Hamsters predisposed to sucrose-induced cholesterol gallstones (LPN strain) are more resistant to excess dietary cholesterol than hamsters that are not sensitive to cholelithiasis induction.

We compared the effects of cholesterol feeding in male hamsters from two strains with different propensities to sucrose-induced cholelithiasis; Laboratoire de Physiologie de la Nutrition (LPN) hamsters are predisposed to developing biliary cholesterol gallstones, whereas Janvier (JAN) hamsters are not. When fed a basal control diet, LPN hamsters had a lower cholesterolemia (-21%, P = 0.01) than JAN hamsters, and a higher activity of 3-hydroxy-3-methyl glutaryl coenzyme A reductase in liver (+148%, P = 0.018) and intestine (+281%, P < 0.0001). After feeding the same diet enriched with 0.3% cholesterol for 5 wk, cholesterolemia increased more dramatically in JAN hamsters (+235%, P < 0.001) than in LPN hamsters (+108%, P < 0.001), as did the liver concentration of cholesterol, which reached 152.30 +/- 13.00 and 44.41 +/- 9.06 micromol/g, respectively. Only JAN hamsters displayed hepatomegaly, with an increased cholesterol saturation index of the gallbladder bile (+100%, P < 0.01), due to the cholesterol challenge. In liver, cholesterol feeding reduced cholesterol 7alpha-hydroxylase activity and mRNA level, and stimulated sterol 27-hydroxylase and oxysterol 7alpha-hydroxylase activities. Hepatic levels of LDL receptor decreased by approximately 60% in both strains, whereas HDL receptor scavenger class B type 1 (SR-BI) levels were unaffected by dietary cholesterol. The greater resistance of LPN hamsters to the hypercholesterolemic diet can be explained by a lower capacity to store cholesterol in the liver and greater efficiency in reducing the activity of 3-hydroxy-3-methyl glutaryl coenzyme A reductase in response to cholesterol feeding [from 11263 to 261 pmol/(min x organ) in LPN hamsters and from 4530 to 694 pmol/(min x organ) in JAN hamsters]. These results highlight the usefulness of this two-strain model, which offers some analogy with the inverse association between the predisposition to cholelithiasis and the risk of atherosclerosis in humans.

Accurate separation of vesicles, micelles and cholesterol crystals in supersaturated model biles by ultracentrifugation, ultrafiltration and dialysis.

Gel filtration with bile salts at intermixed micellar/vesicular concentrations (IMC) in the eluant has been proposed to isolate vesicles and micelles from supersaturated model biles, but the presence of vesicular aggregates makes this method unreliable. We have now validated a new method for isolation of various phases. First, aggregated vesicles and - if present - cholesterol crystals are pelleted by short ultracentrifugation. Cholesterol contained in crystals and vesicular aggregates can be quantitated from the difference of cholesterol contents in the pellets before and after bile salt-induced solubilization of the vesicular aggregates. Micelles are then isolated by ultrafiltration of the supernatant through a highly selective 300 kDa filter and unilamellar vesicles by dialysis against buffer containing bile salts at IMC values. Lipids contained in unilamellar vesicles are also estimated by subtraction of lipid contents in filtered micelles from lipid contents in (unilamellar vesicle+micelle containing) supernatant ('subtraction method'). 'Ultrafiltration-dialysis' and 'subtraction' methods yielded identical lipid solubilization in unilamellar vesicles and identical vesicular cholesterol/phospholipid ratios. In contrast, gel filtration yielded much more lipids in micelles and less in unilamellar vesicles, with much higher vesicular cholesterol/phospholipid ratios. When vesicles obtained by dialysis were analyzed by gel filtration, vesicular cholesterol/phospholipid ratios increased strongly, despite correct IMC values for bile salts in the eluant. Subsequent extraction of column material showed significant amounts of lipids. In conclusion, gel filtration may underestimate vesicular lipids and overestimate vesicular cholesterol/phospholipid ratios, supposedly because of lipids remaining attached to the column. Combined ultracentrifugation-ultrafiltration-dialysis should be considered state-of-the-art methodology for quantification of cholesterol carriers in model biles.

Cholesterol, bile acid, and lipoprotein metabolism in two strains of hamster, one resistant, the other sensitive (LPN) to sucrose-induced cholelithiasis.

