Expression of Raf kinase inhibitor protein in human hepatoma tissues by two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight methods.

PURPOSE: Hepatocellular carcinoma (HCC) is the most common malignant liver tumor. To reduce the mortality and improve the effectiveness of therapy, it is important to search for changes in tumor-specific biomarkers whose function may involve in disease progression and which may be useful as potential therapeutic targets. Materials and Mehtods: In this study, we use two-dimensional polyacrylamide gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry to observe proteome alterations of 12 tissue pairs isolated from HCC patients: Normal and tumorous tissue. Comparing the tissue types with each other, 40 protein spots corresponding to fifteen differentially expressed between normal and cancer part of HCC patients. RESULTS: Raf kinase inhibitor protein (RKIP), an inhibitor of Raf-mediated activation of mitogen-activated protein kinase/extracellular signal-regulated kinase, may play an important role in cancer metastasis and cell proliferation and migration of human hepatoma cells. RKIP may be considered as a marker for HCC, because its expression level changes considerably in HCC compared with normal tissue. In addition, we used the methods of Western blotting and real time-polymerase chain reaction to analysis the protein expression and gene expression of RKIP. The result showed RKIP protein and gene expression in tumor part liver tissues of HCC patient is lower than peritumorous non-neoplastic liver tissue of the corresponding HCC samples. CONCLUSION: These results strongly suggest that RKIP may be considered to be a marker for HCC and RKIP are down-regulated in liver cancer cell.

Intestinal transport of weak electrolytes. Evidence in favor of a three-compartment system.

A study has been made of the transmural fluxes of benzoic, phenylacetic, and pentanoic acids, benzylamine, hexylamine, and D-amphetamine across rat jejunum incubated in vitro. The M to S fluxes of the weak acids were greater than their corresponding S to M fluxes, and the S to M fluxes of the weak bases were larger than their M to S fluxes. These patterns of asymmetric movements were observed when the transmural electrical potential difference was clamped at 0 mV, and when the pH values of the mucosal and serosal fluids were identical. The effects of a weak acid on the fluxes of other weak electrolytes were qualitatively similar when the effector weak acid was added to the mucosal fluid, and when it was added to the serosal fluid. But the effects of a weak base on the fluxes of other weak electrolytes were dependent upon its location, and the interactions observed when the effector weak base was added to the mucosal fluid were qualitatively different than those seen when it was added to the serosal fluid. The interactions between weak electrolytes could readily be explained in terms of the function of a system of three compartments in series, in which the pH of the intermediate compartment is greater than that of the bulk phases. But these observations could not be explained in terms of an analogous system involving an intermediate compartment of low pH, or in terms of a carrier mediated system. The transport function of the three-compartment system can be described in the form of an equation, and it is found that a pH difference of less than 0.5 unit may explain our observations on weak electrolyte transport.

Intestinal fatty acid esterification activity in jejunoileal bypass patients.

In four patients undergoing reversal of jejunoileal bypass we compared functional (in continuity) with bypassed intestine in order to determined the effects of luminal contents. Total mucosal thickness, villus height, and crypt depth, as well as in vitro fatty acid esterification activity were determined. Morphological studies in segments exposed to luminal contents revealed that the ileum had a greater mucosal thickness than the jejunum (p less than 0.001) and that the difference was reflected in both villus height and crypt depth (p less than 0.001). The functioning segments of both jejunum and ileum had a greater mucosal thickness than corresponding bypassed segments consequent to a difference in villus height (p less than 0.001) but not crypt depth. Despite similar exposure to luminal contents, total fatty acid esterification was significantly higher (p less than 0.001) in the functioning jejunum than in the ileum. Jejunum incontinuity possessed higher esterification activity than bypassed jejunum. These results indicate that 1) luminal contents are the most important modulator of intestinal fatty acid esterification activity and the absence of luminal contents in bypassed intestine leads to a significant reduction in esterification activity, 2) the jejunum and ileum possess intrinsic differences in esterification activity even when both are exposed to an identical luminal environment.

Bile salt binding properties of commonly used gastrointestinal drugs: maalox, carafate, and questran.

