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Background and Aim: Macrophages and dendritic cells are closely related mononuclear phagocytic cells. Little is known about their in vivo role in acute intestinal bacterial infections in humans. We undertook to evaluate these cells in rectal mucosal biopsies of patients with acute colitis., Methods: All mucosal mononuclear phagocytic cells in rectal biopsies of patients with acute Campylobacter colitis (n = 5), shigellosis (n = 5), and cholera (n = 10) were evaluated ultrastructurally and compared with those in controls (n = 5)., Results: Mononuclear phagocytic cells in the superficial rectal mucosa showed a higher prevalence of ultrastructural features of activation in Campylobacter colitis and cholera than in controls. A lower prevalence of features of activation with increased monocytes was seen in shigellosis. Cells with the ultrastructural morphology of activated dendritic cells constituted 41% and 45% of all mononuclear phagocytic cells in two of five patients with Campylobacter colitis and 4-22% of cells in four of 10 patients with cholera. Their presence in patients with Campylobacter colitis was associated with significant surface epithelial damage and prominent acute inflammatory changes in the mucosa., Conclusions: This is the first ultrastructural study to show activated macrophages and dendritic cells in vivo in acute Campylobacter colitis and cholera. Dendritic cell activation occurred early in the clinical course of these infections. Surface epithelial damage may play a role in the activation of dendritic cells.

Spontaneous bacterial peritonitis is a major cause of mortality after liver cirrhosis. Altered permeability of the mucosa and deficiencies in host immune defenses through bacterial translocation from the intestine due to intestinal bacterial overgrowth have been implicated in the development of this complication. Molecular mechanisms underlying the process are not well known. In order to understand mechanisms involved in translocation of bacteria, this study explored the role of oxidative stress in mediating changes in intestinal mucosal glycosylation and luminal bacterial content during cirrhosis. CCl4-induced cirrhosis in rats led to prolonged oxidative stress in the intestine, accompanied by increased sugar content of both intestinal brush border and surfactant layers. This was accompanied by changes in bacterial flora in the gut, which showed increased hydrophobicity and adherence to the mucosa. Inhibition of xanthine oxidase using sodium tungstate or antioxidant supplementation using vitamin E reversed the oxidative stress, changes in brush border membrane sugar content, and bacterial adherence. In conclusion, oxidative stress in the intestine during cirrhosis alters mucosal glycosylation, accompanied by an increased hydrophobicity of luminal bacteria, enabling increased bacterial adherence onto epithelial cells. This might facilitate translocation across the mucosa, resulting in complications such as spontaneous bacterial peritonitis.

Nonsteroidal anti-inflammatory drugs (NSAIDs), used extensively in clinical medicine, tend to cause adverse effects in the gastrointestinal tract. Earlier work has shown that oral administration of indomethacin produced oxidative damage in the small intestine and attenuation of the glycocalyx layer of the mucosa. The present study assessed, in greater detail, the alterations produced in the glycocalyx of rat small intestinal mucosa in response to indomethacin, with specific reference to surfactant-like particles (SLP) and brush border membranes (BBM). Changes in gut flora in response to the drug were also studied, as it has been shown that luminal bacteria play a role in the pathogenesis of NSAID-induced intestinal damage. The levels of sugars such as sialic acid, fucose, hexose and hexosamine were increased in SLP and decreased in the BBM following indomethacin treatment, with the effects being maximal 24h after the administration of the drug. The composition of lipids in the SLP was also found to be altered. There was a significant increase in the number of bacteria in the luminal contents of the small intestine and caecum in these animals, as compared with controls. The number of bacteria adherent to the intestinal mucosa was also significantly higher in the drug-treated group. In vitro studies revealed that there was an increased tendency for bacteria to adhere to SLP isolated from indomethacin-treated rats. These results suggest that alterations in glycosylation of SLP and BBM in response to indomethacin, along with qualitative and quantitative changes in the luminal bacterial flora, may facilitate translocation of bacteria into the mucosa. These changes may contribute to the enteropathy observed as a result of NSAID treatment.

