An improved purification procedure for conjugated bile acids using octadecyl-bonded silica cartridges.

A rapid procedure for the extraction of conjugated bile acids from human fluids using pre-packed octadecyl-bonded silica cartridges is described. The method was compared with the other procedures reported in the literature and was found to produce a higher degree of sample purification and to achieve satisfactory accuracy and reproducibility. The present procedure is applicable to the high-performance liquid chromatographic assay of bile acid conjugates in human bile, gastric juice, serum and urine.

Use of benzyl 2-acetamido-2-deoxy-3-O-(2-O-methyl-beta-D-galactosyl)-beta- D-glucopyranoside [2'-O-methyllacto-N-biose I beta Bn] as a specific acceptor for GDP-fucose: N-acetylglucosaminide alpha(1----4)-L-fucosyltransferase.

A synthetic substrate, benzyl 2-acetamido-2-deoxy-3-O-(2-O-methyl-beta-D- galactopyranosyl)-beta-D-glucopyranoside, was demonstrated to be a specific acceptor for the Lewis blood group-specified alpha(1----4)-L-fucosyltransferase from human saliva and stomach mucosa. The fucosyl linkage of the product resulting from the use of this substrate isolated by paper chromatography was characterized by hydrolysis with specific alpha(1----3)/(1----4)-L- fucosidase. The product can be separated by adsorption onto the reverse-phase cartridge and recovered by one-step elution with methanol. The enzymatic properties of alpha(1----4)-L-fucosyltransferase from saliva and stomach mucosa have also been examined using this substrate.

Biological activities of glycosylated and nonglycosylated porcine prolactin.

Nonglycosylated and glycosylated porcine prolactin (PRL) were separated using concanavalin A-Sepharose CL-6B column chromatography and tested for mitogenic and lactogenic activities, as well as immunoaffinity and receptor binding characteristics compared to total (nonseparated) porcine PRL. Mitogenic activity, using Nb2 lymphoma cells, was 4- and 50-fold greater (P less than 0.01) for total PRL than nonglycosylated and glycosylated PRL, respectively. Glycosylated PRL had 64% higher (P less than 0.05) lactogenic activity than nonglycosylated or total PRL. In a homologous radioimmunoassay (RIA), displacement was greatest for total, followed by the nonglycosylated and glycosylated forms of PRL. Competitive inhibition of porcine [125I]-(total) PRL by radioinert total, nonglycosylated and glycosylated PRL in a homologous radioreceptor assay (RRA) indicated similar Ka values for total and nonglycosylated PRL, but different receptor numbers, while radioinert glycosylated PRL had a higher Ka, but bound fewer receptors. Therefore, glycosylated porcine PRL has greater lactogenic activity and higher binding affinity despite decreased mitogenicity, while nonglycosylated PRL had characteristics similar to total PRL. Results from the homologous RRA and the Nb2 assay suggest that both forms of PRL are necessary to achieve biological effects similar to those for total PRL. The two forms of PRL may have individual and collective effects, while changes in the ratio between these forms may influence physiologically diverse effects of PRL on target tissues.

[Studies on trace elements in cancerous stomach tissue of the patients with stomach cancer].

This study was performed to find out whether copper, zinc, manganese, selenium and iron concentrations in the cancerous and normal stomach tissues of the patients with stomach cancer vary within the malignant stages and Borrmann classification or not, and to investigate the interaction of copper, zinc, manganese, selenium and iron concentrations in blood of these patients. Copper concentration in cancerous tissues was not statistically significant as compared with normal tissues. Plasma and whole blood copper concentration of Stage IV showed a significant higher level than that of stage I. Zinc concentration in cancerous tissues was not statistically significant as compared with normal tissues. Selenium concentration in cancerous tissues showed a statistically significant high level as compared with that in normal tissues. Plasma selenium concentration of Stage III showed a significant lower level than that of stage I. Iron concentration in cancerous tissues showed a significantly lower level than that in normal tissues at stage IV. Whole blood iron concentration was low levels in proportion to the progress of stomach cancer. The correlation of selenium concentration between in cancerous tissues and in whole blood of these patients was significant with the correlation coefficient of 0.340. The correlation of iron concentration between in cancerous tissues and in whole blood of these patients was significant with the correlation coefficient of 0.423. The correlation between iron concentration in cancerous tissues and hemoglobin concentration in whole blood of these patients was significant with the correlation coefficient of 0.361.

