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1.
New carbene-generating photolabile bile salt derivatives, 3,3-azo-7 alpha,12 alpha-dihydroxy-5 beta [7 beta-3H]cholan-24-oic acid and (3,3-azo-7 alpha,12 alpha-dihydroxy-5 beta [7 beta-3H]cholan-24-oyl)-2- aminoethanesulfonic acid were synthesized with high specific radioactivity. These 3-diazirine-derivatives could be activated to the corresponding carbenes by irradiation with ultraviolet light at 350 nm with a half-life time of 2 min. The 3-diazirine derivatives behaved in enterohepatic circulation like the natural bile salts. The uptake of [3H]taurocholate into isolated hepatocytes was competitively inhibited by (3,3-azo-7 alpha,12 alpha-dihydroxy-5 beta-cholan-24-oyl)-2- aminoethanesulfonic acid indicating that the 3,3-azo-derivative of taurocholate shares the hepatic transport systems for natural bile salts. It was demonstrated that the radioactively labeled 3-diazirine bile salt derivatives are useful probes for photoaffinity labeling of bile salt binding proteins especially in intact cells and tissues. 相似文献
2.
Identification of different transport systems for bile salts in sinusoidal and canalicular membranes of hepatocytes 总被引:3,自引:0,他引:3
The preservation of the functional polarity of hepatocytes in liver snips (1 x 2 x 4 mm) was demonstrated by fluorescent microscopic studies using the sodium salt of (N-[7-(4-nitrobenzo-2-oxa-1,3-diazol)]-3 beta-amino-7 alpha,12 alpha- dihydroxy-5 beta-cholan-24-oyl)-2-aminoethanesulfonic acid. This fluorescent bile salt derivative is not only taken up by hepatocytes of several cell layers at the surface of the snips but also secreted into bile canaliculi. The intact hepatobiliary transport of bile salts by hepatocytes of liver snips demonstrates that they are a useful system for the investigation of those transcellular transport processes which require the integrity of hepatic structure. Photoaffinity labelling of liver snips with the sodium salt of (7,7-azo-3 alpha,12 alpha-dihydroxy-5 beta-[3 beta-3H]cholan- 24-oyl)-2-aminoethanesulfonic acid revealed that the bile-salt-binding membrane polypeptides with apparent Mr values of 54,000 and 48,000 are exclusively located in the sinusoidal membrane, whereas a single bile-salt-binding polypeptide with an apparent Mr of 100,000 is located in the bile-canalicular membrane. Photoaffinity labelling of liver snips at 4 degrees C, when transcellular bile-salt transport is insignificant, resulted in the labelling of the two sinusoidal membrane polypeptides and practically no labelling of the polypeptide with an apparent Mr of 100,000. This latter polypeptide was also not labelled when Ca2 deprivation abolished bile secretion completely. These results indicate that the directed hepatobiliary transport of bile salts in hepatocytes is accomplished by transport systems which are different for sinusoidal uptake and canalicular secretion. 相似文献
3.
G Burckhardt W Kramer G Kurz F A Wilson 《Biochemical and biophysical research communications》1987,143(3):1018-1023
The uptake of a photolabile taurocholate derivative, (7,7-azo-3 alpha, 12 alpha-dihydroxy-5 beta-cholan-24-oyl)-2-aminoethanesulfonate, 7,7-azo-TC, into rat renal brush-border membrane vesicles was stimulated by Na+ and inhibited by taurocholate indicating an interaction with the Na+/bile salt cotransport system. Irradiation of membrane vesicles in the presence of 7,7-azo-TC inhibited Na+-dependent taurocholate uptake irreversibly. Photoaffinity labeling with [3H]7,7-azo-TC resulted in a predominant incorporation of radioactivity into a polypeptide with apparent molecular weight of 99,000. These results suggest that the proteins involved in Na+/bile salt cotransport are similar in renal and ileal brush-border membranes, but differ from those in hepatocytes. 相似文献
4.
5.