The use of NSAIDs (non-steroidal anti-inflammatory drugs), although of great therapeutic value clinically, is limited by their tendency to cause mucosal damage in the gastrointestinal tract. In the small intestine, the effects these drugs have been shown to produce include inhibition of cyclo-oxygenase, mitochondrial dysfunction and free radical-induced oxidative changes, all of which contribute to the mucosal damage seen. Glutamine is a fuel preferentially used by enterocytes and is known to contribute to maintaining the integrity of these cells. In the present study, we investigated the effect of glutamine on indomethacin-induced changes in the small intestinal mucosa. Rats were given 2% glutamine or glutamic acid or isonitrogenous amino acids, glycine or alanine, in the diet for 7 days. Indomethacin was then administered orally at a dose of 40 mg/kg of body weight. After 1 h, the small intestine was removed and used for the measurement of parameters of oxidative stress and mitochondrial and BBM (brush border membrane) function. Evidence of oxidative stress was found in the mucosa of the small intestine of drug-treated rats, as indicated by significantly increased activity of xanthine oxidase (P < 0.001) and myeloperoxidase (P < 0.001), with corresponding decreases in the levels of several free radical scavenging enzymes and alpha-tocopherol (P < 0.001 in all cases). Levels of products of peroxidation were also significantly elevated (P < 0.001 for all the parameters measured). In addition, oxidative stress was evident in isolated intestinal mitochondria and BBMs (P < 0.001 for all the parameters measured), with associated alterations in function of these organelles (P < 0.001 for all the parameters measured). Supplementation of the diet with glutamine or glutamic acid prior to treatment with indomethacin produced significant amelioration in all the effects produced by the drug in the small intestine (P < 0.001 for all the parameters measured). Glycine and alanine were found to be much less effective in these respects.

Cirrhosis is associated with altered gastrointestinal function, and bacterial translocation from the gut plays an important role in the etiology of spontaneous bacterial peritonitis (SBP) seen in this condition. Although alterations in gut motility and intestinal permeability are recognized in cirrhosis, the intestinal damage at the cellular and subcellular levels is not well understood. This study looked at the mucosal alterations in experimental cirrhosis and the role of oxygen free radicals in this process. It was shown that cirrhosis results in oxidative stress in the intestine, as seen by increased xanthine oxidase (XO) activity and altered antioxidant status. Cirrhosis also affects enterocyte mitochondrial function, as assessed by respiratory control ratio, swelling, and calcium flux. Increased lipid peroxidation of the brush border membranes (BBMs) was seen along with altered intestinal transport. In conclusion, this study shows that intestinal mucosal alterations are seen in experimental cirrhosis and are possibly mediated by oxidative stress.

A novel method of brush border membrane vesicle (BBMV) preparation from the small intestinal mucosa using polyethylene glycol (PEG) precipitation has been presented. This preparation is compared with calcium-precipitated BBMVs in marker enzyme enrichment, contamination by other subcellular membranes, transport of glucose, and lipid composition. PEG-precipitated BBMVs are comparable with calcium-precipitated membranes in all parameters except lipid composition and thiol content. PEG-precipitated membranes have more phosphatidylcholine and phosphatidylethanolamine and less lysophosphatidylcholine and lysophosphatidylethanolamine as compared to calcium-precipitated membranes. Diacylglycerol and triacylglycerol content are also high in PEG-precipitated membranes. Alteration in lipid composition indicate the possible activation of lipase and phospholipase by calcium during BBMV preparation, which is not seen in PEG precipitation. Thiol content is almost double in PEG-precipitated membranes as compared to calcium-precipitated membranes. These results indicate that PEG can be used for the preparation of BBMVs in native form from the intestine without any alteration in their structural components, and these membranes show comparable transport activity., (Copyright 2001 Academic Press.)

Background: Surgical stress can lead to translocation of bacteria from the intestine into the systemic circulation. The intestinal brush border membrane (BBM) plays an important role in defense against such invasion by luminal bacteria and endotoxin. Our earlier work has shown the development of oxidative stress in the intestine after surgical stress and since the BBM is sensitive to free radical attack, this study examined the effect of surgical stress on the structure and function of intestinal BBM., Methods: Intestinal BBM were isolated from control and after surgical stress and compared for structural and functional alterations. Surgical stress was also carried out following pretreatment with the xanthine oxidase inhibitor allopurinol or the nitric oxide donor l-arginine, to study the protection offered by these compounds., Results: Surgical stress affected intestinal BBM structure as well as function. A decrease in alkaline phosphatase activity and alpha-tocopherol content, accompanied by an increase in lipid peroxidation, was seen. Surgical stress induced phospholipid degradation with generation of arachidonic acid. Functional impairment with a decrease in glucose transport ability was also seen. These changes are prevented by inhibition of xanthine oxidase by allopurinol pretreatment but not by NO., Conclusion: Surgical stress in the small intestine causes structural and functional alterations in the BBM through oxidative stress. This damage could affect gut barrier integrity and generation of arachidonic acid might mediate distal organ dysfunction., (Copyright 2000 Academic Press.)