Canine gastric muscarinic receptors up-regulate after vagotomy.

Postvagotomy (PV) gastroparesis is an infrequent but troublesome problem. To test the hypothesis that the rarity of the PV syndrome is due to compensatory up-regulation of muscarinic cholinergic receptors (mAChR), we measured changes in stomach mAChR and gastric acid secretion in dogs before and three weeks after truncal vagotomy. Maximum acid output dropped significantly one week PV and then partially recovered by three weeks PV. mAChR density changed in parallel and was significantly increased in body mucosa, body muscle, and antrum mucosa. In the body, changes in mAChR in mucosa correlated positively with changes in muscle, suggesting that mAChR binding in pinch biopsies of gastric mucosa might become useful in evaluating patients for postvagotomy syndrome. PV up-regulation of mAChR in the mucosa of the canine gastric body might explain PV recovery of gastric acid secretion.

Galanin binding sites in rat gastric and jejunal smooth muscle membrane preparations.

Receptors for galanin in membranes from the rat gastric and jejunal smooth muscle were studied using [125I] radioiodinated synthetic porcine galanin. Specific binding was time and temperature dependent. At 32 degrees C radioligand was degraded in the presence of smooth muscle membranes in a time-dependent manner. At optimal experimental conditions, the equilibrium binding analyses showed the presence of a single population of high affinity binding sites in both the rat stomach and jejunum (Kd value of 2.77 +/- 0.78 nM and 4.93 +/- 1.74 nM for stomach and jejunal smooth muscle membranes, respectively). The concentration of the high affinity binding sites was 58.19 +/- 11.04 and 32.36 +/- 5.68 fmol/mg protein, for gastric and jejunal preparations, respectively. Specific binding was completely inhibited by 10(-6) M of nonradioactive galanin; was 75% blocked by 1 microM of galanin(9-29); it was 10% blocked by 1 microM of galanin(15-29). Galanin(1-15) at a concentration of 1 microM was ineffective for inhibiting [125I]galanin binding. Deletion of four C-terminal amino acid residues from galanin(9-29) to give galanin(9-25) also resulted in almost complete loss of affinity. Radioiodinated galanin and N-terminally deleted fragments had receptor binding potency in the following order: galanin(1-29) greater than galanin(9-29) greater than galanin(15-29). We conclude that the C-terminal part of the galanin chain is important for the rat gastric and jejunal smooth muscle membrane receptor recognition and binding and that N-terminal amino acid sequences are probably not so important, since galanin(1-15) was not active but galanin(9-29) retained most of the receptor binding activity.

[The characteristics of the accumulation and elimination of organophosphorus compounds in rats]. / Osobennosti nakopleniia i vyvedeniia fosfororganicheskikh soedinenii u krys.

Peculiarities of accumulation and excretion of different phosphor-organic pesticides from stomach wall at peroral introduction in LD50 dose. The data on dependence of pesticide LD50 on the rate of accumulation is stomach wall, the time of their biological lifetime, the direction of metabolism, and content of intact pesticides in liver tissues have been presented.

Sex hormone receptors in gastric cancer.

Gastric adenocarcinoma that originates from mucosal tissue invades submucosa, muscle, and serosa in different stages. The level of progesterone receptors (PgR), estrogen receptors (ER), and androgen receptors (AdR) in the superficial part of gastric cancer tissues (CAs) from 16 patients was determined and compared with that of the corresponding normal gastric mucosal tissues (NLm). There were PgR in all CAs (100%) with values that ranged from 20.5 to 548.4 fmol/mg protein. Eight CAs (50%) had ER values that ranged from 6.8 to 325.1 fmol/mg protein. AdR was found in two CAs with values of 14.7 and 16.4 fmol/mg protein. In NLm, 15 (93.8%) had PgR values that ranged from 7.3 to 473.2 fmol/mg protein and ten (62.5%) had ER values that ranged from 0.9 to 87.9 fmol/mg protein. AdR were present in two NLm with values of 1.5 and 73.5 fmol/mg protein. There was no statistical difference in levels of PgR and ER between CAs and NLm. There were PgR in all gastric cancers and in 93.8% of NLm. The results suggest that gastric mucosa may be the target tissues for progesterone action. Furthermore, the lack of correlation between the levels of ER and PgR in gastric cancer tissue suggests that the PgR in gastric cancers are probably estrogen independent.

Distribution of calcitonin gene-related peptide and calcitonin-like immunoreactivity in trout.

