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J D Ostrow

Publications and source records attributed to J D Ostrow.

At least 37 records · Page 2Linked to original sources
PubMed1996

Albumin binding of unconjugated [3H]bilirubin and its uptake by rat liver basolateral plasma membrane vesicles.

Using highly purified unconjugated [3H]bilirubin (UCB), we measured UCB binding to delipidated human serum albumin (HSA) and its uptake by basolateral rat liver plasma membrane vesicles, in both the absence and presence of an inside-positive membrane potential. Free UCB concentrations ([Bf]) were calculated from UCB-HSA affinity constants (K'f), determined by five cycles of ultrafiltration through a Centricon-10 device (Amicon) of the same solutions used in the uptake studies. At HSA concentrations from 12 to 380 microM, K'f (litre/mol) was inversely related to [HSA], irrespective of the [Bf]/[HSA] ratio. K'f was 2.066 x 10(6) + (3.258 x 10(8)/[HSA]). When 50 mM KC1 was isoosmotically substituted for sucrose, the K'f value was significantly lower {2.077 x 10(6) + (1.099 x 10(8)/[HSA])}. The transport occurred into an osmotic-sensitive space. Below saturation ([Bf] < or = 65 nM), both electroneutral and electrogenic components followed saturation kinetics with respect to [Bf], with K(m) values of 28 +/- 7 and 57 +/- 8 nM respectively (mean +/- S.D., n = 3, P < 0.001). The Vmax was greater for the electrogenic than for the electroneutral component (112 +/- 12 versus 45 +/- 4 pmol of UCB. mg-1 of protein. 15 s-1, P < 0.001). Sulphobromophthalein trans-stimulated both electrogenic (61%) and electroneutral (72%) UCB uptake. These data indicate that: (a) as [HSA] increases, K'f decreases, thus increasing the concentration of free UCB. This may account for much of the enhanced hepatocytic uptake of organic anions observed with increasing [HSA]. (b) UCB is taken up at the basolateral membrane of the hepatocyte by two systems with K(m) values within the range of physiological free UCB levels in plasma. The electrogenic component shows a lower affinity and a higher capacity than the electroneutral component. (c) It is important to calculate the actual [Bf] using a K'f value determined under the same experimental conditions (medium and [HSA]) used for the uptake studies.

Animals↗
PubMed1996

New concepts in bilirubin and jaundice: report of the Third International Bilirubin Workshop, April 6-8, 1995, Trieste, Italy.