Background: Surgical stress affects intestinal permeability and our earlier study using a rat model indicated that oxidative stress plays an important role in this process. Proteases are important mediators of cellular damage and are known to be activated in oxidative stress. This study looked at protease activity in enterocytes after surgical stress., Methods: Surgical stress was induced by opening the abdominal wall and handling the intestine as done during laparotomy, in normal and xanthine oxidase-deficient rats. Enterocytes at various stages of differentiation were isolated and protease activity and protection offered by xanthine oxidase inhibitors were determined. Mitochondria and cytosol were prepared from total isolated enterocytes at different periods after surgical stress and protease activation was studied., Results: Surgical stress induced activation of proteases in both the villus and crypt cells. Protease activation is seen in both mitochondria and cytosol, and similar to the other alterations in mucosal cells, protease activation was maximum 60 min after stress, returning to normal by 24 h. Thiol compounds modulate protease activity in both mitochondria and cytosol and the activation is not seen in xanthine oxidase-deficient animals., Conclusions: Surgical stress induces activation of proteases in villus and crypt cells of the small intestine. Both mitochondrial and cytosolic proteases are activated and free radicals generated by xanthine oxidase may mediate protease activation after surgical stress in the intestine., (Copyright 2000 Academic Press.)

Apoptosis is now recognized as an important process responsible for maintenance of the cellular balance between proliferation and death. Apoptosis is distinct from necrosis in that it is a programmed form of cell death and occurs without any accompanying inflammation. This form of cell death can be induced by a wide range of cellular signals, which leads to activation of cell death machinery within the cell and is characterized by distinct morphological changes. Apoptosis is especially relevant in the gastrointestinal tract, as the mammalian intestinal mucosa undergoes a process of continual cell turnover that is essential for maintenance of normal function. Cell proliferation is confined to the crypts, while differentiation occurs during a rapid, orderly migration up to the villus. The differentiated enterocytes, which make up the majority of the cells, then undergo a process of programmed cell death (apoptosis). Although apoptosis is essential for the maintenance of normal gut epithelial function, dysregulated apoptosis is seen in a number of pathological conditions in the gastrointestinal tract. The cellular mechanisms regulating this tightly regimented process have not been clearly defined and this topic represents an area of active investigation as delineation of this process will lead to a better understanding of normal gut mucosal growth.

Our earlier studies have shown apoptosis in the villus tip cells of the monkey small intestinal epithelium. Because mitochondria have been implicated in the apoptotic process, this study looked at the function and lipid composition of mitochondria isolated from apoptotic villus tip cells and compared it with middle and crypt cells. Decreased MTT reduction and respiratory control ratio, increased swelling and altered mitochondrial enzyme activities were seen in the villus tip cell mitochondria when compared to other cells. The lipid composition of the villus tip mitochondria were different from the other mitochondria. A decrease in phosphatidylethanolamine and phosphatidyl-inositol and an increase in phosphatidic acid was seen in these mitochondria. Fatty acid composition analysis showed more unsaturated fatty acids in the free fatty acid and phospholipid fraction in villus tip cell mitochondria as compared to other cells. These studies suggest that in the monkey small intestinal epithelium, apoptotic process is associated with functional and structural alterations in the mitochondria.

The apoptotic process in the normal gastrointestinal mucosa is of interest due to its possible role in physiological cell renewal. The aim of this study was to identify the apoptotic process in the monkey small intestine and the association of glutathione level and its efflux in this process. Monkey small intestinal epithelial cells were separated in to different fractions consisting of villus, middle and crypt cells. Apoptosis was identified by DNA ladder pattern and Hoechst staining. The level of glutathione, its efflux and the enzymes involved in its metabolism were quantitated in these fractions. Apoptotic cells were identified predominantly in the villus tip cell fractions by both DNA ladder pattern and Hoechst dye staining. Glutathione level was 7 fold higher in the crypt cells as compared to villus tip cells the middle cells showing a gradual decrease. A similar pattern was seen in mitochondrial content of glutathione. As the cells mature from crypt to villus, there is increased efflux of GSH, which may be responsible for the decreased level of GSH in apoptotic villus cells. In the monkey small intestine, apoptotic cells are seen in the villus tip fractions and the glutathione level and its efflux may play a role in this process.