Radioimmunoassay and chromatography were used to study the occurrence of calcitonin gene-related peptide in various tissues of the rainbow trout, Salmo gairdnerii. The highest concentrations of the peptide were found in gill (1.68 +/- 0.09 ng/mg protein) and in intestine (1.06 +/- 0.4 ng/mg protein). Significant concentrations were also found in heart and stomach. The level in brain was very low. In trout, the plasma concentration accounted for 283 +/- 82 pg/ml. Chromatographic analysis of the calcitonin gene-related peptide (CGRP)-like immunoreactivity occurring in gills showed that two molecular forms cross-reacted with the anti-human CGRP antibody, one co-eluting with the synthetic human CGRP. In addition, calcitonin in fish is not confined to the ultimobranchial organ but is also present in organs as heart, intestine, kidney, spleen and stomach. The evidence of CGRP in fish emphasizes the role of this hormone in evolution and leads us to investigate its physiological role in this species.

Physical-chemical behavior of dietary and biliary lipids during intestinal digestion and absorption. 2. Phase analysis and aggregation states of luminal lipids during duodenal fat digestion in healthy adult human beings.

Following the feeding of a triacylglycerol-rich meal to healthy adult human beings, duodenal contents were aspirated for ex vivo chemical and physical-chemical analyses. The aspirates were collected during established lipid digestion and absorption into a "cocktail" of chemical inhibitors that rapidly inhibited ex vivo lipolysis. Following ultracentrifugation, the lipids separated into a floating oil layer, several interfacial layers, a "clear" or turbid "subphase", and a precipitated "pellet". By chemical and phase analyses, the floating layer was composed of oil-in-water emulsion particles with cores of triacylglycerol (TG), diacylglycerols (DG), and cholesteryl esters (CE) emulsified with a surface coat of partially ionized fatty acids (FA), monoacylglycerols (MG), diacylphosphatidylcholine (PL), and bile salts (BS). The interfacial layers contained similar emulsion particles dispersed among excess emulsifier which adopted a lamellar liquid-crystalline structure. Precipitated pellets were composed principally of emulsifying lipids, with smaller amounts of crystalline calcium soaps and BS. Relative lipid compositions of all but three subphases fell within a two-phase region of the condensed ternary phase diagram (Staggers et al., 1990, companion paper) where saturated mixed micelles composed of BS, FA "acid-soaps", MG, PL, cholesterol (Ch), and traces of DG (and TG) coexisted with unilamellar liquid-crystalline vesicles composed of the same lipids. Attempts to achieve clean separation of vesicles from micelles by repeat ultracentrifugation failed. Compared with the structure and sizes of lipid particles in equilibrated model systems (Staggers et al., 1990), quasielastic light scattering (QLS) analysis revealed that ex vivo micellar sizes (mean hydrodynamic radii, Rh) were similar (less than or equal to 40 A), whereas unilamellar vesicle sizes (Rh = 200-600 A) were appreciably smaller. Two-component QLS analysis of the subphases showed that much larger proportions of lipids were solubilized by micelles than were dispersed as unilamellar vesicles. When followed as functions of time, vesicles frequently dissolved spontaneously into mixed micelles, indicating that, in the nonequilibrium in vivo conditions, the constituent micellar phase was often unsaturated with lipids. These results are consistent with the hypothesis that, during hydrolysis of emulsified DG and TG by luminal lipases, unilamellar vesicles originate in lamellar liquid crystals that form at emulsion-water interfaces in the upper small intestine. In a BS-replete environment, unilamellar vesicles probably represent the primary dispersed product phase of human fat digestion and facilitate the dissolution of lipolytic products into unsaturated mixed micelles.(ABSTRACT TRUNCATED AT 400 WORDS)

cDNA cloning and tissue distribution of mRNAs for two proteins that are related to the band 3 Cl-/HCO3- exchanger.