U Schramm A Dietrich S Schneider H P Buscher W Gerok G Kurz 《Journal of lipid research》1991,32(11):1769-1779
Interaction of unconjugated and taurine-conjugated NBD-amino-dihydroxy-5 beta-cholan-24-oic acids bearing the fluorophor in the 3 alpha, 3 beta, 7 alpha, 7 beta, 12 alpha, or 12 beta position with albumin results in a small hypsochromic shift of the emission maximum and an increase in quantum yield, suggesting binding by hydrophobic interactions. The different unconjugated fluorescent bile salt derivatives are metabolized by intact rat liver in different ways. The unconjugated 3 beta-NBD-amino derivative is completely transformed to its taurine conjugate and secreted as such, whereas the 3 alpha-NBD-amino derivative is completely transformed to a polar fluorescent compound not identical with its taurine conjugate. The unconjugated 7 alpha- and 7 beta-NBD-amino derivatives are only partially conjugated with taurine and mainly secreted in unmetabolized form. The unconjugated 12 alpha- and 12 beta-NBD-amino derivatives are not at all transformed to their taurine conjugates, but are partially metabolized to unidentified compounds. They are predominantly secreted as the unmetabolized compounds. In contrast to the unconjugated derivatives, all taurine-conjugated fluorescent bile salt derivatives are secreted into bile unmetabolized. With the exception of the 3 alpha-compound, all synthesized taurine-conjugated fluorescent derivatives interfere with the secretion of cholyltaurine. Differential photoaffinity labeling studies using (7,7-azo-3 alpha,12 alpha- dihydroxy-5 beta-cholan-24-oyl)-2'-[2'-3H(N)]aminoethanesulfonate as a photolabile derivative revealed that in liver cells all fluorescent bile salt derivatives interact with the same polypeptides as the physiological bile salts. The hepatobiliary transport of taurine-conjugated NBD-amino bile salt derivatives is, due to hydrophobic interactions, accompanied by an increase in fluorescence intensity which is favorable for the study of biological bile salt transport by fluorescence microscopy. 相似文献
6.
7 beta-Methyl-chenodeoxycholic acid (7-MeCDC, 3 alpha, 7 alpha-dihydroxy-7 beta-methyl-5 beta-cholan-24-oic acid), 7 alpha-methyl-ursodeoxycholic acid (7-MeUDC, 3 alpha, 7 beta-dihydroxy-7 alpha-methyl-5 beta-cholan-24-oic acid), 7 xi-methyl-lithocholic acid (7-MeLC, 3 alpha-hydroxy-7 xi-methyl-5 beta-cholan-24-oic acid) and ursodeoxycholylsarcosine (UDCS) were tested as inhibitors of bacterial bile acid 7 alpha-dehydroxylase activity. At a concentration of 50 microM, 7-MeCDC and 7-MeUDC inhibited enzyme activity by 66% and 12%, respectively. 7 alpha-Dehydroxylase activity was not inhibited in the presence of 7-MeLC and UDCS. None of the four bile acid analogs tested inhibited the growth of Eubacterium sp. V.P.I. 12708 at concentrations up to 100 microM. 相似文献
7.
The effect of bile salts on human vascular endothelial cells 总被引:7,自引:0,他引:7
The uptake and release of radiochromium from adult human vascular endothelial cells in culture was employed to determine the relative toxicity of different bile salts. Endothelial cells after pre-incubation with 51Cr for 18 h were incubated with bile salts for 24 h and percentage chromium release was taken as a measure of toxicity to cells. Lithocholic acid (LC) (potassium salt) was cytotoxic at concentrations greater than 50 microM. However, LC glucuronide, sulfate and the beta-epimer were progressively less toxic with toxicity seen at concentrations of 60, 110 and 180 microM, respectively. The greatest cytotoxic effect was observed with glycolithocholic acid (GLC) (potassium salt) which was toxic at every concentration tested (20-200 microM). Sulfation abolished the toxic effect of GLC. At the concentrations employed for the assay (between 20 and 240 microM) GLC sulfate (disodium salt), taurolithocholic acid sulfate (disodium salt), cholic acid (sodium salt), glycocholic acid (sodium salt), deoxycholic acid (sodium salt) and ursodeoxycholic acid (sodium salt) were not cytotoxic. The 51Cr release cytotoxicity assay was validated with lactate dehydrogenase leakage from endothelial cells with a good correlation (r = 0.87). These data confirm in a human cellular system that LC and its conjugates were the most toxic of the bile salts tested and explains its pathophysiological importance in hepatobiliary disease. It also suggests that biotransformation by either sulfation or beta-epimerisation of bile salts especially of LC, as occurs in patients with intrahepatic or extrahepatic biliary obstruction or severe cholestasis, is hepatoprotective. 相似文献
8.