Recent findings suggest that intracellular oxidants are involved in the induction of apoptosis and this type of cell death can be inhibited by various antioxidants. In our accompanying paper, we have shown apoptosis in the villus tip cells of the monkey small intestinal epithelium. The aim of the present study was to evaluate the possible relationship between oxidative stress, antioxidant levels and the apoptotic process in the monkey small intestinal epithelium. Monkey small intestinal epithelial cells were isolated into different fractions consisting of villus, middle and crypt cells. Mitochondrial function was assessed by the reduction of the tetrazolium dye, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), with and without succinate. The extent of lipid peroxidation was assessed by measuring the formation of conjugated diene, depletion of polyunsaturated fatty acids and alpha-tocopherol. Level of antioxidant enzymes like, superoxide dismutase (SOD), catalase, glutathione S-transferase (GST), glutathione peroxidase (GPx) and glutathione reductase were also quantitated in various cell fractions. MTT reduction was significantly decreased in villus cells as compared to the cells from other fractions and this was evident even in presence of the respiratory substrate, succinate. Increased formation of conjugated diene and depletion of polyunsaturated fatty acids were seen in villus and crypt cells as compared to middle fraction cells. The alpha-tocopherol level was decreased in both villus and crypt cells as compared to cells from middle region. Significant decrease of SOD activity was seen in the villus tip cells and a slight decrease was seen in the crypt fractions. Glutathione dependent enzymes like GST, GPx and GSH reductase showed higher activity in the villus fractions. A similar observation was also seen in the catalase activity. This study has shown that although oxidative stress is seen in both villus and crypt cells, decreased mitochondrial function was seen in villus tip cells which may be responsible for apoptotic process in the intestinal epithelium.

Inflammation is associated with oxidative stress and altered cellular calcium homeostasis. Our earlier studies have shown that, increased phosphatidic acid (PA) formation occurred in enterocyte mitochondria when exposed to superoxide, divalent metal ions or polyamines resulting in altered lipid composition. Since aminosalicylates are the drug of choice for gut inflammation, we have tested the effect of aminosalicylates on PA formation by enterocyte mitochondria. When stimulated by superoxide, Ca2+ or spermine, phosphatidyleth-anolamine (PE) degradation and PA formation occurred in enterocyte mitochondria which can be inhibited by aminosalicylates. The inhibition was 50-60% at 0.5-mM concentration and at 1- or 2-mM final concentration, complete inhibition was observed. Both 5-aminosalicylate (5-ASA) and 4-aminosalicylate (4-ASA) showed similar effects. The stimulation of PA formation by calcium or spermine was not due to increased generation of superoxide by mitochondria which was confirmed by measurement of superoxide production by the mitochondria. These studies suggest that in addition to other cellular effects, aminosalicylates may prevent the enterocyte mitochondrial damage by inhibition of PA formation and PE degradation and alteration of mitochondrial lipid composition.

Mitochondrial swelling and calcium cycling occurs during oxidative stress and can be prevented by cyclosporin A (CysA). Our earlier work has shown that enterocyte mitochondria contains a phospholipase D (PLD) which can be activated by superoxide or calcium. In this study, we have shown that enterocyte mitochondrial PLD activated by these agents can be inhibited by cyclosporin A. This was clearly shown by the absence of phosphatidic acid (PA) formation and phosphatidylethanolamine (PE) degradation. Since this PLD specifically utilizes PE as substrate, PLD activity was also assessed by ethanolamine formation which was inhibited by CysA. CysA also inhibited the cabbage PLD activity as judged by phosphatidylethanol formation. These results suggest that cyclosporin A is an inhibitor of PLD and this may be one of the mechanism by which CysA protects enterocyte mitochondria from oxidative stress.