The workshop covered three major areas: Unconjugated bilirubin (UCB) chemistry and physical chemistry; UCB transport and intracellular trafficking; and evaluation and therapy of neonatal and congenital hyperbilirubinemias. Findings of studies in the chemistry and physical chemistry area were as follows. (1) Nuclear magnetic resonance (NMR) studies of highly enriched 13COOH mesobilirubin in water-dimethyl sulfoxide systems indicated that the pKa values of the carboxyl groups are 4.2 and 4.9, respectively. This finding differs from some reports that suggest that the two pKa values in aqueous systems are near or above pH 7.0. (2) Contrasting views of the hydrophobic interactions of UCB with bile salts were presented: one suggested that multiple bile salt monomers bind to one UCB molecule; the other suggested that UCB binds to the nonpolar surface of helical bile salt micelles. (3) Structures were proposed for the varied calcium and copper bilirubinate salts formed at various pH values and cation/UCB ratios. (4) Studies of binding of UCB to human serum albumin (HSA) showed marked diminution of UCB-binding affinity as albumin and chloride concentrations increased. (5) A unique UCB derivative, bilirubin-C10-sulfonic acid, was identified as the major bile pigment in bullfrog bile. (6) New methods were presented for removal of impurities from preparations of bile salts and UCB. Findings of studies in the transport area were as follows. (1) Four putative basolateral and two putative canalicular hepatocytic transporters of UCB and related organic anions were described. Special emphasis was given to the adenosine triphosphate (ATP)-dependent canalicular multi-specific organic anion transporter that is defective in three strains of mutant rats with congenital conjugated hyperbilirubinemia. (2) The roles of the classical and newer molecular biological approaches to identification of these transporters were contrasted, and their limitations were discussed. (3) The relative roles of the multiple carriers in UCB transport under different conditions and substrate concentrations were discussed. (4) Cytosolic UCB-binding proteins (e.g., ligandin) were shown to promote transcellular movement of UCB by solubilizing and transporting the pigment in the aqueous phase while limiting binding of UCB to the relatively immobile membranes of cell organelles. (5) Mechanisms were presented for translocation of UDP-glucuronic acid (UDPGA) into the lumenal location of UDPGA transferase in the endoplasmic reticulum, as well as the enhancement of this process by N-acetyl-glucosamine. Studies in the neonatal and congenital jaundice area were as follows. (1) Criteria were reviewed for initiating treatment of neonatal jaundice, emphasizing the primacy of serum bilirubin levels, gestational age, and hemolysis as risk factors for kernicterus. (2) New methods were presented for frequent, automated monitoring of serum bilirubin levels and breath CO levels as an index of rates of formation of UCB from heme. (3) The current status and limitations of new approaches to treatment of severe unconjugated hyperbilirubinemia were discussed: hepatocyte transplantation and gene therapy, still in the stage of development in animal models, have provided only partial and temporary relief of hyperbilirubinemia; extracorporeal liver assist devices have had some success in initial human studies; and inhibition of heme oxygenase (HO) with metalloporphyrins, especially tin mesoporphyrin, which markedly decreases bilirubin production for prolonged periods, is a new alternative to phototherapy. (4) The ontogeny of the two HO isozymes was contrasted in the liver, spleen, kidney, and lung.

Animals↗
PubMed1996

Distinct immuno-localization of mucin and other biliary proteins in human cholesterol gallstones.

BACKGROUND/AIMS: Cholesterol gallstones consist of cholesterol crystals and smaller amounts of pigments and calcium salts, arrayed on a mucin plus protein matrix. The localization of the various biliary proteins in the stones has not been characterized. We aimed to localize several biliary proteins in gallstones in order to determine their possible role in stone formation and growth. METHODS: The distribution of several matrix proteins and their relationships to the minerals were determined using immunostaining and EDAX microanalysis on hemisected cholesterol gallstones. RESULTS: Pigment areas were rich in calcium and contained Cu, P and S. These elements were absent in cholesterol regions. Mucin was identified in a three-dimensional network intercalated between cholesterol crystals and as septa between deposits of pigments and cholesterol; APF/CBP and ApN coated only the pigment deposits. No specific topographical localization was found for albumin or IgA. CONCLUSIONS: This suggests a role for mucin, APF/ CBP and ApN in the formation of cholesterol gallstones. We propose that cholesterol crystals bind directly to mucin, whereas calcium salts and pigments deposit on APF/CBP and ApN bind to the mucin.

Bile↗
PubMed1995

Interaction of bovine gallbladder mucin and calcium-binding protein: effects on calcium phosphate precipitation.

BACKGROUND & AIMS: Gallstones consist of calcium salts and cholesterol crystals, arrayed on a matrix of gallbladder mucin (GBM), and regulatory proteins like calcium-binding protein (CBP). To determine if interactions between CBP and GBM follow a biomineralization scheme, their mutual binding and effects on CaHPO4 precipitation were studied. METHODS: Binding of CBP to GBM was assessed by inhibition of the fluorescence of the complex of GBM with bis-1,8-anilinonaphthalene sulfonic acid (bis-ANS). The effects of the proteins on precipitation of CaHPO4 were assessed by nephelometry and gravimetry. Precipitates were analyzed for calcium, phosphate, and protein. RESULTS: CBP and bis-ANS competitively displaced each other from 30 binding sites on mucin, with a 1:1 stoichiometry and similar affinity. The rate of precipitation of CaHPO4 was retarded by mucin and CBP. Precipitate mass was unaffected by GBM alone but decreased with the addition of CBP. Complexing CBP with GBM abolished or moderated this latter effect, altered precipitate morphology, and changed the stoichiometric ratios of Ca to PO4 in the precipitates from 1:1 to 3:2. Mucin and CBP were incorporated into the precipitates. CONCLUSIONS: These studies suggest that the formation of calcium-containing gallstones is a biomineralization process regulated by both GBM and CBP.