A comprehensive study of cholesterol, bile acid, and lipoprotein metabolism was undertaken in two strains of hamster that differed markedly in their response to a sucrose-rich/low fat diet. Under basal conditions, hamsters from the LPN strain differed from Janvier hamsters by a lower cholesterolemia, a higher postprandial insulinemia, a more active cholesterogenesis in both liver [3- to 4-fold higher 3-hydroxy 3-methylglutaryl coenzyme A reductase (HMG-CoAR) activity and mRNA] and small intestine, and a lower hepatic acyl-coenzyme A:cholesterol acyltransferase activity. Cholesterol saturation indices in the gallbladder bile were similar for both strains, but the lipid concentration was 2-fold higher in LPN than in Janvier hamsters. LPN hamsters had a lower capacity to transform cholesterol into bile acids, shown by the smaller fraction of endogenous cholesterol converted into bile acids prior to fecal excretion (0.34 vs. 0.77). In LPN hamsters, the activities of cholesterol 7alpha-hydroxylase (C7OHase) and sterol 27-hydroxylase (S27OHase), the two rate-limiting enzymes of bile acid synthesis, were disproportionably lower (by 2-fold) to that of HMG-CoAR. When fed a sucrose-rich diet, plasma lipids increased, dietary cholesterol absorption improved, hepatic activities of HMG-CoA reductase, C7Ohase, and S27OHase were reduced, and intestinal S27OHase was inhibited in both strains. Despite a similar increase in the biliary hydrophobicity index due to the bile acid enrichment in chenodeoxycholic acid and derivatives, only LPN hamsters had an increased lithogenic index and developed cholesterol gallstones (75% incidence), whereas Janvier hamsters formed pigment gallstones (79% incidence). These studies indicate that LPN hamsters have a genetic predisposition to sucrose-induced cholesterol gallstone formation related to differences in cholesterol and bile acid metabolism.

Developing a tobacco survey for deaf youth.

Formidable barriers hinder use of standard data collection methods among deaf youth. Culturally and linguistically sensitive data collection strategies are needed to identify the unmet health and programming needs of this population. Unfortunately, researchers often fail to describe the issues involved in developing such targeted methods. The authors describe development of a culturally appropriate data collection instrument for a study of tobacco-related knowledge, attitudes, and practices among deaf youth. The instrument uses interactive multimedia technology to administer a questionnaire translated into the primary languages used by the Deaf. The procedures taken to accommodate this technology to these languages and to Deaf culture are described. This process yielded useful insights with respect to data collection not only among the Deaf, but among other frequently overlooked and underserved populations as well.

Asymmetric distribution of phosphatidylcholine and sphingomyelin between micellar and vesicular phases. Potential implications for canalicular bile formation.

Both phosphatidylcholine (PC) and sphingomyelin (SM) are the major phospholipids in the outer leaflet of the hepatocyte canalicular membrane. Yet, the phospholipids secreted into bile consist principally (>95%) of PC. In order to understand the physical;-chemical basis for preferential biliary PC secretion, we compared interactions with bile salts (taurocholate) and cholesterol of egg yolk (EY)SM (mainly 16:0 acyl chains, similar to trace SM in bile), buttermilk (BM)SM (mainly saturated long (>20 C-atoms) acyl chains, similar to canalicular membrane SM) and egg yolk (EY)PC (mainly unsaturated acyl chains at sn-2 position, similar to bile PC). Main gel to liquid-crystalline transition temperatures were 33. 6 degrees C for BMSM and 36.6 degrees C for EYSM. There were no significant effects of varying phospholipid species on micellar sizes or intermixed-micellar/vesicular bile salt concentrations in taurocholate-phospholipid mixtures (3 g/dL, 37 degrees C, PL/BS + PL = 0.2 or 0.4). Various phases were separated from model systems containing both EYPC and (EY or BM)SM, taurocholate, and variable amounts of cholesterol, by ultracentrifugation with ultrafiltration and dialysis of the supernatant. At increasing cholesterol content, there was preferential distribution of lipids and enrichment with SM containing long saturated acyl chains in the detergent-insoluble pelletable fraction consisting of aggregated vesicles. In contrast, both micelles and small unilamellar vesicles in the supernatant were progressively enriched in PC. Although SM containing vesicles without cholesterol were very sensitive to micellar solubilization upon taurocholate addition, incorporation of the sterol rendered SM-containing vesicles highly resistant against the detergent effects of the bile salt. These findings may have important implications for canalicular bile formation.

Lipid solubilization in human gallbladder versus hepatic biles.

