with emphasis on its changes during postnatal development. Liver slices were prepared from Charles River rats and incubated in Krebs-bicarbonate buffer with (8-14C) T under 95% O2-5% CO2 in a metabolic shaker at 37°C. T metabolites were analysed by tlc and hplc. T and 6 main metabolites were recognized in adult liver: 1 methyluric acid; 1 methylxanthine; 1,3 dimethyluric acid; caffeine, a uracil derivative and an unknown polar compound. Preincubation with caffeine and theobromine inhibited T metabolism. SKF 525-A and allopurinol inhibited overall T metabolism; in particular allopurinol inhibited formation of 1 methylxanthine. The specific activity of the enzyme system was 3.9 ± SE 0.4 nm (gm liver)−1. hr−1 in the 4-day-old and increased to a peak of 25.3±1.7 in the 28-day-old rat; Km was similar (67.5 vs. 132.1 μM) but Vmax was two-fold greater in the 28-day-old (23.9 vs. 52.1 nmol [gm liver]−1. hr−1). T and the same metabolites were identified in young and adult rats but the developmental pattern was not uniform. Peak age related activity and involvement of mixed function oxidase system are common features to caffeine (Bioch. Pharmacol. 30: 3145, 1981) and T biotransformation, Xanthine oxidase was involved in T metabolism. As opposed to previous suggestions formation of caffeine from T was not dependent on lack of activity of other pathways.

1227 UREA CYCLE ENZYMES DURING FASTING IN THE OVINE FETUS

Activities of five urea cycle enzymes were measured in maternal and fetal sheep liver during the normal fed state and following five days of fasting. Six ewes and 10 fetuses were studied in both the fed and fasted periods at 132 days gestation (term = 147 days) for liver protein, DNA and enzyme levels. Results indicated that protein and DNA contents remained unchanged during fasting in both the maternal and fetal liver. Fetal liver weight was decreased during fasting from 108 ± 23 to 71 ± 21 gm (mean ± SD). Fed state fetal enzyme levels were at 25-50% of maternal values per mg DNA and 50-125% of maternal levels per gm liver. After fasting, four of the five enzymes increased by 2.3 to sixfold (per mg DNA) in fetal liver; only one enzyme (AS) increased significantly in maternal liver. Activities of each enzyme per whole fetal liver'gave similar results.These data indicate that the in vivo studies which demonstrate a doubling in fetal urea production in the fasted sheep in latter gestation are associated with parallel increases in the fetal hepatic activities of several enzymes responsible for urea synthesis.

1226 THE EFFECT OF FASTING ON OVINE FETAL AND MATERNAL GLUCONEOGENIC ENZYMES

Fetal and maternal sheep were studied to determine whether changes in gluconeogenic enzyme activities could be detected in the liver and/or kidney associated with nutritional deprivation. Thirteen ewes and 16 fetuses were sacrificed in the fed state while 13 ewes with 17 fetuses were sacrificed after five days of fasting, all at 125 days gestation (term = 147 days). Fetal weight was decreased in the fasted versus fed group (2.86 ± .56 versus 3.61 ± .58 kg, p <.001). Weights of fetal liver (66 ± 16 versus 105 ± 27 gm), fetal kidney (9.3 ± 1.9 versus 12.4 ± 1.9 gm) and maternal kidney (74.9 ± 8.3 versus 88.0 ± 9.3 gm) were all significantly decreased with fasting (p < .001). Tissues were analyzed for glucose-6-phosphatase (G-6-P), fructose-1, 6-diphosphatase (FDP), glutamate-oxaloacetate aminotransferase (GOT) and glutamate-pyruvate aminotransferase (GPT). In both fetus and mother, G-6-P and FDP were increased during fasting when measured per gm liver tissue and per mg protein (p <.001). GOT and GPT were also increased in fetal liver but not in kidney with fasting. However, activities of the enzymes per whole fetal liver or kidney were not different between fed and fasted states. Therefore, although gluconeogenic enzyme activities increased in relative activity per unit weight in fetal tissues during fasting, total enzyme activity is unchanged due to a decrease in liver and kidney mass.

Isolation, culture and characterization of adult human hepatocytes from surgical liver biopsies.

A technique is described for isolation and culture of adult human hepatocytes from surgical liver biopsies. The mean cell yield was 1.75 X 10(7) cells per gm liver and viability averaged 80%. Hepatocytes were maintained in primary culture for about 10 days. Cell morphology and histochemical characteristics were similar to hepatocytes in vivo. Bile canaliculi were observed by electron microscopy. Intracellular albumin was demonstrated up to the 7th day of culture; albumin secretion rate was maximal (0.6 +/- 0.33 micrograms per hr per 10(6) cells) 5 days after plating. These studies demonstrate that adult human hepatocytes can be isolated from surgical biopsies with high yield, and differentiated function can be maintained for several days.