We have validated a method to measure bile salt binding by Maalox (aluminum hydroxide and magnesium hydroxide), Carafate (sucralfate), and Questran (cholestyramine) in vitro. The method used in this study involves a correction for adherent water volume and thus provides a correct measure of bile salt binding. With this approach, we described the binding properties of Maalox, Carafate, and Questran. The bile salt binding capacities of Carafate and Maalox are limited and do not have physiological or pharmacological significance. On the other hand, we found that Questran has substantial bile salt binding capacity. At the recommended dosage, Questran could deplete the total bile salt pool. We also found that Carafate, although not used as an antacid, has buffering capacity (maintaining a pH of solution in the range 4.2-4.8) which might contribute to its effectiveness as an ulcer treatment drug.

Mechanisms of intestinal fat absorption.

Even though most of the major controversies regarding intestinal fat absorption have been resolved over the past three decades, our concepts of fat digestion and absorption continue to be modified, and new concepts have emerged. The purpose of this review is to summarize advances in our understanding of fat digestion and absorption since the topic was last reviewed by Johnston in 1968. The discussion will emphasize 1) the role of colipase and its interactions with lipase, bile salt micelle, and triglyceride substrate; 2) the importance of the unstirred water layer in fat absorption; 3) micellar formation and dissociation; 4) the role of fatty acid binding protein; 5) factors influencing the reesterification mechanisms in the intestine; and 6) intestinal contribution to lipoprotein and apoprotein production. The importance of these new concepts and the remaining gaps in our understanding of these complex digestive and absorptive processes are discussed.

Clinical and laboratory approaches to evaluate diarrheal disorders.

Diarrheal disorders are the result of excessive fluid and electrolyte loss through the gastrointestinal tract. Many different underlying mechanisms are known to cause diarrhea. Fordtran suggested that in secretory diarrhea the osmolality of stool water should be accounted for by its electrolyte contents. Therefore, the osmotic gap between the measured osmolality and that estimated from electrolyte contents should be small. In osmotic diarrhea, due to the presence of the osmotic agent, there should be a greater gap between the measured and the estimated osmolalities. Osmotic gaps varying from 100 to 40 mOsm have been used arbitrarily in literatures to define the underlying pathogenesis. Because of the uncertainty, the usefulness of these measurements remains in question. In this article, methods used to measure stool osmolality and electrolyte contents are reviewed. Limitations of these measurements are discussed. Measurements derived from various diarrheal disorders revealed that the basic concepts put forward by Fordtran are corrected. However, we found that the osmotic gaps (measured osmolality - 2 [Na + K] in secretory diarrheal disorders are frequently negative numbers. In osmotic diarrhea, the osmotic gap (greater than 160 mOsm) is substantially greater than the figures used in the literature. In many diarrheal disorders the osmotic gap falls between the two extremes and the pathogenesis is multifactorial in origin. Under these circumstances, stool osmolality and electrolyte measurements provide little insight into the underlying mechanism causing the diarrhea. Furthermore, stool contains many biologically active organisms which can alter the stool osmolality. Unless these effects are appreciated, an inaccurate interpretation of these measurements may result.

Mechanism of intestinal fatty acid uptake in the rat: the role of an acidic microclimate.

1. Micellar solubilization of lipolytic products is an important step in lipid absorption. However, micelles are not absorbed intact; dissociation of lipolytic products from bile salt micelles must occur. The dissociation of micelles has been postulated to occur in an acidic microclimate. 2. The effect of an acidic microclimate on the uptake of micellar fatty acid was examined in the rat intestine. We reported that the presence of a lower pH microclimate is associated with a higher fatty acid uptake, suggesting that a lower pH enhances fatty acid uptakes from the micelles. 3. Fatty acid uptake from solutions containing a constant amount of bile salt (10 mM) and varying amounts of fatty acid (3.3-26.4 mM) revealed a saturation phenomenon which reflects the fatty acid carrying capacity of a 10 mM-taurocholate solution. 4. There was a linear relationship between fatty acid uptake and fatty acid concentration when the micellar solutions contained a constant ratio of fatty acid and taurocholate (1.32). 5. Our results indicate that the fatty acid carrying capacity of the micelle and the number of micelles in the solution are both important determinants for the amount of fatty acids delivered to the microclimate. The amount of fatty acids derived from the dissociation of micelles within the microclimate determines fatty acid uptake by the intestine.

pH dependence of micellar diffusion and dissociation.