BACKGROUND/AIMS: Cholesterol crystallizes more rapidly in gallbladder than in hepatic biles, supposedly due to formation of cholesterol-supersaturated vesicles in concentrated gallbladder biles because of preferential micellization of phospholipids compared to cholesterol. We therefore aimed to compare lipid solubilization in hepatic and gallbladder biles. METHODS: Mixed micellar and vesicular phases were separated from hepatic and associated gallbladder biles of seven cholesterol gallstone patients by using state-of-the-art gel filtration with bile salts at intermixed micellar/intervesicular compositions and concentrations in the eluant. RESULTS: Vesicles were found in 6 out of 7 hepatic biles, but only in 2 of the corresponding gallbladder biles. Both percentage (7.8+/-5.1 vs. 36.3+/-7.6%; p = 0.01) and amount (0.9+/-0.2 vs. 1.7+/-0.3 mM; p = 0.06) of vesicular cholesterol were lower in gallbladder biles. Similar results were found for vesicular phospholipids (1.3+/-0.8 vs. 11.6+/-6.0%; p = 0.05; and 0.3+/-0.1 vs. 1.1+/-0.5 mM; p = 0.07). The vesicular cholesterol/ phospholipid ratio was 1.7+/-0.5 in hepatic bile but 4.3 and 1.8 in the 2 gallbladder biles which contained vesicles. Mixed micelles in gallbladder biles had a higher cholesterol saturation index than mixed micelles in hepatic biles (1.43+/-0.11 vs. 1.15+/-0.07; p = 0.02). CONCLUSIONS: Concentration of bile in the gallbladder leads to decreased vesicular lipid contents. The finding of supersaturated mixed micelles in the absence of vesicles in a significant number of patients points to the possibility of non-vesicular modes of crystallization.

The diagnostic interview schedule for deaf patients on interactive video: a preliminary investigation.

OBJECTIVE: The authors investigated the feasibility of translating the National Institute of Mental Health Quick Diagnostic Interview Schedule-III, Revised, computer version, for deaf individuals. METHOD: The study involved translation of selected scales into American Sign Language, Signed English, and speech reading; review by an advisory panel and back translator; and collection and analysis of deaf individuals' reactions to translations. RESULTS: Focus groups responded favorably, translation problems were revealed, and solutions were suggested. CONCLUSIONS: The findings support the feasibility of translation of the Quick Diagnostic Interview Schedule-III, Revised, into American Sign Language, Signed English, and speech reading for deaf patients.

Cholesterol crystallization in human gallbladder bile: relation to gallstone number, bile composition, and apolipoprotein E4 isoform.

Patients with multiple cholesterol gallstones are at increased risk of recurrence after nonsurgical therapy, possibly because of fast biliary cholesterol crystallization. Serum apolipoprotein E4 (apo E4) is a risk factor for primary cholesterol gallstone formation as well as recurrence. We examined potential effects of stone number and apolipoprotein E genotype on crystallization and on various crystallization-influencing factors in gallbladder biles of 36 cholesterol stone patients (25 multiple stones: 10 carrying the epsilon4 allele). Biliary cholesterol saturation, bile salt composition or concentrations of total protein, immunoglobulin (Ig)A, IgG, alpha1-acid glycoprotein, haptoglobin, or mucin--all crystallization promoters--did not differ between multiple and solitary stone patients, apparently not explaining different speed of crystallization (crystal observation time 3.5 +/- 0.6 days vs. 12.7 +/- 2.4 days, respectively; P = .0003). In contrast, biliary aminopeptidase-N activities (2,607 +/- 592 mU/mL vs. 947 +/- 185 mU/mL; P = .04) were higher and IgM levels (179 +/- 39 vs. 65 +/- 8 mg/L; P = .09) tended to be higher in the case of multiple stones. Although patients carrying the epsilon4 allele had similar stone numbers and crystallization as patients without the epsilon4 allele, their cholesterol saturation index (CSI) was lower (1.08 +/- 0.09 vs. 1.54 +/- 0.13; P = .01), whereas total protein and bile salt concentrations tended to be higher with preferential taurine-conjugation. In conclusion, fast cholesterol crystallization is associated with multiple stones but not with apolipoprotein E4. Whereas fast crystallization may contribute to high recurrence rates after nonsurgical therapy in case of multiple gallstones, the mechanism for increased risk of gallstone formation in patients carrying the epsilon4 allele remains unknown.

Quantitation of cholesterol-carrying particles in human gallbladder bile.