Complementary DNAs encoding two proteins that are related to the Band 3 Cl-/HCO3- exchanger, designated B3RP2 and B3RP3, have been isolated from rat stomach, brain, and kidney libraries. B3RP2 is 1234 amino acids in length and has an Mr of 136,644. B3RP3 is 1227 amino acids in length and has an Mr of 135,405. B3RP2 and B3RP3 exhibit 52 and 50% amino acid identity to Band 3, respectively, and 56% identity to each other. The N-terminal cytoplasmic regions of B3RP2 and B3RP3, which span about 700 amino acids, are more extensive than that of Band 3. The C-terminal hydrophobic regions of the three proteins exhibit a high degree of amino acid identity (64-69%) and have very similar hydropathy profiles, suggesting that they have the same transmembrane organization. The tissue distribution of mRNAs encoding B3RP2, B3RP3, and the Band 3 Cl-/HCO3- exchanger were examined by Northern blot hybridization using poly(A)+ RNAs from a broad range of muscle and non-muscle tissues and from sections of the gastrointestinal tract. B3RP2 mRNAs are expressed in all tissues examined. The highest levels, which include at least three different transcripts, occur in stomach. B3RP3 mRNAs, which also consist of several different transcripts, have a more limited tissue distribution. The highest levels occur in heart, and in gastrointestinal sections the highest levels are in the forestomach. Band 3 mRNAs were observed in many tissues but high levels of expression occurred only in spleen and kidney. Five Band 3 transcripts, ranging in size from 3.6 to 4.9 kilobases, were detected, including three that are expressed in heart.

Effect of cold-restraint stress on glutathione and lipid peroxide levels in the liver and glandular stomach of rats.

The effect of starvation and cold-restraint stress on glutathione and lipid peroxide levels in the liver, stomach and plasma of rats was investigated. Hepatic and gastric glutathione levels were significantly decreased in starvation and cold-restraint groups when compared with values obtained from the control group. In both tissues, lipid peroxide levels were significantly decreased after starvation but were not significantly different from control values after cold-restraint treatment. However, when compared with the values obtained from the starvation group there was a significant increase in both hepatic and gastric lipid peroxide levels after cold-restraint. Plasma lipid peroxide levels were slightly decreased in the starvation group and significantly increased in the cold-restraint group. Our results suggest that pathological consequences of stress on different tissues could be due to stimulation of lipid peroxidation.

The histopathological and biochemical response of the stomach of male F344/N rats following two weeks of oral dosing with ethyl acrylate.

Male F344/N rats were dosed with ethyl acrylate (EA) either by daily gavage or in the drinking water for 2 weeks. The gavage dose levels were 0, 2, 10, 20, 50, 100, and 200 mg/kg; the drinking water dose concentrations were 0, 200, 1,000, 2,000, and 4,000 ppm (corresponding to 0, 23, 99, 197, and 369 mg/kg/day, respectively). In those animals dosed by gavage, irritation of the forestomach increased in incidence and severity over the 20-200 mg/kg dose range. In those animals dosed with EA in the drinking water, a much lower incidence of forestomach irritation and less severe lesions were observed at corresponding dose levels. No lesions were observed in the glandular stomach from either of the 2 modes of oral administration. Following 2 weeks of gavage dosing with EA, the total non-protein sulfhydryl (NPSH) content of the forestomach and glandular stomach, and the NPSH concentration of the liver were determined 2-24 hr after the last gavage dose. Animals dosed at 200 mg/kg reached approximately 11% of the initial NPSH content in the forestomach at 6 hr after dosing. NPSH depletion of this magnitude has been associated with cytotoxicity of other tissues in other studies. By contrast, either the glandular stomach nor liver were depleted of NPSH to levels generally associated with toxicity. These observations are consistent with the conclusion that bolus dosing of EA induces severe depletion of critical cellular thiols in the forestomach with toxic consequences, but not in the glandular stomach or liver. Changing the mode of oral administration for EA to continued small doses in the drinking water allowed efficient detoxification and did not induce sulfhydryl depletion or comparable forestomach toxicity at the same daily body burden.

Expression of ras oncogene p21 protein in early gastric carcinoma and adjacent gastric epithelia.

The expression of the ras gene product p21 in normal gastric mucosa, early gastric carcinoma of diffuse (gastric) and intestinal types, and in adjacent mucosal abnormalities is reported. The analysis was performed on paraffin sections by an immunohistochemical assay using the mouse monoclonal antibody RAP-5 and the rat monoclonal antibody Y13-259. Expression of ras p21 was assessed by staining intensity and percentage of positively stained cells. In comparison to normal gastric mucosa of non-cancer patients, p21 was overexpressed in nearly all early carcinomas of both types and in the dysplastic and/or metaplastic mucosal alterations accompanying intestinal type of gastric cancer. Increased p21 expression was also observed in the normal-appearing mucosa adjacent to early carcinomas of diffuse type, but not in the morphologically normal gastric epithelium adjacent to the intestinal type. The results of this investigation suggest that ras p21 overexpression may be related to early events of human gastric carcinogenesis. The study supports the notion of different pathways in the development of diffuse (gastric) and intestinal types of gastric carcinomas.

