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.

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.

Passive jejunal bile salt absorption alters the enterohepatic circulation in immature rats.

BACKGROUND: Developmental changes in passive bile salt absorption may alter the enterohepatic circulation. METHODS: 14-, 21-, and 40-day-old anesthetized male Sprague-Dawley rats were studied. Jejunum and ileum were isolated, cannulated, and injected or perfused with a taurocholate, [3H]taurocholate, and nonabsorbable marker solution. Bile was collected. RESULTS: Using bolus injection, jejunal taurocholate absorption rates and total taurocholate absorption were nonsaturable, linearly related to taurocholate dose, and decreased from 14 days (1.62 nmol.cm-1.min-1) to 21 days (1.05 nmol.cm-1.min-1) and 40 days (0.54 nmol.cm-1.min-1). While total taurocholate absorption decreased (14 days, 52.4%; 21 days, 43.7%; 40 days, 30.5%), hepatic taurocholate clearance increased (14 days, 18.2%; 21 days, 23.7%; 40 days, 37.3%). Hepatic taurocholate clearance was saturated only at 14 days. Using jejunal perfusion, total taurocholate absorption (14 days, 62.0%; 21 days, 43.1%; 40 days, 45.3%) and taurocholate absorption rate decreased with age (14 days, 941.13 nmol.cm-2.min-1 per micromole of taurocholate; 21 days, 411.28 nmol.cm-2.min-1 per micromole of taurocholate; 40 days, 334.50 nmol.cm-2.min-1 per micromole of taurocholate). CONCLUSIONS: Passive jejunal bile salt absorption and decreased hepatic bile salt clearance could account for the low intraluminal and high serum bile salt levels observed in immature animals and in human neonates.

Acidic mucin layer facilitates micelle dissociation and fatty acid diffusion.

The presence of a mucin layer on the surface of the intestinal epithelium has been suggested as an important factor in maintaining an acidic microclimate. The presence of such a low-pH compartment has been shown to facilitate fatty acid uptake. The mechanisms leading to the enhancement of fatty acid uptake were investigated in a purified acidic mucin layer. Our results indicate that the presence of a low-pH compartment indeed facilitates the dissociation of mixed micelles made of taurocholate and oleic acid. The released fatty acid formed an emulsion at the mucin layer, and this event could be visualized by the naked eye. When the size of the particles in the micelle solution was examined by photon correlation spectroscopy, it was found that acidification alone can lead to the formation of particles with size substantially greater than that of micelles. With the use of labeled fatty acid, the change in optical density can be correlated to the amount of fatty acid appearing in the mucin layer in an asymptotic fashion, suggesting that using the turbidity as an indicator might underestimate fatty acid diffusion. Despite this limitation, the rate of fatty acid diffusion in the mucin layer was estimated to be 400% of that in the buffer solution.

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.

Fecal triglyceride excretion is not excessive in pancreatic insufficiency.

Steatorrhea can result from maldigestion or malabsorption. As the pathophysiology underlying impaired digestion differs from impaired absorption, it is important to differentiate these two disorders. It is generally accepted that patients with maldigestion excrete an excessive amount of triglyceride and patients with malabsorption excrete an excess of the lipolytic product of triglyceride, fatty acid. The two-step Sudan stain has been used as a simple test to differentiate these disorders. The validity of the test has not yet been established. In this study, fecal fatty acid and triglyceride were measured after extraction and thin-layer chromatographic separation. Our results indicate that in adult patients with pancreatic insufficiency, the fecal triglyceride content does not differ from the controls. However, a fivefold to sixfold increase in fecal fatty acid content in patients with pancreatic insufficiency was revealed. As patients with maldigestion do not excrete an excess of undigested triglyceride, it is not possible to differentiate maldigestion from malabsorption by quantifying fecal triglyceride and fatty acid.

Sudan stain of fecal fat: new insight into an old test.