ATP-dependent transport of taurocholate across the hepatocyte canalicular membrane mediated by a 110-kDa glycoprotein binding ATP and bile salt 总被引:7,自引:0,他引:7
M Müller T Ishikawa U Berger C Klünemann L Lucka A Schreyer C Kannicht W Reutter G Kurz D Keppler 《The Journal of biological chemistry》1991,266(28):18920-18926
Direct photoaffinity labeling of liver plasma membrane subfractions enriched in sinusoidal and canalicular membranes using [35S]adenosine 5'-O-(thiotriphosphate) ([35S]ATP gamma S) allows the identification of ATP-binding proteins in these domains. Comparative photoaffinity labeling with [35S]ATP gamma S and with the photolabile bile salt derivative (7,7-azo-3 alpha, 12 alpha-dihydroxy-5 beta-[3 beta-3H]-cholan-24-oyl-2'- aminoethanesulfonate followed by immunoprecipitation with a monoclonal antibody (Be 9.2) revealed the identity of the ATP-binding and the bile salt-binding canalicular membrane glycoprotein with the apparent Mr of 110,000 (gp110). The isoelectric point of this glycoprotein was 3.7. Transport of bile salt was studied in vesicles enriched in canalicular and sinusoidal liver membranes. Incubation of canalicular membrane vesicles with [3H] taurocholate in the presence of ATP resulted in an uptake of the bile salt into the vesicles which was sensitive to vanadate. ATP-dependent taurocholate transport was also observed in membrane vesicles from mutant rats deficient in the ATP-dependent transport of cysteinyl leukotrienes and related amphiphilic anions. Substrates of the P-glycoprotein (gp170), such as verapamil and doxorubicin, did not interfere with the ATP-dependent transport of taurocholate. Reconstitution of purified gp110 into liposomes resulted in an ATP-dependent uptake of [3H]taurocholate. These results demonstrate that gp110 functions as carrier in the ATP-dependent transport of bile salts from the hepatocyte into bile. This export carrier is distinct from hitherto characterized ATP-dependent transport systems. 相似文献
9.
S Schneider U Schramm A Schreyer H P Buscher W Gerok G Kurz 《Journal of lipid research》1991,32(11):1755-1767
In order to visualize bile salt transport, fluorescent bile salt derivatives were synthesized by introduction of the relatively small fluorescent 4-nitrobenzo-2-oxa-1,3-diazol (NBD)-amino group in either the 3-, 7-, or 12-position of the steroid structure, thus providing a complete set of diastereomeric derivatives, 3 alpha-NBD-amino-7 alpha,12 alpha-dihydroxy-5 beta-cholan-24-oic acid, 3 beta-NBD-amino-7 alpha,12 alpha-dihydroxy-5 beta-cholan-24-oic acid, 7 alpha-NBD-amino-3 alpha,12 alpha-dihydroxy-5 beta-cholan-24-oic acid, 7 beta-NBD-amino-3 alpha,12 alpha-dihydroxy-5 beta-cholan-24-oic acid, 12 alpha-NBD-amino-3 alpha,7 alpha-dihydroxy-5 beta-cholan-24-oic acid, 12 beta-NBD-amino-3 alpha,7 alpha-dihydroxy-5 beta-cholan-24-oic acid, as well as their taurine conjugates. Their optical properties with absorption maxima at about 490 nm and emission maxima at 550 nm make them suitable for fluorescent microscopic studies. Fluorescence of the NBD-derivatives is strongly dependent on polarity of the solvent, on the concentration of the bile salt derivatives, and only slightly on temperature. 相似文献
10.