Ischemia/reperfusion of the small intestine can lead to metabolic and structural alterations in the mucosa. Cellular dysfunction occurs when mitochondrial metabolism is compromised, which may ultimately lead to impaired organ function. The aims of this study were to assess the suppression of cellular and mitochondrial oxidative metabolism and involvement of mitochondria in the ischemia/reperfusion injury. The mitochondria were prepared from isolated enterocytes obtained from the small intestine of anesthetized adult rats following different time periods of ischemia and ischemia followed by 5 min reperfusion. Cellular and mitochondrial function were assessed using MTT (3-(4,5-Dimethylthiazol-2-yl) -2,5-diphenyl tetrazolium bromide) reduction assay. Ischemia of increasing time periods caused a progressive decrease in cellular and mitochondrial MTT reduction in enterocytes and reperfusion showed further decrease of MTT formazan formation. Inclusion of 1 mM succinate, as respiratory substrate, showed reversal of suppression of mitochondrial function in 30-60 min ischemia whereas 90 min ischemia or short time period ischemia followed by 5 min reperfusion indicated an irreversible damage to mitochondria. This study indicated that mitochondria are a sensitive target of damage due to oxygen deficiency and possibly due to sudden burst of oxygen free radicals. Mitochondria can withstand short periods of ischemia whereas long duration ischemia or reperfusion results in irreversible damage to mitochondrial function.

The effect of luminal exposure of enterotoxins on the intestinal mucosal glutathione (GSH) was studied in rat. Cholera toxin induced fluid secretion and decreased mucosal GSH by 35% without altering oxidized glutathione (GSSG) level. Toxin induced fluid secretion was tested after mucosal GSH depletion by compounds such as diethyl maleate (DEM) and buthionine sulfoximine (BSO) and thiol supplementation with N-Acetyl cysteine (NAC). Fluid secretion was not altered by prior thiol depletion or supplementation. Exposure of intestinal lumen to bacterial endotoxin resulted in 25% decrease in mucosal GSH with two fold increase in GSSG. Luminal exposure of Shiga toxin did not alter the mucosal thiol. The level of other low molecular weight thiols, cysteine and cystine was not altered by luminal exposure of any of these toxins. These results show that although cholera toxin decreased the mucosal GSH level, prior modulation of thiol status of the mucosa may not have any effect on toxin-induced fluid secretion.

Modification of protein thiol by mixed disulfide formation with low molecular weight cellular thiols has been proposed as one of the post-translational modifications of amino acid side chains and is known to be catalyzed by thioltransferase. Intestinal mucosa is susceptible to oxidative injury and is likely to form protein mixed disulfide during oxidative stress. In the present study thioltransferase was purified from monkey small intestinal mucosa and its role in protein-s-thiolation was investigated. The purified enzyme was homogeneous, as judged by polyacrylamide gel electrophoresis under reducing conditions. The enzyme, with a molecular weight of 52 kDa, was a monomeric protein, which showed optimum activity at pH 8.0 with hydroxyethyl disulfide as substrate. The enzyme specifically cleaved the disulfide bond of the synthetic substrate, hydroxyethyl disulfide, in the presence of reduced glutathione (GSH) with the formation of oxidized glutathione (GSSG) as shown by high performance liquid chromatography. The enzyme also catalyzed protein thiolation of monkey intestinal mitochondria when incubated with glutathione disulfide. These studies have shown that thioltransferase purified from intestinal mucosa could catalyze dethiolation and thiolation.

Oxygen-derived free radicals are known to be generated during ischemia/reperfusion injury and biomembranes are the prime target of these active species. In order to study the effect of in vivo generated free radicals on intestinal mucosal membrane, brush border membranes (BBM) were isolated from rat small intestine after subjecting to ischemia (I) and ischemia/reperfusion (I/R) injury and their lipid composition and marker enzyme activity were compared with BBM prepared from control animals. No significant alteration in the lipid composition of BBM was observed after I or I/R as compared to control. Membrane fluidity measurements showed that I/R increased the fluidity of BBM. Activity of alkaline phosphatase, one of the marker enzymes for BBM was reduced by I or I/R whereas activity of another BBM enzyme, sucrase was not altered. The decrease in alkaline phosphatase activity was more after reperfusion. In vitro fluidization of BBM using benzyl alcohol indicated that the inactivation of alkaline phosphatase was not due to change in fluidity. These results suggest that free radicals generated during I/R inactivate BBM alkaline phosphatase partially without altering the membrane lipid composition.