Fluoride absorption from the gastrointestinal tract of rats.

The contribution of the stomach and the small intestine to absorption of fluoride from the gastrointestinal tract was examined in rats. Fasted adult male rats weighing approximately 350 g were given 50 micrograms of fluoride in 1 ml of water by stomach intubation, with 14C-labeled polyethylene glycol as a marker of water movement through the gastrointestinal tract. Rats were killed at intervals up to 120 min, and the stomach, duodenum, jejunum, ileum, distal ileum and cecum were rapidly clamped and removed for fluoride analysis and 14C counting. Approximately 90% of the fluoride dose was absorbed in 120 min. Peak plasma fluoride concentration occurred 10 min after intubation and began to decline after 40 min as the rate of fluoride absorption slowed. Absorption from the stomach was derived from rates of gastric emptying and remaining fluoride. Even at 10 min after intubation, when the bulk of the fluoride remained in the stomach, only approximately 25% of fluoride absorption had occurred from the stomach and 75% from the small intestine. After 120 min, 19.8% of total fluoride absorption had occurred from the stomach. Although the stomach is unquestionably a significant site for fluoride absorption, its contribution is much smaller than that of the small intestine.

The antiulcer effect of verapamil in relation to gastric calcium levels in stressed rats.

The antiulcer effect of verapamil, and its relationship to stomach calcium levels, were examined in rats restrained at 4 degrees C (stress). Stress for 2 hr significantly increased muscle calcium and induced mucosal ulceration in the gastric glandular segment; calcium concentrations in the glandular mucosa and serum were unaffected. Verapamil or calcium gluconate given 30 min before stress prevented the rise in gastric muscle calcium, and attenuated ulcer severity. Bis(beta-aminoethylether)-NNN'N'-tetra-acetic acid (EGTA) pretreatment, however, further elevated stomach muscle calcium and markedly worsened lesion formation. These findings suggest that increased stomach muscle calcium could be a causal factor in stress-induced gastric glandular ulceration.

Gastric and pancreatic intrinsic factor-mediated absorption of cobalamin in the dog.

The effects of canine gastric and pancreatic intrinsic factors on uptake and subcellular localization of cobalamin have been investigated in vivo to determine whether these proteins could mediate the physiological absorption of cobalamin in the dog. Cyano [57Co]cobalamin was introduced into ileal loops in dogs under general anesthesia, either free (control) or bound to gastric or pancreatic intrinsic factor. At 2 h, total uptake of cobalamin by ileal mucosa was significantly enhanced after prior binding to either gastric or pancreatic intrinsic factor compared with controls. Displacement of receptor-bound cobalamin with EDTA showed that enhanced total uptake reflected increased internalization of cobalamin by both proteins. Findings after reorienting sucrose density gradient centrifugation of ileal mucosa from loops containing intrinsic factor-cobalamin complexes were consistent with a major lysosomal and perhaps endosomal localization of internalized cobalamin, in agreement with results after oral administration of cobalamin. In marked contrast, cobalamin was recovered predominantly in the soluble fractions and was not associated with particulate subcellular organelles in ileal mucosa from control loops. These findings suggest that both gastric and pancreatic intrinsic factors can promote the physiological absorption of cobalamin by receptor-mediated endocytosis in the dog.

The kinetics of papillotoxic doses of 3H-N-phenylanthranilic acid in rats.

N-phenylanthranilic acid (N-PAA; 4 mmol/kg/day p.o.) causes a diffuse renal papillary necrosis and a polyuria in 7 days. A single dose of 3H-N-PAA was widely distributed with second-order elimination kinetics, t1/2 +/- 50 h for stomach, heart, kidney, and bladder and t1/2 greater than or equal to 90 h for liver, spleen, muscle and lung. The estimated plasma t1/2 = 10.2 h, and over 75% was excreted via urine in 36 h and 13% via faeces in 72 h. In chronically cannulated animals 29% of N-PAA-derived material was in bile and 24% in urine at 36 h, which suggests enterohepatic circulation. Bile and urine contained several metabolites but no parent compound. Multiple doses for 8 and 16 days increased urinary N-PAA excretion to 90% in 36 h, but faecal contents decreased to 6-8% in 72 h and plasma t1/2 to less than or equal to 7.5 h.