Absence of negative feedback control of bile acid biosynthesis in cultured rat hepatocytes 总被引:3,自引:0,他引:3
W M Kubaska E C Gurley P B Hylemon P S Guzelian Z R Vlahcevic 《The Journal of biological chemistry》1985,260(25):13459-13463
The effect of individual bile acids on bile acid synthesis was studied in primary hepatocyte cultures. Relative rates of bile acid synthesis were measured as the conversion of lipoprotein [4-14C]cholesterol into 4-14C-labeled bile acids. Additions to the culture media of cholate, taurocholate, glycocholate, chenodeoxycholate, taurochenodeoxycholate, glycochenodeoxycholate, deoxycholate, and taurodeoxycholate (10-200 microM) did not inhibit bile acid synthesis. The addition of cholate (100 microM) to the medium raised the intracellular level of cholate 10-fold, documenting effective uptake of added bile acid by cultured hepatocytes. The addition of 200 microM taurocholate to cultured hepatocytes prelabeled with [4-14C]cholesterol did not result in inhibition of bile acid synthesis. Taurocholate (10-200 microM) also failed to inhibit bile acid synthesis in suspensions of freshly isolated hepatocytes after 2, 4, and 6 h of incubation. Surprisingly, the addition of taurocholate and taurochenodeoxycholate (10-200 microM) stimulated taurocholate synthesis from [2-14C]mevalonate-labeled cholesterol (p less than 0.05). Neither taurocholate nor taurochenodeoxycholate directly inhibited cholesterol 7 alpha-hydroxylase activity in the microsomes prepared from cholestyramine-fed rats. By contrast, 7-ketocholesterol and 20 alpha-hydroxycholesterol strongly inhibited cholesterol 7 alpha-hydroxylase activity at low concentrations (10 microM). In conclusion, these data strongly suggest that bile acids, at the level of the hepatocyte, do not directly inhibit bile acid synthesis from exogenous or endogenous cholesterol even at concentrations 3-6-fold higher than those found in rat portal blood. 相似文献
11.
The availability of different sources of cholesterol for bile acid synthesis by cultured chick embryo hepatocytes was studied. Mevalonolactone was taken up by the cells and converted to cholesterol, cholesterol ester and tauroconjugates of bile acids. The addition of mevalonolactone had little effect on the conversion of endogenous cholesterol to taurocholic acid; however, taurochenodeoxycholic acid synthesis was stimulated. 25-30% of the cholesterol synthesized from mevalonolactone was converted to taurochenodeoxycholic, taurocholic and two so-far unidentified bile acids. All bile acids were secreted into the incubation medium. When cholesterol was added as mixed liposomes with phosphatidylcholine, it was taken up by the cells and converted to bile acids. At low concentrations of liposomes, the greater part of the cholesterol which was taken up by the cells was converted to bile acids. At higher concentrations, considerable amounts of cholesterol and cholesterol ester accumulated inside the cells. When mevalonolactone and cholesterol liposomes was added together, both substrates were used simultaneously for bile acids synthesis. HDL cholesterol was the best substrate tested, yielding large amounts of two, so-far, unidentified bile acids (possibly allo-bile acids) and smaller amounts of taurocholic and taurochenodeoxycholic acid. Addition of HDL suppressed the conversion of endogenous cholesterol to taurocholic acid; taurochenodeoxycholic acid synthesis, however, was stimulated. 相似文献
12.
The effect of cholesterol feeding on gallbladder bile acids of the rabbit. Evidence that lithocholic acid is a primary bile acid in the rabbit.
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The bile acid in gallbladder bile of rabbits fed a normal diet or one containing 2% (w/w) cholesterol have been determined by gas chromatography-mass spectrometry. The predominant bile acids in normally fed rabbits were 3 alpha, 7 alpha, 12 alpha-trihydroxy-5 beta-cholan-24-oic acid (cholic acid), 3 alpha, 12 alpha-dihydroxy-5 alpha-cholan-24-oic acid (allodeoxycholic acid) and 3 alpha, 12 alpha-dihydroxy-5 beta-cholan-24-oic acid (deoxycholic acid) with very much smaller amounts of 3 alpha-hydroxy-5 beta-cholan-24-oic acid (lithocholic acid) and 3 alpha, 12 beta-dihydroxy-5 beta-cholan-24-oic acid. In the cholesterol-fed animals the lithocholate became a predominant bile acid. Sulphated bile acids accounted for less than 1% of the total bile acids. It is proposed that lithocholic acid may be a primary bile acid in the cholesterol-fed rabbit, formed by an alternative pathway of biosynthesis involving hepatic mitochondria. 相似文献
13.