Oxidative stress is associated with the formation of oxidized glutathione (GSSG) in the cells, which can form mixed disulfide with proteins leading to alteration of their function. The present study looks at the effect of in vitro exposure of GSSG on intestinal mitochondria and brush border membrane (BBM). Incubation with 1 mM GSSG increased the protein bound GSH in mitochondria by 15-fold. This was associated with loss of activity of certain mitochondrial enzymes such as succinic dehydrogenase, isocitrate dehydrogenase, total ATPase and NADH dehydrogenase whereas NADH oxidase was not affected. A similar treatment of BBMV with GSSG increased the protein bound GSH by 4.7-fold without altering its enzyme activity. Exposure to GSSG had no effect on the Na(+)-dependent glucose transport by BBMV. These studies suggest that GSSG formed during oxidative stress may modify thiol groups in proteins by forming mixed disulfides leading to functional alteration of certain cellular proteins.

Oxygen-derived free radicals have been implicated in the pathogenesis of ulcerative colitis. Mammalian tissues contain antioxidant systems that offer protection from the damaging effect of these active species. In the present study, the activity of the antioxidant enzymes catalase, glutathione peroxidase, glutathione transferase and glutathione reductase were measured in rectal biopsies from patients with ulcerative colitis and compared with that obtained from normal subjects. A significant decrease in the activity of glutathione transferase was observed in ulcerative colitis (48.32 +/- 6.73 units/mg protein, mean +/- s.e.) compared to normal (68.20 +/- 6.83; P = 0.015). There was no difference in the activity of other antioxidant enzymes between controls and ulcerative colitis. Myeloperoxidase, a marker for neutrophil infiltration, was considerably increased in ulcerative colitis while malonaldehyde, the end product of lipid peroxidation, was not increased. The reduced activity of glutathione transferase in ulcerative colitis may be an additional factor in the pathogenesis of mucosal damage in this disease.

Lipid composition of total membrane fractions prepared from scraped rat intestinal mucosa and isolated epithelial cells were compared. Membranes prepared from mucosa had four times higher nonesterified fatty acids (NEFA) as compared to the epithelial cell membranes. Cholesterol and phospholipid contents were similar in both the membrane preparations but triglyceride content was high and di- and monoglyceride were low in epithelial cell membranes as compared to the mucosal membranes. Inclusion of p-bromophenacyl bromide, a phospholipase inhibitor, in the intestinal lumen wash solution and homogenizing buffer did not reduce the NEFA content of the scraped mucosal membranes whereas inclusion of diethyl p-nitrophenyl phosphate, a lipase inhibitor reduced it by 40%. These results suggest that NEFA are normal constituent of intestinal cell membranes.

This study looks at the role of xanthine oxidase (XO) in ischemia/reperfusion (I/R) induced intestinal mucosal damage using normal and xanthine oxidase deficient rats. Tungstate feeding for 3 days depleted the intestinal mucosal XO by 80%. A ligated loop of the rat small intestine (both normal and XO-deficient) was subjected to 1 h of total ischemia followed by 5 min revascularisation. The ensuing mucosal damage was assessed by biochemical and histological studies. Ischemia or I/R increased the XO levels in normal rats without any change in XO-deficient rats. Myeloperoxidase (a neutrophil marker) level was increased in both group of rats but it was comparatively higher in the XO-deficient rats. Accumulation of peroxidation products such as malondialdehyde, conjugated diene and increased production of hydroxyl radicals by microsomes were seen after ischemia and I/R and were similar in normal and XO-deficient rats. Studies on other parameters of peroxidation showed a decrease in polyunsaturated fatty acids and alpha-tocopherol, an increase in cysteine and cystine levels after I/R and were similar in both normal and XO-deficient rats. Histological results indicated gross morphological changes in the intestinal mucosa due to ischemia and I/R, and the damage was more severe in XO-deficient rats. These observations suggest that oxygen-derived free radicals are involved in the intestinal mucosal damage during I/R and infiltrated neutrophils rather than XO may be the primary source of free radicals under these conditions.

Brush border membranes isolated from monkey intestinal mucosa was found to contain considerable amount of nonesterified fatty acids. Incubation of brush border membranes with fatty acid free albumin selectively removed the free fatty acids more than 80% without altering the level of phospholipids or cholesterol. The sodium dependent D-glucose transport was stimulated by the albumin treatment. Kinetic study showed that albumin treatment did not alter the apparent affinity (Km) of the transporter for glucose whereas the maximal velocity (Vmax) was increased significantly. The sodium dependent D-glucose transport was inhibited by the exogenously added unsaturated fatty acids. Saturated fatty acids and methyl esters of unsaturated fatty acids showed no inhibition. Based on these results, it may be concluded that free fatty acids inhibit the sodium dependent intestinal D-glucose transport either by directly interacting with the transport protein or by abolishing the sodium gradient.

