Bile salt transport in intestinal lymph of the rat

The concentrations of conjugated cholate and chenodeoxycholate have been measured in samples of rat thoracic duct lymph, portal and systemic blood. Both these bile salts were present in lymph. After administration of a corn oil meal the flux of both these bile salts in lymph increased significantly (P less than 0.001; Student's t test), the cholate flux rising from 0.9 +/- 0.6 nmol/h (mean +/- SD) to 3.5 +/- 1.1 nmol/h postprandially and the chenodeoxycholate flux rising from 2.5 +/- 0.8 to 4.6 +/- 1.6 nmol/h. This postprandial increase in bile salt flux was due to both an increase in bile salt concentration and an increased lymph flow which rose significantly (P=0.007) from 0.5 +/- 0.2 to 0.7 +/- 0.2 ml/h. Biliary drainage significantly (P less than 0.001) reduced the flux of cholate and chenodeoxycholate in lymph to 0.08 +/- 0.05 and 0.17 +/- 0.10 nmol/h respectively. These biliary drained animals produced no significant rise (P greater than 0.1) in bile salt flux when fed the corn oil meal. The ratio of the concentrations of conjugated cholate to chenodeoxycholate in systemic and portal blood was 1.9:1 and 3.3:1 respectively. In contrast, this bile salt ratio was only 0.44:1 in lymph. These studies show that bile salts, in particular chenodeoxycholate, can pass directly from the intestine into lymphatics thus establishing an enterolymphatic circulation of bile salts.     

Experimental evidence for in vitro fluid transport in the presence of a traditional medicinal fruit extract across rat everted intestinal sacs

The present study was designed to investigate the effects of aqueous fruit extract of Momordica charantia (MC), a traditional medicinal plant, on the transport of fluid in vitro. Everted intestinal sacs from rats were mounted in an organ bath containing Krebs solution. We compared the effect of MC extract on water transport with increasing inorganic phosphate concentration with or without D-glucose in the buffer. In the control experiments, fluid uptake was enhanced significantly (P < 0.05) at high inorganic phosphate concentration (8-10 mM) in the presence of 5.5 mM D-glucose. Addition of 3.0 mg/mL MC extract to the serosal side inhibits the uptake of fluid significantly (P < 0.05). At high inorganic phosphate concentration (8-10 mM), fluid uptake was not inhibited (P > 0.05) when incubated with 3.0 mg/mL MC fruit extract. It is hypothesized that an increase in inorganic phosphate enhances oxidative phosphorylation thereby increasing the fluid uptake across everted intestinal sacs of rat. These findings seem to indicate that the MC-induced reduction on intestinal fluid absorption capacity could be mainly the result of an interference with the carrier-mediated coupled entrance of glucose and Na(+) across the brush-border membrane.     

Normobaric hyperoxia in vivo inhibits fatty acid incorporation into sheep erythrocyte phospholipid in vitro

Oxygen toxicity is a major complication of normobaric hyperoxia in therapeutic settings. Because alterations in membrane function occurring as a consequence of peroxidation of membrane phospholipid fatty acids may be an early event in the pathogenesis of oxygen-induced injury, we studied the effects of hyperoxia on the ability of the membrane to repair itself by incorporating fatty acid via the pathway for deacylation and reacylation in situ. Although the lung is the major site of clinically significant injury, the erythrocyte is also directly exposed to elevated PO2 in vivo. In this study, incorporation of [9,10(-3)H]-oleic acid into phospholipid has been measured in sheep erythrocytes in vitro after exposure of four animals to normobaric hyperoxia in vivo. [9,10(-3)H]-Oleic acid incorporation into erythrocyte phospholipid decreased within 24 hours and reached 50% of pre-exposure levels after 70 hours of exposure to 100% O2. No significant change in the fatty acid composition of membrane phospholipid was detected under these conditions. In contrast to the results with intact cells, incorporation of [9,10(-3)H]-oleic acid into phospholipid by isolated erythrocyte membranes prepared from the cells of two animals increased after 70 hours of exposure to 100% O2, indicating that the inhibition of fatty acid incorporation in intact erythrocytes does not result from irreversible inactivation of the enzymes involved in acylation of endogenous lysophospholipid. Because the ability of cells to replace membrane phospholipid fatty acids via deacylation and reacylation in situ could be important in the maintenance of membrane integrity during oxidative stress, the decrease in fatty acid incorporation by erythrocytes in vitro may reflect an early event in the pathogenesis of oxygen-induced cellular injury.     