Photolabile derivatives of bile salts. Synthesis and suitability for photoaffinity labeling 总被引:4,自引:0,他引:4
In an approach to the identification of bile salt-binding carriers, the photoactivable bile acid derivatives A) 3 beta-azido, 7 alpha,12 alpha-dihydroxy-5 beta-cholan-24-oic acid, B) 7,7-azo-3 alpha,12 alpha-dihydroxy-5 beta-cholan-24-oic acid, and C) 11 xi-azido-12-oxo-3 alpha,7 alpha-dihydroxy-5 beta-cholan-24-oic acid were synthesized in unconjugated and taurine-conjugated form. Photolysis of the 3 beta-azido derivatives was studied using a light source with a maximum emission at 300 nm and established a half-life time of 18.5 min. The photochemistry of the 7,7-azo derivatives was investigated using light with a maximum at 350 nm and had a half-life time of 2.2 min. The 11 xi-azido-12-oxo derivatives were photolyzed with light having a maximum at 300 nm resulting in a half-life time of 8.5 min. The suitability of the 7,7-azo derivatives for photoaffinity labeling was demonstrated by photolyses in 14C-labeled methanol and acetonitrile. The generated carbene reacted with the solvents under covalent bond formation of 6 to 12%. The efficiency of all synthesized photolabile derivatives for photoaffinity labeling of bile salt binding proteins was demonstrated. 相似文献
14.
W M Kubaska E C Gurley P B Hylemon D M Heuman E H Mosbach Z R Vlahcevic 《Biochimica et biophysica acta》1987,920(3):195-204
The effect of individual 7 beta-hydroxy bile acids (ursodeoxycholic and ursocholic acid), bile acid analogues of ursodeoxycholic acid, combination of bile acids (taurochenodeoxycholate and taurocholate), and mixtures of bile acids, phospholipids and cholesterol in proportions found in rat bile, on bile acids synthesis was studied in cultured rat hepatocytes. Individual steroids tested included ursodeoxycholate (UDCA), ursocholate (UCA), glycoursodeoxycholate (GUDCA) and tauroursodeoxycholate (TUDCA). Analogues of UDCA (7-methylursodeoxycholate, sarcosylursodeoxycholate and ursooxazoline) and allochenodeoxycholate, a representative of 5 alpha-cholanoic bile acid were also tested in order to determine the specificity of the bile acid biofeedback. Each individual steroid was added to the culture media at concentrations ranging from 10 to 200 microM. Mixtures of taurochenodeoxycholate (TDCA) and taurocholate in concentrations ranging from 150 to 600 microM alone and in combination with phosphatidylcholine (10-125 microM) and cholesterol (3-13 microM) were also tested for their effects on bile acid synthesis. Rates of bile acid synthesis were determined as the conversion of added lipoprotein [4-14C]cholesterol or [2-14C]mevalonate into 14C-labeled bile acids and by GLC quantitation of bile acids secreted into the culture media. Individual bile acids, bile acid analogues, combination of bile acids and mixture of bile acids with phosphatidylcholine and cholesterol failed to inhibit bile acid synthesis in cultured hepatocytes. The addition of UDCA or UCA to the culture medium resulted in a marked increase in the intracellular level of both bile acids, and in the case of UDCA there was a 4-fold increase in beta-muricholate. These results demonstrate effective uptake and metabolism of these bile acids by the rat hepatocytes. UDCA, UCA, TUDCA and GUDCA also failed to inhibit cholesterol-7 alpha-hydroxylase activity in microsomes prepared from cholestyramine-fed rats. The current data confirm and extend our previous observations that, under conditions employed, neither single bile acid nor a mixture of bile acids with or without phosphatidylcholine and cholesterol inhibits bile acid synthesis in primary rat hepatocyte cultures. We postulate that mechanisms other than a direct effect of bile acids on cholesterol-7 alpha-hydroxylase might play a role in the regulation of bile acid synthesis. 相似文献
15.