The 72-h fecal fat determination is used as the gold standard to document the presence of steatorrhea. Although the Sudan stain for fecal fat is advocated as a sensitive screening test, a quantitative correlation between the 72-h fecal fat quantitation and the fecal Sudan stain is lacking. This study was designed to examine the staining properties of different classes of purified lipids in an experimentally defined artificial matrix, and to elucidate the reasons for the lack of quantitative correlation between these two tests. Our results indicate that the "neutral fat" stain without acidification or heating identifies triglyceride; and at an appropriate pH, the "neutral stain" also identifies fatty acid. The "split fat" stain with acidification and heating identifies both triglyceride and fatty acid. After acidification, fatty acid soaps are converted to the nonionized fatty acid. Thus, fatty acid soaps can be identified indirectly as fat droplets that are stained by the split fat stain. Although cholesterol is stained with Sudan stain after heating, upon cooling, cholesterol forms crystals of anhydrous cholesterol, making its staining pattern distinct. Neither the neutral fat nor the split fat stain can detect phospholipid or cholesteryl ester. The 72-h fecal fat determination is a measure of the total fatty acid content after a specimen is saponified. The resulting fatty acids are derived from a variety of endogenous and exogenous sources, including free fatty acids, soaps of fatty acids, triglycerides, cholesterol esters, and phospholipids. Therefore, the 72-h fecal fat quantitation does not differentiate between the primary sources of the measured fatty acid. It is concluded that the 72-h fecal fat determination is not specific for documenting triglyceride (fat) malabsorption. Until new methods are developed that specifically measure fecal triglyceride and fatty acid, the Sudan stain of fecal fat appears to be a more specific method for detecting the presence of triglyceride and fatty acid in a matrix.

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.

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.

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.

Mechanisms maintaining a low-pH microclimate in the intestine.

The presence of an unequilibrated region adjacent to the mucosal surface of the small intestine has long been suggested. Controversy remains as to the characteristics of this region. This study was designed to further define these characteristics. Our studies demonstrated by two independent methods, pH electrode measurement and dye indicator, that a low-pH compartment does exist at the surface of the intestine. The thickness of this microclimate was estimated to be 700 microns, comparable with that described for the unstirred water layer. Maintenance of the low-pH compartment is due to the presence of a mucus coating rather than hydrogen ion secretion. We postulate that the mucus functions as an ampholyte and restricts hydrogen ion movement in its matrix. The acidic microclimate, unstirred water layer, and the mucus coating might in fact represent a common phenomenon.

Stool electrolyte and osmolality measurements in the evaluation of diarrheal disorders.

Stool osmolality and electrolyte measurements were obtained from 12 patients with diarrheal disorders. Osmolality of diarrheal stool (285 to 330 mosmol) regardless of the cause is less than the reported osmolality of normal stool. Storage of stool at room temperature can artifactually increase stool osmolality as the result of bacterial metabolism. When stool samples are fresh, a negative osmotic gap (measured osmolality - 2 X [Na + K]) is commonly associated with secretory diarrhea, whereas a high osmotic gap (greater than 160 mosmol) is seen in patients with osmotic diarrhea. In many conditions fasting does not resolve diarrhea completely, and when the stool osmotic gap is greater than 50 mosmol, the pathogenesis of diarrhea is difficult to define.

Intestinal triglycerides are derived from both endogenous and exogenous sources.

Although studies have indicated that the small intestine is capable of utilizing endogenous substrates for triglyceride synthesis in the absence of dietary lipid, the importance of the endogenous contribution to total intestinal triglyceride production during absorption has not yet been defined. In this study we have examined the quantitative contribution of endogenous triglyceride production during different luminal lipid loads. By use of a mesenteric lymph fistula rat model with total parenteral nutritional support, mesenteric lymphatic triglyceride transport was investigated. Our results indicate that, during absorption, a substantial fraction (greater than 50%) of total triglyceride is derived from endogenous sources. Increased luminal fatty acid loads lead to an increase in both endogenous and exogenous triglyceride production. Incorporation of luminally infused oleic acid into triglyceride carried by chylomicrons is dependent on the luminal fatty acid load, while incorporation of oleic acid into very low-density lipoprotein (VLDL) triglyceride is saturable. We conclude that both chylomicron and VLDL are involved in transporting triglyceride derived from both endogenous and exogenous sources. The different patterns in the partition of endogenous and exogenous triglyceride into chylomicrons and VLDL suggest that these two lipid-carrying lipoproteins are probably packaged differently in the small intestine.

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.

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.

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.

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.

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.

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.