Bile acid binding to dietary casein: a study in vitro and in vivo

1. Studies were carried out in vitro using an ultracentrifugation method to quantify bile acid binding to the different components of a Lundh test meal, and to determine what factors influence bile acid binding to one of the components (casein). We validated the ultracentrifugation method by showing good agreement with the equilibrium dialysis method. Studies were carried out in vivo on jejunal aspirate from 10 ileal resection patients in order to determine whether bile acid binding to casein could be demonstrated, and whether this influenced aqueous-phase bile acid and fatty acid concentrations. 2. In vitro, the Lundh test meal was found to adsorb bile acid. The protein content of the meal (casein) alone accounted for this binding, which was abolished by use of casein hydrolysate. The binding to casein was a saturable process. Both binding affinity and binding capacity were significantly greater for taurocholate at pH 4.5 than at pH 6.5, and for dihydroxylated than for trihydroxylated bile acid, suggesting that hydrophobic bonding was involved. 3. In vivo, jejunal samples aspirated at pH greater than 6 from 10 ileal resection patients showed 25% binding of bile acid to protein. On substitution of amino acids for casein, mean binding was reduced to 16% (P less than 0.05), residual binding being attributed to endogenous protein. This was associated with an increase in fatty acid solubilization from 28% to 60% (P less than 0.025).(ABSTRACT TRUNCATED AT 250 WORDS)     

Fatty acid binding protein. Role in esterification of absorbed long chain fatty acid in rat intestine.

Fatty acid binding protein (FABP) is a protein of 12,000 mol wt found in cytosol of intestinal mucosa and other tissues, which exhibits high affinity for long chain fatty acids. It has been suggested that FABP (which may comprise a group of closely related proteins of 12,000 mol wt) participates in cellular fatty acid transport and metabolism. Although earlier findings were consistent with this concept, the present studies were designed to examine its physiological function more directly. Everted jejunal sacs were incubated in mixed fatty acid-monoglyceride-bile acid micelles, in the presence or absence of equimolar concentrations of either of two compounds which inhibit oleate binding to FABP:flavaspidic acid-N-methyl-glucaminate and alpha-bromopalmitate. Oleate uptake, mucosal morphology, and oxidation of [14C]acetate remained unaffected by these agents, but oleate incorporation into triglyceride was inhibited by 62-64% after 4 min. The inhibition by flavaspidic acid was reversible with higher oleate concentrations. The effect of these compounds on enzymes of triglyceride biosynthesis was examined in intestinal microsomes. Neither flavaspidic acid nor alpha-bromopalmitate inhibited acyl CoA:monoglyceride acyl-transferase. Fatty acid:coenzyme A ligase activity was significantly enhanced in the presence of partially purified FABP, probably reflecting a physical effect on the fatty acid substrate or on the formation of the enzyme-substrate complex. Activity of the enzyme in the presence of 0.1 mM oleate was only modestly inhibited by equimolar flavaspidic acid and alpha-bromopalmitate, and this effect was blunted or prevented by FABP. We conclude that in everted gut sacs, inhibition of triglyceride synthesis by flavaspidic acid and alpha-bromopalmitate could not be explained as an effect on fatty acid uptake or on esterifying enzymes in the endoplasmic reticulum but rather can be interpreted as reflecting inhibition of fatty acid binding to FABP. These findings lend further support to the concept that FABP participates in cellular fatty acid transport and metabolism. It is also possible that FABP, by effecting an intracellular compartmentalization of fatty acids and acyl CoA, may play a broader role in cellular lipid metabolism.     

p-Hydroxymercuribenzoic acid inhibits arachidonic acid esterification into two distinct pools and stimulates insulin release in intact rat islets

