Interaction of bilirubin with human erythrocyte membranes.

The kinetics of [3H]bilirubin binding to human erythrocyte ghost membranes was investigated. The binding occurred rapidly and was saturable with respect to [3H]bilirubin and membrane concentration. The apparent dissociation constant (Kd) and maximum binding (Bmax.) for bilirubin of the membranes were 2.3 microM and 0.93 nmol/mg of protein respectively. Low-affinity binding, non-saturable at 400 microM, was observed. Thermal dependency of the saturable binding showed a U-shaped curve with the lowest value around 37 degrees C. Affinity labelling of the membrane proteins using [3H]bilirubin-Woodward's reagent K complex did not define individual proteins. The Kd (12 microM) and Bmax. (4.4 nmol/mg of protein) for bilirubin of the tryptic membranes increased 5.0 and 5.2 times the respective control values (2.4 microM and 0.85 nmol/mg of protein). Heat-treatment of the membranes for 3 min at 100 degrees C increased the saturable binding as much as by 222%. These results indicate that there exist saturable bilirubin-binding sites on the erythrocyte membranes and also suggest that they are not composed of proteins.     

The excretion pattern of biliverdin and bilirubin in bile of the small skate (Raja erinacea)

Summary Bile pigment composition (biliverdin, bilirubin and their conjugates) was analyzed in stored gallbladder bile and newly synthesized hepatic bile from the small skate (Raja erinacea). During a five day period of captivity, gallbladder volume remained relatively constant while bilirubin and biliverdin content increased two to three fold. Biliverdin which accounted for 50% of the pigments did not increase as a percentage of tetrapyrroles during this period. The relative proportion of bilirubin and its conjugates also remained constant, averaging 65% for bilirubin monoglucuronide, 30% for bilirubin diglucuronide and 5% for unconjugated bilirubin as measured by HPLC methods. Intravenous administration of biliverdin resulted in significant increases in the biliary excretion of both biliverdin and all bilirubin tetrapyrroles. Insignificant quantities of³H-biliverdin were detected in hepatic bile following the intravenous administration of³H-bilirubin. These studies indicate that the small skate excreted both biliverdin and bilirubin conjugates in bile and that the biliverdin was not produced by in vitro oxidation of bilirubin or its metabolites.     

Biliary excretion of flavopiridol and its glucuronides in the isolated perfused rat liver: role of multidrug resistance protein 2 (Mrp2)

Flavopiridol (FLAP) is a novel anticancer agent that is extensively glucuronidated in patients. Biliary excretion is the main elimination pathway of FLAP conjugates responsible for enterohepatic recirculation and for the main side effect diarrhea. To investigate the hepatic transport system for FLAP glucuronides, livers of Wistar and Mrp2-deficient TR- rats were perfused with FLAP (30 microM) in a single pass system. Biliary excretion and efflux into perfusate during a 60 min period greatly differ in TR- rats. While cumulative biliary excretion of M1 and M2 was significantly reduced to 4.3% and 5.4% efflux into perfusate was increased by 1.5 and 4.2-fold. This indicates that in control rats, M1 and M2 are almost exclusively eliminated into bile by Mrp2. Cumulative FLAP secretion into bile and perfusate, however, was non-significantly reduced by 36.7% and 43.2% in the mutant rat strain, suggesting that besides Mrp2, other transporters might also be involved in FLAP elimination. FLAP stimulates bile flow up to 24% in control rats, but secretion is nearly absent in TR- rats further supporting an efficient transport of FLAP glucuronides by Mrp2. FLAP (30 microM) also reversibly inhibited the Mrp2-mediated biliary elimination of bilirubin and bromsulphthalein in Wistar rats by 54% and 51%, respectively, indicating a competition with the elimination of Mrp2-specific substrates. In summary, we found that FLAP glucuronides are substrates of Mrp2 effectively inhibiting the biliary excretion of bilirubin. This may explain the increased serum bilirubin levels observed in cancer patients during FLAP therapy.     