Anilino Naphthalenesulfonates↗
PubMed1995

Method for removal of surface-active impurities and calcium from conjugated bile salt preparations: comparison with silicic acid chromatography.

Some commercial preparations of common natural conjugated bile salts contain impurities (e.g., amines, lipids, and calcium) that are likely to affect their physicochemical properties. A method was developed for purifying commercial preparations of sodium salts of glycine- and taurine-conjugated bile acids. The method consists of passage of a dilute aqueous solution of the sodium bile salt through three columns in sequence: graphitized carbon, a hydrophobic bonded octadecylsilane (C18) cartridge, and a calcium-chelating resin. The final solution was extracted with chloroform, and the purified bile salt was then isolated by freeze-drying, with a yield of 65-75%. Each bile salt purified by this method was compared with the corresponding bile salt purified by conventional adsorption chromatography on a silicic acid column, using a mixture of methanol and chloroform as eluant. Purity was assessed by visible spectra, by surface tension measurements (using the maximum bubble-pressure method and a Wilhelmy wire method), by chloroform extractability of impurities in the conjugated bile acid, by liposome solubilization, and by chemical analysis of the calcium content. Both purification methods removed colored and surface-active impurities, but the new method was always as or more effective than silicic acid column chromatography. Calcium ion, present in commercial bile salts in concentrations up to 16 mmol/mol bile salt, was removed completely by the three-column method, but not by silicic acid chromatography. The new method is thus a simple, rapid, and efficient procedure for purification of the sodium salts of glycine- and taurine-conjugated bile acids for physicochemical measurements, in which elimination of surface-active impurities and polyvalent cations is desired.

Bile Acids and Salts↗
PubMed1992

Isolation of a calcium-regulatory protein from black pigment gallstones: similarity with a protein from cholesterol gallstones.

We have previously isolated from 13 cholesterol gallstones a low molecular weight acidic bili-protein that inhibited the precipitation of calcium carbonate in vitro. We now report the isolation of a similar protein from seven black pigment gallstones. Cholesterol was removed from the stones by Soxhlet apparatus with methyl t-butyl ether, and bile acids were extracted with methanol. The protein was purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after demineralization of the stones with ethylenediaminetetraacetate. Structural and functional properties of the protein from the black stones that were similar to the protein from the cholesterol stones included the following: (a) an apparent molecular weight of about 5 kD; (b) a high content of acidic (19.8%) and hydrophobic (50.1%) amino acids with a low content of basic residues (8.4%) and little sulfide-containing amino acids (1.9%); (c) an inhibitory effect on both the initiation and growth of calcium carbonate crystals in vitro; and (d) very tight (possibly covalent) binding of a diazo-positive yellow pigment, presumably bilirubin, with maximum spectral absorbance at 410 nm. The structural and functional similarities of these bili-proteins from black pigment and cholesterol gallstones and their striking effects on calcium carbonate precipitation in vitro suggest that they play a common role in the regulation of precipitation of calcium salts during the formation of both types of gallstones.

Calcium Carbonate↗
PubMed1992

Epitope mapping of the human biliary amphipathic, anionic polypeptide: similarity with a calcium-binding protein isolated from gallstones and bile, and immunologic cross-reactivity with apolipoprotein A-I.