Activation of an islet phospholipase A2 may contribute to glucose-induced insulin release. In order to simulate the accumulation of the resultant hydrolytic products (arachidonic acid, AA; its lipoxygenase-derived oxygenation product 12-hydroxyeicosatetraenoic acid; and lysophospholipids) without many of the other concomitants of beta cell activation, we studied the effects on intact rat islets of p-hydroxymercuribenzoic acid (PHMB), which inhibits the reacylation of lysophospholipids with AA in other cell types. PHMB inhibited in a dose-responsive fashion (-90% at 500 microM) the incorporation of [3H]AA into a "basal" pool or pools whose release and reuptake mechanisms appeared to be largely energy- and Ca++-independent (resistant to inhibition by mannoheptulose, antimycin A or CoCl2); reciprocally, islets prelabeled with [3H]AA accumulated an increased amount of [3H]-12-hydroxyeicosatetraenoic acid (twice basal at 200 microM PHMB and three times basal at 500 microM) when reacylation of any [3H]AA released basally at 1.7 mM glucose was inhibited. PHMB also blocked (by up to 99% at 500 microM) the incorporation of [3H]AA into a functionally defined, glucose-stimulated compartment of fatty acid (tightly coupled to the islet 12-lipoxygenase) whose release and reuptake required metabolic energy and Ca++. It was also demonstrated that PHMB inhibited the esterification of [3H]AA (at low or high glucose concentrations) into specific phospholipids in islet membranes. In parallel with these alterations in lipid metabolism, PHMB caused rapid, potent and reversible increments in insulin release with a threshold concentration (about 25 microM) identical to that inhibiting AA fluxes. PHMB both initiated release (at 1.7 mM glucose) and potentiated the effects of islet fuels (16.7 mM glucose or 15 mM alpha-ketoisocaproic acid). Thus, pharmacologic manipulation of the AA reuptake mechanism is a new approach to unmask potential roles in insulin release of phospholipid hydrolysis products from different lipid pools and in the absence or presence of phospholipase A2 activation.     

Intestinal surface acid microclimate in vitro and in vivo in the rat

The presence of the intestinal surface acid microclimate (ISAM) has long been suggested. Controversy, however, still exists as to the existence, general characteristics, and requirements of this microclimate. We examined the existence and distribution of the ISAM along the rat small intestine and colon and determined its general characteristics. The results clearly demonstrate the existence of the ISAM in vitro and in vivo. Glucose and sodium in the incubation medium, as well as normal intracellular metabolism, are essential requirements for the formation and maintenance of the ISAM. We conclude that the ISAM is a normal physiologic phenomenon of the luminal surface of the intestine.     

The roles of P-glycoprotein and intracellular metabolism in the intestinal absorption of methadone: in vitro studies using the rat everted intestinal sac

Methadone is used as a treatment for opiate detoxification in methadone maintenance programs. Intra- and inter-patient variations in methadone bioavailability have been observed after oral methadone treatment and this makes it difficult to predict a dosing regimen. Intestinal absorption and metabolism could explain these variations. The in vitro gut sac model was used to study the intestinal absorption of methadone, and it confirmed that methadone is a substrate for P-glycoprotein. The transport of methadone was increased in presence of P-gp inhibitors verapamil and quinidine. The appearance of a major metabolite of methadone, 2-ethylidene-1, 5-dimethyl-3, 3-diphenyl pyrrolidine (EDDP) in the gut sac contents also demonstrated the existence of intestinal metabolism of methadone.     

Identification, isolation, and partial characterization of a fatty acid binding protein from rat jejunal microvillous membranes.

The mechanisms by which FFA are absorbed by the gut are unclear. To examine these processes, binding of [14C]oleate to isolated rat jejunal microvillous membranes (MVM) was studied in vitro. When [14C]oleate alone or compounded with bovine serum albumin at various molar ratios was incubated with MVM aliquots, binding was time- and temperature-dependent, inhibitable by addition of excess cold oleate, and decreased by heat denaturation or trypsin digestion of the membranes. When [14C]oleate binding to heat denatured MVM, which increased continuously as a function of the free oleate concentration and was taken as a measure of nonspecific binding, was subtracted from total binding to native MVM, a curve suggestive of saturable specific binding was observed. In contrast to fatty acids, there was no specific binding of [14C]taurocholate or [35S]sulfobromophthalein to jejunal MVM. After MVM solubilization with 1% Triton X-100, affinity chromatography over oleate-agarose and elution with 7 M urea yielded a single 40,000-mol-wt protein. This Sudan Black/periodic acid-Schiff-stain-negative protein co-chromatographed on Sephadex G-100 with [14C]oleate, [14C]palmitate, [14C]arachidonate, and [14C]linoleate, but not with the [14C]oleate ester of cholesterol, [14C]phosphatidylcholine, [14C]taurocholate, or [35S]sulfobromophthalein. A rabbit antibody to the previously reported hepatic membrane fatty acid binding protein (FABP) gave a single line of immunologic identity between the FABPs of rat jejunum and rat liver membrane. It inhibited the binding of [14C]oleate to native MVM but not heat denatured MVM, and, in immunohistochemical studies, demonstrated the presence of the FABP in the apical and lateral portions of the brush border cells of the jejunum, but not on the luminal surface of esophagus or colon. These data are compatible with the hypothesis that a specific FABP plays a role in fatty acid absorption from the gut.     