Inhibition of the biosynthesis of uroporphyrinogen and heme in rat liver during obstructive jaundice produced by bile duct ligation

Altered hepatic microsomal drug metabolism has been reported to occur in afflicted with hyperbilirubinemia. Similarities of the chemical structures of hydroxymethylbilane, an intermediate in the biosynthesis of uroporphyrinogen, to bilirubin prompted investigations of the effect of bilirubin on the activity of uroporphyrinogen I synthase (porphobilinogen deaminase, EC 4.3.1.8) and the biosynthesis of heme. Bilirubin was found to be a reversible, noncompetitive inhibitor of uroporphyrinogen I synthase. The inhibition constant (Ki) for bilirubin was 1.5 microM. Bile acids had no effect on rat hepatic uroporphyrinogen I synthase activity. Hyperbilirubinemia was achieved in rats by biliary ligation in order to investigate whether elevated levels of bilirubin impair the biosynthesis of hepatic heme in vivo. The relative rate of heme biosynthesis, as measured by the rate of incorporation of delta-[4-14C]aminolevulinic acid into heme, was decreased 59% 24 h after biliary obstruction. The levels of hepatic microsomal heme and cytochrome P-450 were decreased by 43 and 40%, respectively, 72 h after biliary obstruction. The activities of hepatic delta-aminolevulinic acid synthase and uroporphyrinogen I synthase were increased by 39 and 46%, respectively, 72 h after biliary obstruction. During the 48- to 72-h period following biliary obstruction, the urinary excretion of porphobilinogen and uroporphyrin was increased 3.0- and 3.5-fold, respectively, whereas, the urinary excretion of delta-aminolevulinic acid was not altered. During this 48-to 72-h time interval following biliary obstruction, 100% of the uroporphyrin was excreted as isomer I. These results indicate that bilirubin is capable of depressing the biosynthesis of rat hepatic heme and thus cytochrome P-450-mediated drug metabolism by inhibition of the formation of uroporphyrinogen. These findings are a plausible mechanism for reports of impaired clearance of various drugs in patients afflicted with hyperbilirubinemic disease states.     

Mechanism of hepatocellular uptake of albumin-bound bilirubin

We previously demonstrated that unconjugated bilirubin spontaneously diffuses through phospholipid bilayers at a rate which exceeds albumin dissociation, suggesting that solvation from albumin represents the rate-limiting step in hepatic bilirubin clearance. To further examine this hypothesis, we studied the uptake of bovine serum albumin (BSA)-bound bilirubin by cultured hepatoblastoma (HepG2) cells. Uptake of bilirubin was saturable, with a K(m) and V(max) of 4.2+/-0.5 microM (+/-S.E.M.) and 469+/-41 pmol min(-1) mg(-1) at 25 degrees C. Substantial bilirubin uptake also was observed at 4 degrees C (K(m)=7.0+/-0.8 microM, V(max)=282+/-26 pmol min(-1) mg(-1)), supporting a diffusional transport mechanism. Consistent with reported solvation rates, the cellular uptake of bilirubin bound to human serum albumin was more rapid than for BSA-bound bilirubin, indicative of dissociation-limited uptake. Counterintuitively, an inverse correlation between pH and the rate of bilirubin flip-flop was observed, due to pH effects on the rate of dissociation of bilirubin from albumin and from the membrane bilayer. The identification of an inflection point at pH 8.1 is indicative of a pK(a) value for bilirubin in this range. Taken together, our data suggest that hepatocellular uptake of bilirubin is dissociation-limited and occurs principally by a mechanism involving spontaneous transmembrane diffusion.     

The cytoprotective effects of bilirubin and biliverdin on rat hepatocytes and human erythrocytes and the impact of albumin.

The hypothesis that unconjugated bilirubin and biliverdin are cytoprotective antioxidants has been examined for the first time in systems containing cells. In primary rat hepatocytes exposed to xanthine oxidase and hypoxanthine, bilirubin (0-60 microM) failed to prolong cell survival. In contrast, biliverdin (20-100 microM) markedly delayed hepatocyte necrosis in a concentration-dependent manner. When 0.3 mM of albumin was present, bilirubin (0-50 microM) became protective of hepatocytes, while biliverdin was less dramatically enhanced in its cytoprotective effect. In human erythrocytes exposed to peroxyl radicals, bilirubin and biliverdin inhibited 50% cell lysis at lower concentrations than Trolox and ascorbate, respectively. Albumin alone appeared less cytoprotective in red cells than in hepatocytes, but its presence enhanced the effects of both pigments on erythrocytes. Of probable physiologic relevance, bilirubin with albumin present or biliverdin alone protected hepatocytes substantially (and to a lesser extent red cells) at the normal blood levels of bilirubin (3.4-26 microM). Moreover, the fact that the pigments are cytoprotective at higher bilirubin levels (e.g., 50-100 microM) tempts the speculation that they may be circulating cytoprotectors of overlooked importance in jaundice.     