Biliary amphipathic anionic polypeptide (APF) the major protein of the pigment-lipoprotein complex in bile, and calcium-binding protein (CBP) from gallstones are both small (less than 10 kDa), highly acidic, amphipathic proteins present in bile and closely associated also with pigmented areas in human gallstones. Polyclonal antibodies against APF have shown cross-reactivity with plasma high density lipoproteins (HDL). This study examines the hypothesis that APF and CBP might be closely related or even identical, and might also share common epitopes with the larger apoA-I (23 kDa). To assess this, immunoreactivity of the three delipidated, highly purified proteins was determined against a panel of 12 monoclonal antibodies (MAbs) prepared against APF and a panel of 4 MAbs against apoA-I. APF was isolated from bile by zonal ultracentrifugation. CBP was isolated from proteins precipitated from bile by CaCl2, as well as from the calcium bilirubinate shells of cholesterol gallstones, by extraction successively with methyl-t-butyl ether, methanol, and Na2EDTA, followed by Sephadex G-25 chromatography and two-stage preparative SDS-PAGE. ApoA-I was prepared by two types of chromatography: Sephacryl S200 chromatography and heparin-chromatographic immunoaffinity. Specific polyclonal antibodies to APF and apoA-I were prepared from immunized rabbits. MAbs to APF and apoA-I were prepared by immunization of mice, using standard hybridoma technique. Western blotting of APF and CBP in 15% SDS-PAGE yielded one band with an apparent molecular weight of 6.5 kDa, which, along with apoA-I, was immunostained by polyclonal antibodies to APF and apoA-I. Using 12 MAbs against APF with three types of ELISA (direct antigen binding, competitive antigen displacement, and epitope competition between antibodies), it was shown that APF and delipidated apoA-I shared six epitopes, three of which were detected also on the surface of intact HDL particles. Six other epitopes were present in APF but not apoA-I, four of which were exposed on the surface of HDL. Four MAbs against apoA-I reacted with APF and CBP. Amino acid analyses of APF and CBP were similar with 20-23% acidic and 7-11% basic amino acids and low contents of cysteine, methionine, and tyrosine; both differed from apoA-I in containing isoleucine and cysteine. Using ELISA and one MAb (no. 32) against APF, this polypeptide was detected in human plasma HDL, the pigment-lipoprotein complex in the bile of humans, dogs, and rats, and in both pigment and cholesterol gallstones.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1992

Ionization and self-association of unconjugated bilirubin, determined by rapid solvent partition from chloroform, with further studies of bilirubin solubility.

Our studies of equilibrium solubilization of crystals of unconjugated bilirubin (UCB) in buffered aqueous NaCl (1988. J. Lipid Res. 29: 335-348) suggested that the two carboxylic pKa values were 6.8 and 9.3 and the solubility of UCB diacid was 0.1 microM. These data, however, were not ideal, due to possible effects of crystal size, metastability, 96-h incubation times with formation of polar derivatives, impurities in the bilirubin, and imprecision of analyses at low concentrations of UCB ([UCB]). In the present study, designed to determine the pKa values and self-association of UCB, these problems were minimized by solvent partition of UCB from solution in CHCl3 into buffered aqueous NaCl. There was no crystal phase. Equilibrium was attained rapidly (10 min); UCB and CHCl3 were highly purified; and accurate diazo assay of low [UCB] in the aqueous phase, [Bw], was achieved by concentrating the UCB through back-extraction into a small volume of CHCl3. By determining effects on partition rations of varying the [UCB] in the CHCl3 phase, [Bc], we could assess also the self-association of UCB species in the aqueous phase. Partition ratios (P = Bw/Bc) did not differ between initial and repeat extractions, indicating insignificant concentrations of polar UCB derivatives. Similar P ratios were obtained when equilibrium was approached from a supersaturated aqueous phase. At 21-25 degrees C, mu = 0.15, the data (n = 76) fit the equation: log P = log Po + log[1 + 10(pH-A) + 10(2pH-B) + Bc.10(4pH-D)]; the bracketed terms reflect P for H2Bo (diacid), HB- (monoanion), B= (dianion), and (B=)2 dimer, respectively. Computer-fitted values for constants (+/- SD) were: Po = P for H2Bo = 5.79 x 10(-5); A = pK1 = 8.12 +/- 0.23; B = pK1 + pK2 = 16.56 +/- 0.10; pK2 = 8.44 +/- 0.33; D = pk22 + 2(pK1 + pK2) -log(2Po) = 37.64 +/- 0.07, and k22 = 0.26 microM-1 [formation constant of (B=)2 dimer]. In ancillary studies, multiple cycles of direct dissolution of UCB crystals revealed a progressive decrease in aqueous solubility of UCB as fine crystals were removed; this effect was minimal in CHCl3. Unlike in water, moreover, varied UCB crystal forms had similar solubilities in CHCl3, with [Bc] = 1.14 mM at saturation. As determined from [Bc]sat.Po, the aqueous solubility of H2Bo was 66 nM.(ABSTRACT TRUNCATED AT 400 WORDS)