The intestinal epithelium is likely to be exposed to oxidants derived from diet, bacterial metabolic products or endogenously formed cellular metabolites. Glutathione, being the major intracellular thiol, provides protection against oxidative injury. The present study looks at the effect of luminal exposure to oxidants on intestinal thiol status. Different oxidants were placed in the lumen of anesthetized rats for 30 min and the mucosal non-protein and protein-bound thiols were quantitated using HPLC. Oxidant exposure altered the thiol redox status and increased the oxidized glutathione (GSSG) level 6-7-fold and protein-bound reduced glutathione (GSH) 2-fold, whereas the non-protein GSH level remained the same or increased slightly. This could be due to either increased mucosal GSH synthesis or transport from circulation. After incubation with buthionine sulfoximine, a specific inhibitor of GSH synthesis, prior to oxidant exposure, the same increase in GSSG and GSH in the mucosa was seen. Oxidant exposure also decreased the plasma GSH level by 50%. The other cellular non-protein thiols, cysteine and cystine, did not change after luminal exposure to oxidants. These results suggest that oxidant exposure stimulates uptake of GSH from the circulation to maintain mucosal GSH.

This study looks at the effect of oxidant exposure on changes in structural components and functional properties of monkey intestinal brush-border membrane vesicles (BBMV). These membranes were found resistant to iron-dependent lipid peroxidation as judged by measurement of various parameters such as formation of malonaldehyde (MDA) and conjugated diene and depletion of total arachidonic acid, tocopherol and membrane-associated protein thiol groups. Free radicals generated by thermal decomposition of 2,2'-azobis(2-amidinopropane) dihydrochloride (ABAP) which does not require iron, were capable of inducing lipid peroxidation in this membrane. Fluorescence polarisation studies used to assess the physical state of the membrane lipids after exposure to various free radical generating systems showed that ABAP could decrease the fluidity of BBMV whereas other systems had no effect. Exposure of BBMV to ABAP or cumene hydroperoxide decreased the glucose and amino acid transport. BBMV had a high content of nonesterified fatty acids as part of the total lipids and removal of these free fatty acids by treatment with fatty acid free albumin made the membranes susceptible to iron-dependent peroxidation. These studies suggest that intestinal epithelial cell membranes are resistant to iron-dependent lipid peroxidation due to the presence of membrane-associated free fatty acids. Possibly lipid peroxidation may play a less significant role in damage to these cells.

The lumen of the gastrointestinal tract is likely to have free iron and prooxidants that might generate free radicals, resulting in structural and functional damage to the absorptive cells. In the present study the effect of luminal exposure of different oxidants on intestinal mucosal lipid peroxidation and absorptive function in anaesthetized animals has been studied. Oxidants that require iron for free radical generation did not have any effect on the mucosa, whereas free radicals generated by using 2,2'azobis (2-amidino propane) dihydrochloride or menadione, which do not require iron, resulted in lipid peroxidation as assessed by malonaldehyde and conjugated diene formation and depletion of the antioxidant alpha-tocopherol. These oxidants also induced water and electrolyte secretion as studied by luminal perfusion. This observation is physiologically significant since the presence of prooxidants along with iron in the lumen may not have a damaging effect on the mucosa.

CuZn superoxide dismutase from monkey (Macaca radiata) intestinal mucosa was purified to homogenity. The enzyme showed a subunit molecular weight of 16000. The enzyme preparation from intestinal mucosa of rat, rabbit, guinea-pig and monkey was distinctly different in electrophoretic mobility and in elution profile on ion-exchange chromatography, possibly due to their difference in charge. The difference may not be due to glycosylation, since the enzyme was not stained for glycoprotein. Polyclonal antibody against purified monkey enzyme inhibited the activity of intestinal CuZn superoxide dismutase from rat, rabbit and guinea-pig. Thus it appears that intestinal CuZn superoxide dismutases from different sources, despite being similar in immunological and other properties, differ in certain amino acids and hence in charge.