Mouse protoporphyrinogen oxidase. Kinetic parameters and demonstration of inhibition by bilirubin.

The penultimate step of haem biosynthesis, the oxidation of protoporphyrinogen to protoporphyrin, was examined with purified murine hepatic protoporphyrinogen oxidase (EC 1.3.3.4) in detergent solution. The kinetic parameters for the two-substrate (protoporphyrinogen and oxygen) reaction were determined. The limiting Km for protoporphyrinogen when oxygen is saturating is 6.6 microM, whereas the Km for oxygen with saturating concentrations of protoporphyrinogen is 125 microM. The kcat. for the overall reaction is 447 h-1. The ratio of kcat. to the Km for protoporphyrinogen is approx. 20-fold greater than the kcat./Km,O2 ratio. The ratio of protoporphyrin formed to dioxygen consumed is 1:3. Ubiquinone-6, ubiquinone-10 and dicoumarol stimulate protoporphyrinogen oxidase activity at low concentrations (less than 15 microM), whereas coenzyme Q0 and menadione show no activation at these concentrations. Above 30 microM, all five quinones inhibit the enzyme activity. FAD does not significantly affect the activity of the enzyme. Bilirubin, a product of haem catabolism, is shown to be a competitive inhibitor of the penultimate enzyme of the haem-biosynthetic pathway, protoporphyrinogen oxidase, with a calculated Ki of 25 microM. The terminal enzyme of haem-biosynthetic pathway, namely ferrochelatase, is not inhibited by bilirubin at concentrations over double the Ki value for the oxidase. In contrast with other enzymic systems, the toxicity of bilirubin is not reversed by binding to albumin.     

Effects of food deprivation on expression of growth hormone receptor and proximate composition in liver of black seabream Acanthopagrus schlegeli

The effects of food deprivation on the hepatic level growth hormone receptor (GHR) were investigated in black seabream (Acanthopagrus schlegeli) both at the protein level (by radioreceptor assay) and at the mRNA level (by ribonuclease protection assay). Serum levels of growth hormone (GH) and triiodothyronine (T₃) were also measured. Condition factor and hepatic proximate composition of the fish were also assessed. Significant decrease in hepatic GHR binding was recorded as early as on day 2 of starvation. On day 30 this decrease was even more pronounced, with the level in the starved fish reaching less than 20% the fed control level. A concomitant decrease in the hepatic GHR mRNA content was also noted during this period, with a progressive decrease from day 2 to day 30 of starvation. The extent of decrease in the mRNA content was less pronounced than the decrease in receptor binding, with the hepatic GHR mRNA content in the day 30 starved fish representing approximately 30% of the level in the fed control. In large contrast, serum GH level increased progressively during starvation. After 30 days of starvation, serum GH levels in the starved fish were more than three times the concentration found in the fed control. Serum T₃ levels, on the other hand, decreased during starvation, with the difference reaching significance on day 15 and day 30. After 30 days of starvation, serum T₃ levels in the starved fish were only approximately 40% the concentration found in the fed control. The hepatic lipid content exhibited an increasing trend during starvation. On day 30 the hepatic lipid content of the starved fish had doubled the level found in the fed control. However, the hepatic protein content did not exhibit much change during starvation. There was also a minor decrease in the moisture content of the liver during starvation, but the condition factor of the fish as a whole registered a gradual decrease during the course of food deprivation.     

Bilirubin production and conjugation from newly formed heme in isolated rat hepatocytes.