Bilirubin↗
PubMed1991

Canine bile contains anticrystallization factors that inhibit precipitation of calcium carbonate.

Previous studies have strongly suggested that human bile contains a substance(s) that interferes with the precipitation of calcium phosphate and carbonate from solution. These studies, however, did not distinguish between calcium binding by biliary constituents resulting in decreased calcium carbonate saturation (alterations in solution thermodynamics) and true inhibition of calcium salt precipitation by kinetic factors. Because our recent studies have shown that canine common duct bile is always supersaturated with calcium carbonate (thermodynamically at risk for precipitation), we hypothesized that it must contain kinetic factors that inhibit formation and/or growth of calcium carbonate crystals. Effects of canine bile, bovine albumin and the bile salt taurocholate on calcium carbonate precipitation were studied in highly supersaturated solutions of calcium carbonate that spontaneously undergo rapid precipitation. Measured free ionized calcium concentrations, [Ca++], and calculated calcium carbonate saturation indices were compared in test solutions and controls to evaluate the thermodynamic effects of test solutions on the degree of saturation in the assay system. It is shown that addition of only 0.2 ml of normal canine gallbladder bile to the assay system (a 1:101 dilution of biliary components) abolished precipitation. A lesser inhibitory effect (a decrease in the rate of precipitation) was observed when gallbladder bile was diluted but was lost after 10-fold dilution. Canine common duct bile caused a decrease in the rate of precipitation similar to diluted gallbladder bile. In contrast, sodium taurocholate (250 mmol/L), the major bile salt in the dog, and albumin (1.5 gm/L), the most abundant protein in bile, had only a minimal inhibitory effect.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Natural history and significance of esophageal squamous cell dysplasia.

Balloon-mesh cytologic screening for esophageal cancer done in 255 asymptomatic high-risk United States veterans (age greater than 40 years, ethanol abuse for greater than 20 years, and cigarette smoking greater than 20 pack years) identified 37 patients with squamous cell dysplasia. Of the 37 patients with dysplasia, 28 were re-evaluated prospectively at 6-month intervals for up to 36 months by balloon-mesh cytology, esophagoscopy with vital staining and biopsies, chest radiographs, oropharyngeal examination, and indirect laryngoscopy. During prospective follow-up evaluation, cytology specimens were repetitively normal in 16 patients (57%), showed inflammatory changes in eight patients (29%), persisted as dysplasia in two patients (7%) (both had endoscopic and histologic evidence of esophagitis), and progressed to carcinoma in two patients (7%) (one esophageal, one laryngeal). Although histologic findings concurred with the resolution of dysplasia, biopsy specimens were characterized by a similar difficulty in distinguishing dysplasia from inflammation. Erroneous histologic diagnoses of carcinoma in situ were made in two patients with reflux esophagitis evident endoscopically and confirmed during the course of a 24-36 month follow-up period. The authors conclude that squamous cell dysplasia detected by balloon-mesh cytology is seldom a precursor of esophageal cancer in the high-risk U.S. population but, rather, is often related to esophagitis. Thus, balloon-mesh cytology has limited use as a screening method for the early detection of esophageal cancer in the United States.

Adult↗