Synthesis and transport characteristics of photoaffinity probes for the hepatocyte bile acid transport system 总被引:1,自引:0,他引:1
In an effort to characterize the hepatocyte bile acid transport system, a photoreactive derivative of taurocholate, (7,7-azo-3 alpha,12 alpha-dihydroxy-5 beta-cholan-24-oyl)-2-aminoethanesulfonic acid (7-ADTC) has been synthesized and its transport properties compared to those of the natural substrate. Both the bile acid and its synthetic analog were shown to be transported against an electrochemical gradient as well as a chemical gradient. Transport as a function of concentration and the presence of sodium indicated that both substrates were taken up by a sodium-dependent and a sodium-independent route. Taurocholate had Km values of 26 and 57 microM and Vmax values of 0.77 and 0.15 nmol/mg of protein/min, respectively. In comparison, 7-ADTC had very similar kinetic properties with Km values of 25 and 31 microM and Vmax values of 1.14 and 0.27 nmol/mg of protein/min. Each compound was shown to inhibit competitively the transport of the other, suggesting that these substrates utilized a common membrane carrier. The transport properties of the photoreactive anion transport inhibitor, N-(4-azido-2-nitrophenyl)-2-aminoethylsulfonate (NAP-taurine) were also characterized in the hepatocyte system. Transport occurred via a sodium-dependent and a sodium-independent route with Km values of 210 and 555 microM and Vmax values of 0.57 and 1.62 nmol/mg of protein/min. As in the case of 7-ADTC, NAP-taurine and taurocholate were also shown to be mutual competitive inhibitors. In the absence of light, 7-ADTC was a reversible inhibitor of taurocholate uptake. Upon irradiation, irreversible photoinactivation of the taurocholate uptake system was observed. These results indicate that 7-ADTC and NAP-taurine can be utilized as photoaffinity probes for the identification of the bile acid carrier protein(s) in hepatocyte plasma membranes. 相似文献
16.
1. The compound trans-1,4 bis-(2-dichlorobenzylaminomethyl)cyclohexane dihydrochloride (AY9944) blocks cholesterol synthesis at a late stage. This leads to a decrease in cholesterol and accumulation of cholesta-5,7-diene-3-beta-ol (7-dehydrocholesterol) in tissues and plasma. 2. The effect of AY9944 on bile salt synthesis in rat liver was studied. The synthesis of conjugated cholic and chenodeoxycholic acids was measured in hepatocytes isolated from rats 2 h, 24 h and 48 h after administration of a single oral dose of AY9944. Production of the two bile salts was inhibited by 70-80% in hepatocytes from AY9944-treated as compared to untreated animals. 3. When AY9944 was added to the incubation medium in vitro of hepatocytes prepared from untreated rats the synthesis of conjugated cholic and chenodeoxycholic acids was not inhibited during the first hour of incubation, probably because of the presence of endogenous cholesterol. However when hepatocytes from untreated rats were incubated with AY9944 for periods of 2 h or longer, bile salt production was decreased markedly. 4. Bile salt synthesis is stimulated when rats are subjected to total biliary drainage for 24 h. The effect of AY9944 on this stimulation was studied. The content of conjugated cholic and chenodeoxycholic acid in the bile was measured as an indicator of bile salt synthesis. 5. In control animals the rate of secretion of biliary bile salts began to increase after about 24 h of total biliary drainage and reached a maximum after approximately 36 h. A single oral dose of AY9944 given 2 h after the start of total biliary drainage delayed and reduced this response. 6. The results show that inhibition of cholesterol synthesis by AY9944 resulting in the replacement of cholesterol by 7-dehydrocholesterol decreases but does not completely prevent bile salt synthesis. 相似文献
17.
The taurocholate transport system in normal and transformed hepatocytes has been characterized using transport kinetics and photoaffinity labeling procedures. A photoreactive diazirine derivative of taurocholate, (7,7-azo-3 alpha,12 alpha-dihydroxy-5 beta-cholan-24-oyl)-2-amino [ 1,2-3H ]ethanesulfonic acid (7-ADTC), which has been shown to be a substrate for the bile acid carrier system, was photolyzed in the presence of intact hepatocytes, hepatoma tissue culture (HTC) cells, and plasma membranes derived from the hepatocyte sinusoidal surface. Irradiation of membranes in the presence of 7-ADTC resulted in the incorporation of the photoprobe into two proteins with Mr = 68,000 and 54,000. The specificity of labeling was confirmed by the significant inhibition of labeling observed when photolysis was carried out in the presence of taurocholate. The 68,000-Da protein was easily extracted with water and was shown to exhibit electrophoretic properties identical with rat serum albumin. The 54,000-Da protein required Triton X-100 for solubilization, indicating a strong association with the plasma membrane. Labeling of intact hepatocytes also resulted in specific labeling of the 54,000-Da protein. In contrast to hepatocytes, HTC cells derived from Morris hepatoma 7288C as well as H4-II-E cells derived from Reuber hepatoma H-35 exhibited a total loss of mediated bile acid uptake. Photolysis of 7-ADTC in the presence of HTC cells did not result in the labeling of any proteins, a result consistent with the loss of transport activity, and further supporting the specificity of the labeling reaction. The anion transport inhibitor N-(4-azido-2-nitrophenyl)-2-aminoethyl-[ 35S ]sulfonate, which has been shown to be a substrate for the bile acid carrier system also labeled the 54,000-Da plasma membrane protein when photolyzed in the presence of intact hepatocytes. These results suggest that the 54,000-Da protein is a component of the hepatocyte bile acid transport system and that the activity of this system is greatly reduced in several hepatoma cell lines. 相似文献
18.