Urea synthesis after protein feeding reflects hepatic mass in rats.

Urea synthesis is an exclusive biosynthetic function of the liver. Since the exact relationship between urea synthesis in vivo and functional liver mass remains unclear, we established an animal model using oral protein loading and measurement of resultant urea synthesis in rats. We studied rats subjected to sham operation, 40% hepatectomy, 66% hepatectomy, portacaval shunt and CCl4-induced cirrhosis. Urea synthesis was calculated as the sum of urinary urea excretion and accumulation of urea in body water during the 6-hr period after oral administration of a casein protein load equivalent to 20 gm per kg body weight. Peak urea synthesis rate in the sham-operated group of rats was 142 +/- 11 mumoles per hr per gm wet liver weight (mean +/- 1 S.D.), 473 +/- 34 mumoles per hr per gm liver protein and 80 +/- 5 mumoles per hr per mg liver DNA. This rate closely matched those of the other groups for each type of liver mass measurement. Marked reduction (p less than 0.01) of urea synthesis on a DNA basis was noted only in the CCl4-cirrhotic livers, related to the significantly higher (p less than 0.01) DNA content of the cirrhotic livers. Similarly, increased (p less than 0.05) liver protein content of the sham-operated rats when compared with the other groups was reflected in slightly lower urea synthesis rates expressed on the basis of liver protein (p less than 0.05) when compared to that of all other groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Hepatic glutathione homeostasis in the rat: efflux accounts for glutathione turnover.

Hepatic glutathione turnover and the efflux of glutathione from the liver into bile and blood were measured in male Sprague-Dawley rats in vivo. In fed rats the efflux of glutathione into blood, calculated from the hepatic arteriovenous concentration gradient and hepatic blood flow, amounted to 12.4 +/- 1.4 nmoles min X gm liver. Together with the excretion of glutathione into bile (3.4 +/- 0.4 nmoles per min X gm liver) total efflux accounted for the hepatic turnover of glutathione of 15.2 +/- 0.9 nmoles per min X gm liver. Fasting animals for 48 hr markedly increased hepatic glutathione turnover to 26.4 +/- 1.2 nmoles per min X gm liver. Increased efflux into blood rather than increased intrahepatic catabolism accounted for this increased turnover. The systemic clearance of glutathione was 3.22 +/- 0.51 ml per min X 100 gm body weight. The efflux of glutathione from liver therefore was calculated to contribute over 90% of total glutathione inflow into the circulation, as determined from the clearance and the arterial concentration of glutathione. Thus, the liver is the major source of plasma glutathione, and turnover of hepatic glutathione in the basal state is accounted for almost entirely by efflux of glutathione from the liver. During fasting, the plasma clearance of exogenous glutathione increased to 5.32 +/- 0.35 ml per min X 100 gm body weight, and the utilization of methionine for glutathione synthesis increased markedly. The increased extrahepatic catabolism during fasting results in a decrease in plasma glutathione, which in turn may account for the observed increase in sinusoidal glutathione efflux with concomitant stimulation of the rate of hepatic glutathione turnover and of synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)

A nonhuman primate model of Gilbert's syndrome.

A Bolivian population of squirrel monkeys, Saimiri sciureus, exhibits several features of Gilbert's syndrome in man, and is proposed as a nonhuman primate model of the condition. The Bolivian population was found to have higher fasting (40.6 +/- 2.7 microM; mean +/- S.E.) and postcibal (9.9 +/- 0.9 microM) plasma unconjugated bilirubin concentrations (p less than 0.001) than a closely related Brazilian population (fasting 5.5 +/- 0.7 microM); postcibal (2.4 +/- 0.7 microM). After intravenous administration of [3H]bilirubin as a tracer dose or at 3.4 mumoles per kg body weight, there was delayed plasma clearance in the Bolivian monkeys. Hepatic UDP-glucuronyl transferase activity for bilirubin (164 +/- 25 nmoles per 30 min per gm liver) and biliary bilirubin diglucuronide to monoglucuronide ratios (2.9 +/- 0.2) were lower in Bolivian monkeys than in Brazilians (421 +/- 36 nmoles per 30 min per gm liver--p less than 0.01 and 4.1 +/- 0.1--p less than 0.02, respectively). Hepatic cytosol glutathione-S-transferase B activity (ligandin) levels were similar for the two populations. After phenobarbital therapy, fasting (11.1 +/- 0.9 microM) and postcibal (5.3 +/- 1 microM) plasma bilirubin concentrations in Bolivian monkeys were significantly reduced (p less than 0.001). Sulfobromophthalein clearance was slightly slower in the Bolivian than in the Brazilian monkeys. SGOT, lactate dehydrogenase, gamma-glutamyl transpeptidase and alkaline phosphatase activities were not increased in Bolivians. Fasting serum conjugated bile salt concentrations in Bolivian monkeys were lower than that in Brazilian monkeys (p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of bile flow in the isolated perfused rat liver by a synthetic parenteral amino acid mixture: associated net amino acid fluxes.