1. Heme synthesis from delta-aminolevulinic acid (delta-ALA) in freshly isolated rat hepatocytes was maximal at 100 microM with a rate of approx. 7 nmol being synthesized per g wet weight cells. 2. Approximately 8% of synthesized heme was converted to bilirubin and 50% of the newly synthesized bilirubin was conjugated. 3. The ratio of di to monoconjugate was approx. 2.5. Incorporation of delta-ALA into bilirubin was increased by additional delta-ALA, heme and was also doubled in cells isolated from animals treated with CoCl2. 4. Bilirubin formation was inhibited approx. 90% by in vitro treatment with heme oxygenase inhibitors zinc and tin protoporphyrin.     

Hepatic microsomal bilirubin UDP-glucuronosyltransferase. The kinetics of bilirubin mono- and diglucuronide synthesis.

Hepatic biotransformation of bilirubin to the hydrophilic species bilirubin mono- (BMG) and diglucuronide (BDG) by microsomal bilirubin UDP-glucuronosyl-transferase (GT) is a prerequisite for its physiologic excretion into bile. The reaction mechanism of bilirubin-GT and the access of bilirubin and BMG (the intermediate substrate) to the active site of bilirubin-GT are undefined. Highly purified [14C]bilirubin and [3H] BMG were coincubated with rat liver microsomes, and the initial rates of radiolabeled bilirubin glucuronide synthesis were measured. Although these substrates differ markedly in their hydrophilicity, no significant differences were observed in [14C]- and [3H]BDG rates of formation from equimolar [14C]bilirubin and [3H] BMG, in the absence or presence of soluble binding proteins (albumin and hepatic cytosol). In further kinetic studies, [14C]bilirubin and [3H]BMG exhibited mutually competitive inhibition of [3H]- and [14C]BDG synthesis, respectively, and [3H]BMG also inhibited [14C]BMG formation. Finally, unlabeled BMG and BDG inhibited the glucuronidation of [14C]bilirubin, with all three pigments yielding virtual Michaelis-Menten dissociation constants in the 10-20 microM range. These findings indicate that: 1) bilirubin-GT follows Michaelis-Menten kinetics for both bilirubin and BMG glucuronidation over the range of substrate concentrations employed; 2) the findings are consistent with a single active site for the enzymatic synthesis of both BMG and BDG; 3) bilirubin, BMG, and BDG bind competitively to this active site with comparable affinities; and 4) access of both bilirubin and BMG substrates to the enzymatic active site is reduced by soluble binding proteins.     

Relation between glutathione redox changes and biliary excretion of taurocholate in perfused rat liver

The influence of the intracellular glutathione status on bile acid excretion was studied in the perfused rat liver. Perturbation of the thiol redox state by short term additions of diamide (100 microM) or hydrogen peroxide (250 microM) or t-butyl hydroperoxide (250 microM) led to a reversible inhibition of biliary taurocholate release without affecting hepatic uptake; inhibition amounted to 45% for diamide and 90% for the hydroperoxides. Concomitantly, the bile acid accumulated intracellularly. Bile flow increased from 1.3 to 2.0 microliters X min-1 X g liver-1 upon infusion of taurocholate (10 microM); the latter value was suppressed to 1.2 microliters X min-1 X g liver-1 by the addition of t-butyl hydroperoxide (250 microM). Similarly, the hepatic disposition of another bile constituent, bilirubin, was suppressed by 70% upon addition of hydrogen peroxide. While the addition of hydrogen peroxide inhibited also the endogenous release of bile acids almost completely, endogenous bile flow was much less affected, decreasing from 1.3 to 1.0 microliters X min-1 X g liver-1. Measurement of [14C]erythritol clearance showed bile/perfusate ratios of about unity both in the absence and presence of hydrogen peroxide, suggesting canalicular origin of the bile under both conditions. In livers from Se-deficient rats low in Se-GSH peroxidase (less than 5% of controls), hydrogen peroxide inhibited taurocholate transport substantially less, providing evidence for the involvement of glutathione in mediating the inhibition observed in normal livers. The percentage inhibition of taurocholate release and intracellular glutathione disulfide (GSSG) content were closely correlated. The addition of t-butyl hydroperoxide caused a several-fold increase of biliary GSSG release, whereas biliary GSH release was even decreased. The results establish a role of glutathione in canalicular taurocholate disposition.     