Cytotoxic effects of polymorphonuclear leukocytes and macrophages in patients undergoing lymph depletion via thoracic duct drainage

It has been previously reported that both human peripheral blood monocyte derived macrophages and polymorphonuclear leukocytes acquire enhanced cytotoxicity for tumor cells. Lymphocyte depletion by thoracic duct cannulation prior to renal transplantation has been shown to suppress allograft rejection. However, the effects of thoracic duct drainage on macrophage and polymorphonuclear leukocyte function is not known. When the macrophages obtained from thoracic duct drainage patients were studied prior to cannulation, four of the five patients possessed cytotoxic macrophages. At 1 to 2 weeks post thoracic duct drainage, cytotoxicity was significantly depressed whereas by 3 weeks post thoracic duct cannulation most of the patients' macrophages exhibited maximal cytotoxicity. Approximately 3 1/2 weeks after cannulation these five patients received cadaveric renal transplants. The cytotoxic effects of the macrophages were tested again after transplantation and it was found that the macrophages became incapable of killing the tumor targets. In contrast to our findings with macrophage mediated cytotoxicity, the polymorphonuclear leukocytes generally retained their cytotoxic capabilities at all time points tested. However, it was noted that cytotoxic activity reached maximal levels within the first 3 weeks after cannulation but fell to low cytotoxic levels at approximately 4 to 5 weeks after cannulation. When tested several months post cannulation and transplantation, polymorphonuclear leukocyte mediated cytotoxicity increased dramatically in four of the five patients studied.     

Bile acid and phospholipid fatty acid composition in bile of patients with cholesterol and pigment gallstones.

It has been previously reported that patients with cholesterol gallstones have increased biliary deoxycholate and arachidonate content as compared with normal subjects without gallstones. Increased biliary deoxycholate and arachidonate content might be a primary factor in the pathogenesis of cholesterol gallstones or merely an epiphenomenon due to the presence of gallstones. We therefore compared biliary bile acid composition in 46 patients with cholesterol gallstones and 22 patients with pigment stones. In addition, biliary phospholipid fatty acid composition was determined in 44 of these patients (30 cholesterol and 14 pigment stone patients). No significant differences were detected. In particular, the percentage deoxycholic acid (mean +/- SD: 20.3 +/- 8.8% and 21.5 +/- 10.9% respectively) and the percentage arachidonic acid (4.4 +/- 2.0% and 4.5 +/- 2.2%, respectively) were very similar. A significant correlation between age and biliary cholesterol saturation index was found only for the group of patients with pigment stones (R = 0.52, p less than 0.02). In conclusion, the present study does not support a primary role for increased biliary deoxycholic acid or arachidonic acid in the pathogenesis of cholesterol gallstones.     