The inter-mixed micellar/vesicular (non-phospholipid-associated) bile salt concentration (IMC) can be rapidly measured in model biles by centrifugal ultrafiltration, thus allowing reliable separation of vesicular and micellar cholesterol carriers by gel filtration with an elution buffer containing bile salts at the correct IMC (Donovan, J. M., and A. A. Jackson. 1993. J. Lipid Res. 34: 1121-1129). We adapted this method to the more complex human gallbladder bile and examined the relationship between cholesterol solubilization and crystallization in gallbladder biles from 10 cholesterol gallstone patients. The IMC (mean +/- SEM) was 9.67 +/- 1.97 (range 3.56-35.02) mM with significant enrichment with hydrophilic bile salt species. Upon gel filtration of these biles with an eluant buffer containing 10 major bile salts at concentrations according to their IMC, cholesterol was found to be solubilized mainly in mixed micelles. Vesicles were detected in all 10 biles after separation by KBr density gradient ultracentrifugation but in only 5 of these biles with the IMC method. Biles without vesicles had a lower CSI (1.15 +/- 0.12 vs. 1.90 +/- 0.28, P < 0.05), a higher total lipid concentration (11.9 +/- 2.3 vs. 5.9 +/- 1.1, P < 0.05), and a higher bile salt/ (bile salt + phospholipid) ratio (0.83 +/- 0.01 vs. 0.74 +/- 0.04, P = 0.07). For both IMC and ultracentrifugation methods, vesicular cholesterol concentration showed a negative correlation with crystal observation time and a positive correlation with cumulative crystal score during 21 days. Our data indicate that methods such as density gradient ultracentrifugation overestimate vesicular cholesterol solubilization in human biles.

Ursodeoxycholic acid reduces protein levels and nucleation-promoting activity in human gallbladder bile.

BACKGROUND & AIMS: Ursodeoxycholic acid prevents gallstone formation in selected patients. The aim of this study was to examine whether decreased concentration and nucleation-promoting activity of various proteins contribute to this beneficial effect. METHODS: Gallbladder bile of 13 patients with cholesterol gallstones treated with ursodeoxycholic acid (10 mg/kg(-1)/day(-1)) and of 13 untreated patients were compared. RESULTS: Total protein concentration in gallbladder bile (2.8 +/- 0.6 vs. 6.7 +/- 1.3 mg/mL; P=0.008) and concanavalin A-binding fraction (0.16 +/- 0.03 vs. 0.42 +/- 0.07 mg/mL; P=0.003) were strongly decreased by ursodeoxycholic acid therapy. Significant decreases were also found for gallbladder bile alpha1-acid glycoprotein, haptoglobin, immunoglobulin (Ig) A, IgG, gamma-glutamyl transpeptidase, and aminopeptidase N but not for IgM, mucin, or beta-glucuronidase. Decreases were most pronounced for proteins of canalicular membrane origin. Gallbladder bile total protein correlated with cholesterol saturation index (r=0.54; P=0.0047) but not with bile salt hydrophobicity index. Crystallization-promoting activity of the concanavalin A-binding fraction (assessed by nephelometry and microscopic examination) was also significantly decreased by ursodeoxycholic acid. CONCLUSIONS: Ursodeoxycholic acid strongly decreases levels of various proteins and nucleation-promoting activity in bile.

Stimulation of osmoregulating processes in the perfused gill of the crab Pachygrapsus marmoratus (Crustacea, Decapoda) by a sinus gland peptide.

Isolated posterior gills of the hyper-hyporegulating crab Pachygrapsus marmoratus were perfused with extracts of homologous sinus glands. Sinus gland extracts stimulated the influx of Na+ ions and increased the transepithelial potential difference in the gills in a dose-dependent and reversible fashion. The bioactivity of extracts prepared from crabs that had been acclimated to 10/1000 salinity for at least 1 week was not significantly different from that of extracts prepared from seawater (36/1000 salinity) crabs. The perfusion experiments with both extracts containing two sinus glands significantly increased Na+ influx by about 150% and transepithelial potential difference by about 45%. Sinus gland extracts also increased the Na+/K(+)-ATPase activity by 54% in incubated posterior gills. The bioactivity of extracts was reduced by pronase and trypsin, but not by heating for 10 min at 100 degrees. The molecular weight of the responsible factor(s) was > 5000 Da. Thus, the sinus gland of P. marmoratus is concluded to be involved in the neuroendocrine control of osmoregulation and to contain a peptide(s) that directly influences brachial function.

Expression of tilapia prepro-melanin-concentrating hormone mRNA in hypothalamic and neurohypophysial cells.