Conjugation metabolism of acetaminophen and bilirubin in extrahepatic tissues of rats

An anhepatic rat model was used to explore the extrahepatic conjugating metabolism of acetaminophen and serum bilirubin. The recovery of glucuronide- and sulfate-acetaminophen was 47.5% in normal control and 13.4% in model rats in the urine collected for 6 h after administration of acetaminophen 20 mg kg(-1). Following the increase of acetaminophen dose to 150 mg kg(-1), the recovery of urinary glucuronide-acetaminophen increased by 53.9% in normal control; but it decreased by 36.4% in model rats. In contrast to normal control, the pretreatment with phenobarbital did not affect acetaminophen and its metabolite levels in plasma and urine in model rats. After the establishment of anhepatic model the serum direct bilirubin rose dramatically. Urinary bilirubin test was positive in model rats, but not in normal control. No changes were observed in serum total bilirubin and ratio of direct/total bilirubin after the pretreatment with ranitidine or phenobarbital 50 mg kg (-1), i.p. for 5 days in model rats. The results indicate that the glucuronide- and sulfate-acetaminophen formed in the extrahepatic tissues of model rats is 28.2% of normal control, serum free bilirubin can be transformed into conjugated bilirubin in extrahepatic tissues, and the regulation mechanism of phase II conjugating enzymes is different between the hepatic and extrahepatic tissues.     

Hepatic bilirubin and UDP-glucuronate levels in Bolivian squirrel monkeys exhibiting fasting hyperbilirubinemia

1. Bolivian squirrel monkeys (BoSMs), which are animal models for Gilbert's syndrome, have 40% less hepatic bilirubin UDP-glucuronyltransferase (BR-UPPG-T) activity than Brazilian squirrel monkeys (BrSMs). 2. Although fasting results in similar decreases in hepatic UDP-glucose and UDP-glucuronate levels in both simian subspecies, increased activities (55%) of BR-UDPG-T are induced only in the fasted control BrSMs, which do not exhibit the marked fasting hyperbilirubinemia (FH). 3. Total hepatic bilirubin (BR) concentrations were 50% greater in both fed and fasted BoSMs when compared to BrSMs. 4. Hepatic unconjugated BR levels increase upon fasting only in Gilbert-like BoSMs, reaching concentrations twice that observed in BrSMs. 5. Elevated hepatic BR levels in fasted BoSMs may reflect BR overproduction or inadequate glucuronidation. 6. The increased BR-UDPG-T activity induced in BrSMs during fasting could compensate in-part for the UDPGA depletion and prevent the marked FH as observed in BoSMs.     

Hepatoprotective activity of quercetin against acrylonitrile-induced hepatotoxicity in rats

Acrylonitrile is a potent hepatotoxic, mutagen, and carcinogen. A role for free radical-mediated lipid peroxidation in the toxicity of acrylonitrile has been suggested. The present study was designed to assess the hepatoprotective effect of quercetin against acrylonitrile-induced hepatotoxicity in rats. Liver damage was induced by oral administration of acrylonitrile (50 mg/kg/day/5 weeks). Acrylonitrile produced a significant elevation of malondialdehyde (138.9%) with a marked decrease in reduced glutathione (72.4%), and enzymatic antioxidants; superoxide dismutase (81%), and glutathione peroxidase (53.2%) in the liver. Serum aspartate aminotransferase, alanine aminotransferases, direct bilirubin, and total bilirubin showed a significant increase in acrylonitrile alone treated rats (115.5%, 110.8%, 1006.8%, and 1000.8%, respectively). Pretreatment with quercetin (70 mg/kg/day/6 weeks) and its coadministration with acrylonitrile prevented acrylonitrile-induced alterations in hepatic lipid peroxides and enzymatic antioxidants as well as serum aminotransferases and bilirubin. Histopathological findings supported the biochemical results. We suggest that querectin possess hepatoprotective effect against acrylonitrile-induced hepatotoxicity through its antioxidant activity.     

Synthesis, biological evaluation and molecular modelling studies of novel ACD- and ABD-ring steroidomimetics as inhibitors of CYP17

Two novel classes of non-steroidal substrate mimetics were synthesised and examined for their potency as inhibitors of human CYP17. Selected compounds were tested for inhibition of hepatic CYP enzymes 3A4, 1A2, 2C9 and 2C19. The most promising compound 15 showed a good inhibition of the target enzyme (31% and 66% at 0.2 and 2 microM, respectively), and little inhibition of the most important hepatic enzyme CYP3A4 (6% and 19% inhibition at 0.2 and 2 microM, respectively) and the key enzyme of glucocorticoid biosynthesis CYP11B1 (3% and 23% inhibition at 0.2 and 2 microM, respectively). Docking studies revealed that this compound does not assume the same binding mode as steroidal ligands.     