Colonic absorption of insulin: an in vitro and in vivo evaluation

Rectal administration of insulin as a suppository or p.o. administration of a proteolysis-resistant insulin analog lowers serum glucose levels, suggesting uptake and/or diffusion of intact insulin across the epithelium of the large intestine; however, no rigorous studies of insulin degradation and/or transepithelial flux in vitro have been reported with isolated colonic epithelium. Everted and noneverted sacs of rat distal colonic epithelium were prepared and incubated at 37 degrees C with porcine [125I]insulin and less than 1% of the [125I]insulin crossed the epithelial barrier under these in vitro conditions. The apical surface degraded [125I]insulin at a rate of 0.071 fmol/mg of dry wt./min, whereas the submucosal surface degraded insulin at a rate of 0.045 fmol/mg of dry wt./min. Over 60% of the available [125I]insulin was degraded by the apical surface of the distal colon during a 15-min incubation, whereas approximately 40% of the radioligand was degraded by the submucosal surface under identical conditions. Degradation of [125I]insulin was inhibited partially by the addition of excess unlabeled insulin and inhibited completely by the addition of bacitracin (1 mg/ml). These results indicate that the mammalian colonic epithelium is an effective barrier to transepithelial flux of insulin and identify insulin-degrading activity on both the apical and submucosal surfaces of the colonic epithelium. Although the colonic epithelium represents a significant physiological barrier to the uptake of insulin from the intestinal lumen, paradoxically, administration of large doses of unlabeled insulin into the distal colon or into the entire large intestine caused a dose-dependent increase in serum insulin levels during a 15-min experiment in vivo.     

Bile acid N-acetylglucosaminidation. In vivo and in vitro evidence for a selective conjugation reaction of 7 beta-hydroxylated bile acids in humans.

The aim of this study was to define whether N-acetylglucosaminidation is a selective conjugation pathway of structurally related bile acids in humans. The following bile acids released enzymatically from N-acetylglucosaminides were identified: 3 alpha,7 beta-dihydroxy-5 beta-cholanoic (ursodeoxycholic), 3 beta, 7 beta-dihydroxy-5 beta-cholanoic (isoursodeoxycholic), 3 beta,7 beta-dihydroxy-5 alpha-cholanoic (alloisoursodeoxycholic), 3 beta,7 beta-dihydroxy-5-cholenoic, 3 alpha,7 beta,12 alpha-trihydroxy-5 beta-cholanoic, and 3 alpha,6 alpha,7 beta-trihydroxy-5 beta-cholanoic acids. The selectivity of conjugation was studied by administration of 0.5 g ursodeoxycholic (UDCA) or hyodeoxycholic (HDCA) acids, labeled with 13C, to patients with extrahepatic cholestasis, and of 0.5 g of 13C-labeled chenodeoxycholic acid (CDCA) to patients with extra- or intrahepatic cholestasis. After administration of [24-13C]-CDCA, labeled glucosides, and the glucuronide of CDCA were excreted in similar amounts. Labeled N-acetylglucosaminides of UDCA and isoUDCA were also formed. When [24-13C]-UDCA was given, 13C-label was detected in the N-acetylglucosaminide, the glucosides, and the glucuronide of UDCA, and in the N-acetylglucosaminide of isoUDCA. In the patient studied, 32% of the total UDCA excreted in urine was conjugated with N-acetylglucosamine. In contrast, 96% of the excreted amount of [24-13C]HDCA was glucuronidated, and 13C-labeled glucosides but no N-acetylglucosaminide were detected. The selectivity of N-acetylglucosaminidation towards bile acids containing a 7 beta-hydroxyl group was confirmed in vitro using human liver and kidney microsomes and uridine diphosphate glucose (UDP)-N-acetylglucosamine. These studies show that N-acetylglucosaminidation is a selective conjugation pathway for 7 beta-hydroxylated bile acids.     

The class of surface immunoglobulin on cells carrying IgG memory in rat thoracic duct lymph: the size of the subpopulation mediating IgG memory.

The fluorescence activated cell sorter was used to determine the class of immunoglobulin on the thoracic duct lymphocytes (TDL) which carried IgG memory. Although only about 3% of all TDL carried membrane IgG these cells accounted for most, if not all, of the adoptive IgG anti-DNP response. It is concluded that both CR+ and CR- cells mediating IgG memory in rat TDL bear the same class of membrane immunoglobulin as that secreted by their differentiated progeny. The class of membrane immunoglobulin on CR+ and CR- rat TDL was also examined. It was found that IgM+ cells, which made up over 80% of all Ig+ cells, were virtually all CR+. In contrast, the few percent of IgG+ and IgA+ cells present were to be found in both subpopulations. There was no evidence of a large population of B cells bearing exclusively heavy chains other than IgA, IgG, of IgM. The observation that some IgG+ cells as well as IgM+ cells possess a receptor for C3 appears to rule out the hypothesis that this receptor is involved in blocking a switch from IgM to IgG synthesis.