To identify a role for amino acids in cholestasis associated with total parenteral nutrition, we measured bile formation by the isolated perfused rat liver in the presence and absence of added amino acids. All livers were infused constantly with sodium [14C]taurocholate (0.28 mumoles per min) for 90 min. At 40 min, a primed-constant infusion of a synthetic L-amino acid mixture (121 + 19.3 mumoles of N per min) was administered for an additional 50 min. Mean bile flow rates during the amino acid infusion were reduced from 15.4 microliter per min per 10 gm liver weight to 10.4 microliter per min per 10 gm (p less than 0.005). There was no significant change during saline infusion of control livers, and there was no significant difference in perfusate osmolalities in the two groups. Although biliary recovery of infused taurocholate was slightly lower in the experimental perfusions than in controls (95.3% vs. 101.7%, p less than 0.05), there was no significant reduction in taurocholate excretion rate during the infusion in either group. Bile flow changes were related to ambient concentrations and net fluxes of individual amino acids in the perfusate. Of the 14 infused amino acids, glycine and arginine achieved levels greater than 3 times greater than reported physiological postprandial portal venous concentrations in the rat, and together constituted about 25% of the 90-min perfusate amino acids (8.3 mM). The highest net hepatic uptake was for glycine (125 mumoles per hr per 10 gm), which was almost 50% of its infusion rate.(ABSTRACT TRUNCATED AT 250 WORDS)

Increased synthesis of rat liver alkaline phosphatase by bile duct ligation.

The following studies were done to determine the mechanism of the increase in rat liver alkaline phosphatase activity after bile duct ligation. Antiserum was raised in rabbits to highly purified rat liver alkaline phosphatase. In immune titration experiments, the 350% increase in rat liver alkaline phosphatase activity caused by bile duct ligation was paralleled by a similar increase in immunoprecipitated alkaline phosphatase protein. In a second set of experiments, rat liver alkaline phosphatase was labeled with L-[3H]leucine injected into portal veins. Alkaline phosphatase was purified by antibody affinity chromatography followed by immunoprecipitation. The incorporation of L-[3H]leucine into alkaline phosphatase was significantly higher in bile duct-ligated rats than in controls, 68,357 +/- 7,144 vs. 19,297 +/- 3,076 dpm per gm liver (p less than 0.001) and 349 +/- 36 vs. 104 +/- 17 dpm per gm protein (p less than 0.001). In a third set of experiments, the incorporation of L-3H-amino acid into highly purified rat liver alkaline phosphatase was measured. Rat liver alkaline phosphatase was purified by means of sequential N-butanol extraction, antibody affinity column chromatography, preparative polyacrylamide gel electrophoresis, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. There was significantly more L-3H-amino acid incorporated into alkaline phosphatase in bile duct-ligated rats compared to sham-operated rats, 3,565 vs. 704 dpm per gm liver and 19,656 vs. 3,843 dpm/gm protein. The data suggest that bile duct ligation increases the synthesis of rat liver alkaline phosphatase.

Toxic doses of acetaminophen suppress hepatic glutathione synthesis in rats.

The effect of a toxic dose of acetaminophen on hepatic glutathione turnover was studied in fed and fasted rats. Following the administration of 1 gm per kg of acetaminophen, the fractional rate of glutathione turnover increased from 0.19 to 0.28 hr-1 in fed rats and from 0.43 to 0.50 hr-1 in rats fasted for 48 hr. The increase in the fractional rate of turnover was proportionally much less than the decrease in hepatic glutathione concentration resulting from the toxic dose of acetaminophen. Thus, the estimated hepatic synthesis of glutathione decreased from 0.86 and 1.50 mumole per gm liver . hr to 0.59 and 0.53 mumole per gm . hr in fed and fasted rats, respectively. The excretion of acetaminophen-sulphate was significantly decreased in fasted rats. The fraction of administered acetaminophen metabolized to the mercapturic acid, however, was similar in both groups. Nevertheless, all fasted animals had massive centrilobular necrosis whereas virtually no necrosis was observed in fed animals. The administration of sulfate did not protect against liver injury in fasted rats. In view of the similar synthesis of glutathione in fed and fasted rats following a toxic dose of acetaminophen, an inability to synthesize adequate quantities of glutathione or a decreased availability of sulfate cannot account for the increased susceptibility to liver injury in the fasted state. However, a toxic dose of acetaminophen depletes the hepatic glutathione to a lower nadir before glutathione is repleted by synthesis, and a greater amount of tissue arylation occurs during this brief period of critically depleted glutathione.