A simplified model system was used to examine the effect of pH on the diffusion and dissociation of mixed micelles across an interphase. Diffusion of radioactively labeled taurocholate and oleic acid from a micellar solution into a phosphate buffer of varying pH (5.0-8.0) was measured. The proportion of bile salt and fatty acid as monomers and aggregates in both solutions was determined by ultrafiltration. Results indicate that all of the oleic acid existed as aggregates in the micellar solution, whereas taurocholate was in equilibrium between aggregate and monomer forms. Between pH 5.0 and 7.0, oleic acid and taurocholate diffusion was inversely related to pH and due to the differences in aggregate but not monomer diffusion. Fatty acid diffusion across the interface was always less than taurocholate diffusion, because taurocholate monomers could also diffuse across the interphase. Calculation of the proportion of taurocholate diffusion from aggregate and monomers based on micellar composition was similar to measured values at pH greater than or equal to 6.5, indicating a lack of micelle dissociation. However, at pH less than or equal to 6 measured values for bile salt monomers exceeded calculations, indicating that a low pH microclimate favors dissociation of micelles. A hypothesis is advanced to explain these events occurring in a disequilibrium area adjacent to the epithelial cell membrane.

Helicobacter pylori and gastrointestinal disease.

Helicobacter pylori infection is quite common. In the United States, prevalence varies considerably with race, nationality, socio-economic status and location of residence. In Western countries, the prevalence of the infection has shown a steady increase with increasing age. H. pylori has been shown to cause chronic gastritis. Most patients infected with H. pylori are asymptomatic and require no therapy. The precise role of the infection in the pathogenesis of peptic ulcer disease is unknown. However, H. pylori infection is associated with a high recurrence rate of both gastric and duodenal ulcers. Eradication of the infection reduces the recurrence rate. Once H. pylori infection is acquired, it usually persists for years, possibly for the patient's lifetime. Although a causative role in gastric cancer has not been proved, evidence suggests an association between H. pylori infection and well-differentiated gastric adenocarcinoma and gastric lymphoma.

Effect of insulin on in vitro intestinal fatty acid esterification in the rat.

We have previously shown that glucose metabolism plays an important role in modifying intestinal fatty acid esterification. Because it is well known that glucose metabolism is under insulin regulation, we examined the effect of insulin on intestinal fatty acid esterification. Insulin pretreatment led to a marked decrease in in vitro intestinal fatty acid esterification, but this decrease was abolished by maintaining blood glucose above 80 mg/dl. Addition of insulin to the incubation medium failed to produce any effect on intestinal fatty acid esterification. The decreased fatty acid esterification on hypoglycemic rats was not associated with changes in fatty acid uptake or lipid esterifying enzyme activities. However, there was a significant increase in the production of volatile metabolites of fatty acid. We conclude that 1) insulin itself has no effect on intestinal fatty acid esterification, 2) the effects observed in this study are due to insulin-induced hypoglycemia, 3) hypoglycemia does not alter intestinal fatty acid uptake or intrinsic esterification activity, but leads to preferential oxidation rather than esterification of fatty acid by the small intestine, and 4) the critical blood glucose concentration needed to maintain normal esterification in the rat was approximately at 80 mg/dl.

Jejunum is more important than terminal ileum for taurocholate absorption in rats.

The terminal ileum, with its active transport system, is considered the major site of bile salt absorption. However, earlier studies used bile salt concentrations below physiological levels and may not apply in vivo. Analysis of these studies shows that ileal active transport cannot account for total bile salt recovery. To reevaluate bile salt absorption in rats, we used four preparations and physiological bile salt concentrations. Studies with intestinal sacs showed that, above critical micellar concentration, uptake of taurocholate (TC) was equal in both jejunum and ileum and linear with respect to concentration. A similar pattern was observed in studies of mucosal-to-serosal TC transport using a flux chamber. In vivo studies in anesthetized rats showed approximately 30% of TC absorbed from proximal jejunum and appearing in bile when the bolus had traversed only half the intestine. In unanesthetized fed rats, 60% of TC appeared in bile before the bolus reached distal ileum. Because luminal concentrations of TC are highest proximally, passive absorption by the proximal intestine is mainly responsible for conserving TC within the enterohepatic circulation. Ileal active transport is more efficient at low concentrations and absorbs the TC remaining after proximal absorption.

Effects of luminal nutrition and metabolic status on in vivo glucose absorption.