Reduction of the C2 and C4 vinyl groups of Sn-protoporphyrin to form Sn-mesoporphyrin markedly enhances the ability of the metalloporphyrin to inhibit in vivo heme catabolism

Sn (tin)-mesoporphyrin (Sn-protoporphyrin in which the vinyl groups at C2 and C4 have been reduced to ethyl groups) when incubated with rat splenic microsomal heme oxygenase proved to be a potent competitive inhibitor of enzyme activity in vitro, with a Ki of 0.014 microM. Sn-mesoporphyrin (1 mumol/kg body wt) also inhibited hepatic, renal, and splenic heme oxygenase activity in vivo in adult animals for extended periods of time. Sn-mesoporphyrin (1 mumol/kg body wt) prevented the transient increase in serum bilirubin 24 h after birth in the rat neonate and substantially reduced the levels of serum bilirubin in ALA (delta-aminolevulinic acid) induced hyperbilirubinemia in the 7-day-old suckling neonate. Tissue heme oxygenase activity was decreased in both animal models of hyperbilirubinemia. Sn-mesoporphyrin administration led to a prolonged increase in the heme saturation of hepatic tryptophan pyrrolase indicating an increase in the "heme pool" related to tryptophan pyrrolase and the compound also suppressed chemically induced hepatic porphyria. The administration of Sn-mesoporphyrin to bile duct-cannulated rats was followed by a prompt and sustained decrease in bilirubin output in bile. In addition the excretion of heme in bile was enhanced in these animals. These studies indicate that Sn-mesoporphyrin, like Sn-protoporphyrin, decreases serum bilirubin by inhibiting the production of bilirubin in vivo and its mode of action is through a sustained competitive inhibition of heme oxygenase. However, when a direct comparison of Sn-protoporphyrin and Sn-mesoporphyrin was made, these studies clearly established that the reduction of the C2 and C4 vinyl groups of the porphyrin macrocycle to ethyl groups increases the effectiveness of the Sn-mesoporphyrin derivative 10-fold or more as compared with Sn-protoporphyrin in inhibiting heme catabolism in the animal model systems examined. Thus alterations in the side chain substituents as well as of the central metal atom can influence in a significant manner the potency of the resultant synthetic heme analog as an agent capable of inhibiting heme degradation in vivo.     

Lithocholate binding by Y and Y' proteins in bovine small intestine.

Cytosolic proteins may play an important role in the intracellular transport of bile acids in enterocytes. The lithocholate binding properties of cytosolic protein from bovine small intestine were studied. Lithocholate binding was observed in the Y (45-50 kDa), Y' (30-35 kDa), and Z fractions (10-15 kDa) following gel filtration of cytosol. A Y protein with glutathione S-transferase activity (46 kDa) was purified by S-octyl-glutathione affinity chromatography and chromatofocusing (eluted at pH 7.5) of the Y fraction. Two Y' bile acid binding proteins with dihydrodiol dehydrogenase activity were partially purified from the Y' fraction by chromatofocusing and hydroxyapatite-HPLC. The lithocholate binding affinity of Y' protein (Kd < 0.35 microM) was higher than that of Y protein (Kd = 2 microM) and was comparable to that of Z protein (Kd = 0.2 microM). The binding affinity of Y protein was higher for bilirubin (Kd = 2.5 microM) than that for BSP (Kd = 200 microM). This was comparable to the binding affinity of bovine hepatic Y protein. These data indicate that Y' and Z proteins participate in the intracellular transport of bile acids from the brush border to the basolateral pole in enterocytes.     

Does bilirubin play a role in the pathogenesis of both cholesterol and pigment gallstone formation? Direct and indirect influences of bilirubin on bile lithogenicity.