Identification of bile acids in the serum and urine in cholestasis. Evidence for 6alpha-hydroxylation of bile acids in man. 总被引:4,自引:1,他引:3
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In this qualitative study of the pattern of bile acid excretion in cholestasis, methods are described for the isolation of bile acids from large volumes of urine and plasma. The bile acids were subjected to a group separation and identified by combined gas chromatography-mass spectrometry. The techniques were developed to allow identification of the minor components of the bile acid mixture. Four bile acids that have not previously been described in human urine and plasma were detected, namely 3beta, 7alpha-dihydroxy-5beta-cholan-24-oic acid, 3alpha, 6alpha-dihydroxy-5beta-cholan-24-oic acid (hyodeoxycholic acid), 3alpha, 6alpha, 7alpha-trihydroxy-5beta-cholan-24-oic acid (hyocholic acid) and 3alpha, 7beta, 12alpha-trihydroxy-5beta-cholan-24-oic acid. In addition three C27 steroids were found; 26-hydroxycholesterol and a trihydroxy cholestane, probably 5 beta-cholestane-3alpha, 7alpha, 26-triol were found in the sulphate fraction of plasma and urine. In the plasma sample, a sulphate conjugate of 24-hydroxycholesterol was found. The presence of these compounds probably reflects the existence of further pathways for bile acid metabolism. It is not yet known whether this is a consequence of the cholestasis or whether they are also present in normal man, at much lower concentrations. 相似文献
19.
Bile acids of snakes of the subfamily Viperinae and the biosynthesis of C-23-hydroxylated bile acids in liver homogenate fractions from the adder, Vipera berus (Linn.).
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1. Analysis of bile salts of four snakes of the subfamily Viperinae showed that their bile acids consisted mainly of C-23-hydroxylated bile acids. 2. Incubations of 14C-labelled sodium cholate (3 alpha, 7 alpha, 12 alpha-trihydroxy-5 beta-cholan-24-oate) and deoxycholate (3 alpha, 12 alpha-dihydroxy-5 beta-cholan-24-oate) with whole and fractionated adder liver homogenates were carried out in the presence of molecular oxygen and NADPH or an NADPH-generating system. The formation of C-23-hydroxylated bile acids, namely bitocholic acid (3 alpha, 12 alpha, 23xi-trihydroxy-5 beta-cholan-24-oic acid) and 3 alpha, 7 alpha, 12 alpha, 23 xi-tetrahydroxy-cholanic acid (3 alpha, 7 alpha, 12 alpha, 23 xi-tetrahydroxy-5 beta-cholan-24-oic acid), was observed mainly in the microsomal fraction and partly in the mitochondrial fraction. 3. Biosynthetic pathways of C-23-hydroxylated bile acids are discussed. 相似文献
20.
The effect of ursodeoxycholic acid analogues bearing modifications at the side-chain moiety of the molecule was tested on cholesterol 7 alpha-hydroxylase and HMG-CoA reductase in rat liver microsomes. The compounds included 23 R,S mixture and the single isomers 23R and 23S of 23 methylursodeoxycholic acid (23-methyl UDCA), the isomeric mixture (cis + trans) of 3 alpha,7 beta-dihydroxy-20,22-methylen-5 beta-cholan-23-oic acid (norcypro-UDCA) and the corresponding single isomers. Each steroid was added to liver microsomes as the sodium salt, at concentrations ranging from 25 to 200 microM. Isomers 23R and 23S of 23-methyl-UDCA inhibited cholesterol 7 alpha-hydroxylase in a concentration-dependent manner. The inhibitory capacity was similar for the two isomers. The extent of inhibition of the analogues was greater than that of the parent compound UDCA. Shortening of the side-chain in norcypro-UDCA resulted in a partial loss of the inhibitory effect, as compared to cypro-UDCA (3 alpha,7 beta-dihydroxy-22,23-methylen-5 beta-cholan-24-oic acid). None of these bile acid derivatives affected the activity of the enzyme HMG-CoA reductase. 相似文献