Melanin-concentrating hormone (MCH) is a neuropeptide involved in background adaptation in teleost fish, and in multiple regulatory functions in mammals and fish. To study the expression of the MCH preprohormone (ppMCH) in teleosts, we first cloned a hypothalamic cDNA encoding the complete ppMCH of tilapia (Oreochromis mossambicus), and a cRNA probe derived from a 270 bp ppMCH cDNA fragment was used for the expression studies. The level of ppMCH mRNA expression in tilapia hypothalamus, measured by dot blot analysis, was significantly higher in fish adapted to a white background than in black-adapted animals, which is in accordance with the reported MCH plasma and tissue concentrations in fish. Northern blot analysis not only revealed a strong ppMCH mRNA signal in the hypothalamus, but also the presence of ppMCH mRNA in the neurointermediate lobe (NIL) of the pituitary. In situ hybridization and immunocytochemistry showed that ppMCH mRNA as well as MCH immunoreactivity are located in perikarya of two hypothalamic regions, namely in the nucleus lateralis tuberis (NLT) and the nucleus recessus lateralis (NRL). Quantitative analysis by dot blot hybridization revealed about eight times more ppMCH mRNA in the NLT than in the NRL and NIL of mature tilapias. ppMCH mRNA in the NIL could be localized to cell bodies of the neurohypophysis, which were also MCH immunoreactive.

Synthetic studies toward pyruvate acetal-containing saccharides: en route to the efficient synthesis of Rhizobium-related exopolysaccharide fragments.

The disaccharide building block benzyl O-(2,3-di-O-benzoyl-4,6-O-[(R)-1-(methoxycarbonyl) ethylidene]-beta-D-galactopyranosyl)-(1-->3)-2-O-benzoyl-4,6-O-[(S)-1- (methoxycarbonyl)ethylidene]-alpha-D-glucopyranoside (13), related to a Rhizobium exopolysaccharide, was prepared by coupling various 4,6-O-[(R)-1-(methoxycarbonyl)ethylidene]-D-galactosyl donors (benzoyl-protected chloride 1, pivaloyl-protected chloride 2, and benzoyl-protected fluorides 3 and 4, and trichloroacetimidate 5) with benzyl 2-O-benzoyl-4,6-O-[(S)-1- (methoxycarbonyl)ethylidene]-alpha-D-glucopyranoside (10) and the corresponding 2,3-O-tetraisopropyldisiloxane-protected glucoside 12. The best results, with respect to beta-selectivity and yield of the coupling, were obtained with 5 and 10 in dichloromethane. The beta-linked (13) and alpha-linked (14) disaccharides were efficiently converted via the 1-OH derivatives 17 and 21 into the corresponding trichloroacetimidates 18 and 22. The latter were used for the synthesis of the disaccharide ligands 4,6-(R)-pyruvate-beta-D-Galp-(1-->3)-4,6-(S)-pyruvate-beta-D-Glcp-O(CH2) 5NH2 (20), and 4,6-(R)-pyruvate-alpha-D-Galp-(1-->3)-4,6-(S)-pyruvate-beta-D-Glcp-O (CH2)5NH2 (24). The corresponding tri- and tetra-saccharide derivatives 4,6-(R)-pyruvate-beta-D-Galp-(1-->3)-4,6-(S)-pyruvate-beta-D-Glcp-(1-->4 )-beta- D-Glcp-O(CH2)5NH2 (28) and 4,6-(R)-pyruvate-beta-D-Galp-(1-->3)-4,6-(S)-pyruvate-beta-D-Glcp-(1-->4 )-beta- D-Glcp-(1-->4)-beta-D-Glcp-O(CH2)25NH2 (36) were obtained similarly.

Synthesis of 5-aminopentyl 4,6-O-[(R)-1-carboxyethylidene]-beta-D-galactopyranoside and its use as a ligand for the affinity chromatography of human serum amyloid P . protein.

A series of 2,3-di-O-benzoyl-D-galactopyranosides, alpha-allyl (5), alpha-benzyl (6), beta-ethyl-1-thio (7), beta-phenyl-1-thio (8), and alpha-methyl (9),were prepared from the corresponding 4,6-O-benzylidene derivatives and were acetalated in acetonitrile with methyl pyruvate, to give diastereoselectively the 2,3-di-O-benzoyl-4,6-O-[(R)-1-methoxycarbonylethylidene]-D- galactopyranosides 10-16. The latter were converted into the 2,3-di-O-benzoyl-4,6-O-[(R)-1-methoxycarbonylethylidene]-D-galacto pyranosyl alpha- and beta-trichloroacetimidates 19 and 20, alpha- and beta-fluorides 21 and 22, the alpha-bromide 23, and the alpha-chloride 24, respectively. These donors, including the phenyl 1-thiogalactoside 14, reacted with 5-[(benzyl-oxycarbonyl)amino]pentanol to give the corresponding protected beta-D-galactoside 27, deblocking of which afforded the title compound 1. Binding of 1 to epoxypropyl-modified acrylamide beads gave an affinity adsorbent that was used to isolate serum amyloid P protein from human serum.