Luminal nutrients, but not metabolic status, maintain active glucose transport by the rat intestine in vitro. The present study was designed to examine the effects of these factors on the in vivo absorption of glucose and 3-O-methylglucose. Rats were fed either intraluminally or by total parenteral nutrition (TPN) for 7 days or fasted for 72 h. Sugar absorption was measured under pentobarbital sodium (Nembutal) anesthesia at concentrations from 7 to 28 mM. Luminally fed rats had a significantly greater mucosal mass and absorption rates per centimeter of both sugars than either TPN or fasted animals. However, TPN rats had significantly greater absorption per milligram protein (i.e., specific activity) for both glucose and 3-O-methylglucose than luminally fed rats. In addition, TPN rats absorbed significantly more glucose per milligram protein, but not 3-O-methylglucose, than fasted animals. These data indicate: 1) luminal nutrients maintain glucose absorption by their trophic effects on mucosal mass; 2) the increase in specific activity for sugar absorption after TPN is unrelated to caloric balance; and 3) intestinal glucose metabolism affects its rate of absorption of glucose, but not 3-O-methylglucose.

Characteristics of intestinal glucose secretion in normal and diabetic rats.

A method was developed to characterize and quantitate the transfer of glucose from the plasma to the intestinal lumen. In normal rats, there was a linear correlation between the blood glucose concentration and the rate of appearance of plasma glucose into the intestinal lumen perfused with Krebs-Ringer buffer (r = 0.88, P less than 0.01). Intestinal perfusion with buffers containing either mannitol, glucose, or phlorizin significantly increased the recovery of secreted glucose compared with plain buffer. Rats perfused with buffer containing mannitol or those undergoing plasma volume expansion with dextran demonstrated a change in water movement from net absorption to secretion coupled with a significant increase in glucose secretion. During luminal perfusion with a buffer containing 21 mM glucose, glucose secretion represented 14% of the net glucose absorption rate. Intestinal perfusion with phlorizin gave the highest measured recovery of glucose, probably by blocking active reabsorption of secreted glucose. A series of simultaneous perfusions performed in the jejunum and ileum revealed similar rates of glucose transfer in both segments of intestine. Measurement of glucose secretion in rats with streptozotocin diabetes gave the highest values for the plasma-to-lumen movement of glucose. Treatment with insulin reduced the blood sugar and glucose transfer rate. These data demonstrate that glucose moves bidirectionally across the rat intestine, and its secretion is a passive process.

Development of fatty acid esterification mechanisms in rat small intestine.

Fatty acid esterification was measured in fetal jejunal and ileal isografts implanted under the kidney capsules of adult host rats and compared to the age-controlled intestine grown in situ. Studies were conducted on the 21st, 35th, 49th, and 63rd postconceptional days, corresponding to prenatal, suckling, weaning, and weaned rats. Substantial fatty acid esterification activity was found in prenatal jejunum but not in ileum. A proximal-distal gradient of fatty acid esterification was observed in all groups grown in situ, but not in isografts. The monoglyceride pathway (MG-P) accounted for about one-third of total fatty acid esterification (TFAE) in jejunum grown in situ and remained constant through the study. In the ileum, MG-P was the major esterification pathway during the first 4 postnatal weeks, but decreased progressively after weaning to become insignificant in adult rats. TFAE fell in the jejunal isografts, whereas it increased in the ileum. MG-P remained as the major pathway in the implanted jejunum and ileum. Our studies suggest that luminal contents are probably the most important modulator for the development and maintenance of intestinal fatty acid esterification, and "fetal programming" manifested by changes in fatty acid esterification mechanisms in the isografts is less important.

Effects of nutrients, endogenous secretions, and fasting on in vitro glucose uptake.

The effect of luminal nutrients, endogenous secretions, and metabolic balance on the initial velocity of glucose uptake was measured in everted rings of rat intestine. Animals were fed either by total parenteral nutrition (TPN), or into the midjejunum for 7 days, or fasted for 72 h. Luminal feeding resulted in higher midjejunal glucose uptake per milligram of protein, DNA, and centimeter gut length, when compared to fasting or TPN. Kinetic analysis revealed a higher "apparent" Vmax in the luminally fed group, whereas the "apparent" Km was similar in all groups. Diversion of the pancreaticobility secretions did not affect glucose uptake in luminally fed or TPN-fed animals. TPN-fed and fasted animals had similar glucose uptake. Luminal feeding did not have a remote effect on glucose uptake in intestine isolated from the nutrient stream. The results indicate that luminal nutrients maintain carrier-mediated glucose transport after direct contact with the mucosa. Overall metabolic balance and pancreaticobiliary secretions do not affect carrier-mediated glucose transport. The nutrient effect appears to involve an increased number of glucose carriers per cell without a change in carrier affinity for glucose.