Bilirubin is found in the center of cholesterol gallstones, but its pathogenic role in their formation is unknown. Bilirubin causes a disproportionate reduction of biliary lipid secretion without affecting bile salt secretion in association with a change of biliary lecithin species, which modulates the cholesterol crystallization process. Therefore, the present study investigated whether bilirubin can influence the cholesterol crystallization procedure, and the mechanism(s) of any such action. Supersaturated model bile was prepared (taurocholate/lecithin/cholesterol at 71:18:11, a total lipid concentration of 9.0 g/dl, and cholesterol saturation index of 1.8), and cholesterol crystallization was monitored over time using a spectrophotometer and video-enhanced differential contrast microscopy in the absence or presence of bilirubin (at a final concentration of 10 microM, 20 microM, 40 microM, and 100 microM). Bilirubin enhanced the onset of cholesterol crystallization by 50%, whereas the crystal growth rate and final crystal mass were reduced at a high concentration of bilirubin. Taken together, these results suggest that bilirubin influences the cholesterol crystallization process, by either a direct interaction with biliary lipids that alters metastability, an indirect alteration of the bile salt-micellar lipid holding capacity, or both. Thus, bilirubin may play a role in the pathogenesis of both cholesterol and pigment gallstones.     

Stimulation of hepatic inositol 1,4,5-trisphosphate kinase activity by Ca2+-dependent and -independent mechanisms.

A cytosolic fraction derived from rat hepatocytes was used to investigate the regulation of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] kinase, the enzyme which converts Ins(1,4,5)P3 to inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4]. The activity was doubled by raising the free Ca2+ concentration of the assay medium from 0.1 microM to 1.0 microM. A 5 min preincubation of the hepatocytes with 100 microM-dibutyryl cyclic AMP (db.cAMP) plus 100 nM-tetradecanoylphorbol acetate (TPA) resulted in a 40% increase in Ins(1,4,5)P3 kinase activity when subsequently assayed at 0.1 microM-Ca2+. This effect was smaller at [Ca2+] greater than 0.5 microM, and absent at 1.0 microM-Ca2+. Similar results were obtained after preincubation with 100 microM-db.cAMP plus 300 nM-vasopressin (20% increase at 0.1 microM-Ca2+; no effect at 1.0 microM-Ca2+). Preincubation with vasopressin, db.cAMP or TPA alone did not alter Ins(1,4,5)P3 kinase activity. It is proposed that these results, together with recent evidence implicating Ins(1,3,4,5)P4 in the control of Ca2+ influx, could be relevant to earlier findings that hepatic Ca2+ uptake is synergistically stimulated by cyclic AMP analogues and vasopressin.     

用siRNA沉默人血红素加氧酶-1基因降低胆红素水平

拟通过RNA干扰技术特异下调人血红素加氧酶-1(human heme oxygenase-1,hHO-1)基因的表达,减少hHO-1的产量从而降低胆红素的产生,探讨在胆红素产生前就阻断其产生,为临床早期防治新生儿高胆红素血症及胆红素中毒性脑病探索一种新的有效手段。针对hHO-1基因设计并化学合成三对小分子干扰RNA(small interfering RNA,siRNA)。采用脂质体转染法将siRNA转染入人肝脏细胞株HL-7702;荧光显微镜检测siRNA转染细胞的效率;转染siRNA1~2天后经RT-PCR和Western印迹方法检测hHO-1表达水平和蛋白质量;并采用HO-1诱导剂血红素诱导或hHO-1表达质粒转染细胞以上调hHO-1表达,检测siRNA干扰后hHO-1产量和酶活性。结果显示:设计的三对siRNA能不同程度的特异下调hHO-1表达,筛选获得抑制效果最佳的siRNA-3。siRNA-3抑制hHO-1呈现浓度与时间依赖性。与非特异对照siRNA及未处理组比较,血红素诱导和hHO-1表达质粒转染均能上调HL-7702细胞内hHO-1表达,提高hHO-1产量,但转染siRNA-3后hHO-1表达明显抑制,同时hHO-1活性随着基因表达下调而下降。实验表明设计合成的siRNA-3抑制效果明显。siRNA-3通过降解hHO-1,减少hHO-1产量,降低酶活性,最终减少胆红素产生,从而使RNA干扰技术成为降低新生儿高胆红素血症和胆红素中毒性脑病发生的一种候选方法。