Role of small intestine in pathogenesis of hyperlipidemia in diabetic rats.

The small intestine can utilize endogenous substrates for triglyceride synthesis. In diabetes mellitus, potential endogenous substrates are elevated. This study was designed to investigate whether intestinal triglyceride production utilizing endogenous substrates contributes to the pathogenesis of hyperlipidemia in diabetes. Intestinal fatty acid esterification as well as activities of acyl-CoA synthetase and acyl-CoA monoglyceride acyltransferase are the same in diabetic and control rats when the results are expressed per milligram protein. However, due to marked intestinal hypertrophy these activities are increased when the results are expressed as per centimeter gut length. In the mesenteric lymph fistula rat model, we found that during fasting diabetic rats have a greater than twofold increase in triglyceride output that is carried mainly by very low-density lipoproteins (VLDL). During lipid infusion, total triglyceride fatty acid output was not different between diabetic and control rats, although there were significant differences in the patterns of partition of endogenous and exogenous triglyceride into chylomicrons and VLDL. Endogenous triglyceride production did not increase in diabetic rats during lipid infusion. In contrast, there was a substantial increase in endogenous triglyceride production in the control group to a level comparable with that of the diabetic rats. There was a significant reduction in incorporation of exogenous triglyceride into chylomicrons in diabetic rats.

Effects of cholecystokinin and secretin on intestinal structure and function.

Cholecystokinin and secretin are believed to be trophic gastrointestinal hormones. Studies were designed to determine whether these hormones exert their effect through stimulation of endogenous secretion. First, four groups of parenterally nourished rats underwent bypass of the proximal two-thirds of the intestine. One group received secretin, another cholecystokinin octapeptide (CCK-OP), another CCK-OP plus secretin, while the fourth group served as control. After 1 wk, animals were killed; pancreas and segments of intestine were removed. First, mucosal weight, protein content, and fatty acid esterification activity were affected only in intestine in continuity with endogenous secretions after hormone administration. Second, the effects of these hormones were tested in chow-fed rats. The hormone-treated group, despite pancreatic hyperplasia, had similar indexes of intestinal mass compared with pair-fed controls. We conclude that CCK-OP and secretin mediate their trophic effects on the small intestine indirectly, probably through stimulation of pancreatic secretion. In addition, the effects of luminal nutrients have complex interactions with these hormones.

Effect of sugar and monoglyceride on fatty acid esterification.

The effect of hexose and monoolein on fatty acid esterification was studied in everted rings of rat jejunum. By use of [3H]oleic acid and [U-14C]glucose, esterification via both phosphatidic acid and monoglyceride pathways was quantified. Our results showed that 1) metabolizable sugars stimulate fatty acids esterification regardless of their mode of transport; 2) in the presence of glucose alone, in vitro fatty acid esterification is mediated entirely by the phosphatidic acid pathway; 3) glucose stimulates both esterification pathways in the presence of monoolein; 4) monoolein stimulates only the monoglyceride pathway and has no effect on the phosphatidic acid pathway; and 5) the rate-limiting factor in the presence of monoolein alone is the formation of acyl-CoA, whereas in the presence of glucose alone, the rate-limiting factor is the formation of alpha-glycerophosphate.

Effects of luminal glucose versus nonnutritive infusates on jejunal mass and absorption in the rat.

These studies were designed to better understand the effects of luminal nutrition on intestinal mass and function. Parenterally nourished rats received a midjejunal infusion of either 0.9% saline, 10% glucose, 10% 3-O-methyl glucose, or 30% glucose. A fifth group underwent sham operation. After 7 days, intestinal mass and in vitro glucose and leucine uptake were measured in the intestine just distal to the infusion site. Luminal infusion led to greater intestinal mass in all groups compared to controls, but only the 10% and 30% glucose groups had significantly greater overall glucose uptake. Kinetic analysis revealed a greater apparent maximal transport rate in both glucose groups. The 30% glucose group had a greater apparent maximal transport rate for leucine and permeability for glucose and leucine. These data confirmed that "work load," in addition to luminal nutrition, maintains intestinal mass. However, adaptation of intestinal transport is more specific and appears to be regulated both by substrate metabolism and caloric density.