Rat liver and intestinal microsomes were exposed to various free radical generating systems and their effect were assessed by studying different parameters such as formation of malonaldehyde (MDA) and conjugated diene, arachidonic acid depletion and alteration in protein thiol groups and tocopherol levels. These studies revealed that liver being highly vulnerable tissue showed all the effects of free radical attack whereas intestinal microsomes were resistant to most oxidants except iron independent generation of free radicals using 2-2'-azobis (2-amidinopropane) dihydrochloride (ABAP). Intestinal microsomes were found to contain considerable amount of non-esterified fatty acids in total lipid fraction as compared to liver microsomes and iron-fatty acid complex may be incapable of participating in peroxidation. In vitro measurement of hydroxyl radical generation showed that intestinal microsomes were incapable of generating these active species. These results suggest that iron dependent free radical mediated lipid peroxidation might not occur in intestinal epithelial cells.

The effect of methylglyoxal on protein -SH and -NH2 groups in cytosolic and membranous fractions of epithelial cells lining the gastrointestinal tract of rat was investigated, using isolated villus and crypt cells (enterocytes) and colonocytes. It was found that 11-12% cytosolic protein -SH and 14-17% membrane protein -SH groups were lost when villus and crypt cells were treated with 2 mM methylglyoxal. In colonocytes, the corresponding loss in protein -SH groups was 46 and 30% under the same treatment. Similarly, 27-37% protein -NH2 group in the cytosolic fraction and 18-19% protein -NH2 group in membranous fractions of the enterocytes were lost by 2 mM methylglyoxal treatment. In colonocytes, the loss of protein -NH2 group was 30 and 15% in cytosolic and membranous fractions, respectively, under the same treatment. Effect of methylglyoxal on activity of various brush border enzymes such as alkaline phosphatase, gamma-glutamyl transpeptidase, leucine aminopeptidase, Mg2(+)-ATPase, sucrase and lactase was also studied. Alkaline phosphatase and gamma-glutamyl transpeptidase activities were inhibited to the extent of 30 and 15% respectively. There was no significant change in the activities of other enzymes after treating the brush border vesicles with 2 mM methylglyoxal. These findings show that methylglyoxal can cause loss of protein thiol and amino groups and enzyme activity in mucosal cells of rat gastrointestinal tract and the effect is more pronounced in colonocytes, which are in constant contact with bacterial metabolites.

Glyoxalase I ((R)-S-lactoylglutathione methylglyoxal-lyase (isomerizing), EC 4.4.1.5) from monkey intestinal mucosa was purified to homogeneity. The purified enzyme had a molecular weight of 48,000, composed of two apparently identical subunits. Active-site modification was carried out on the purified enzyme in presence and absence of S-hexylglutathione, a reversible competitive inhibitor of glyoxalase I. Modification by tetranitromethane and N-acetylimidazole caused inactivation of the enzyme. Inactivation by N-acetylimidazole was reversible with hydroxylamine treatment, suggesting the importance of tyrosine residues for the activity of the enzyme. The enzyme was inactivated by 2-hydroxy-5-nitrobenzyl bromide, N-bromosuccinimide, 2,4,6-trinitrobenzenesulphonic acid, pyridoxal phosphate and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, indicating the importance of tryptophan, lysine and glutamic acid/aspartic acid residues for the activity of the enzyme. The enzyme was inactivated by diethyl pyrocarbonate and the activity was not restored by hydroxylamine treatment, suggesting that histidine residues may not be important for activity. Modification by N-ethylmaleimide and p-hydroxymercuribenzoate did not affect its activity, indicating that sulphydryl groups may not be important for activity. These studies indicated that the amino acids present in the active site of glyoxalase I from intestinal mucosa which may be important for activity are tyrosine, tryptophan, lysine and glutamic acid/aspartic acid residues.

The antioxidant previously isolated from intestinal mucosa has been subjected to further purification and identification. Although this inhibitor moved as a single spot on thin-layer chromatography in a number of different solvent systems, it proved to be a mixture of free carboxylic acids whose relative composition was similar in different batches. Detailed studies involving the use of high-pressure liquid chromatography, combined gas chromatography-mass spectrometry, high-field 360 MHz proton nuclear magnetic resonance spectroscopy, fast atom bombardment mass spectrometry and other techniques established that the inhibitor was a mixture of carboxylic acids of the following identity and relative composition (the major components comprising 92% of the total fatty acids): palmitic acid, 14.8%; palmitoleic acid, 3.6%; stearic acid, 7.0%; oleic acid, 21.0%; linoleic acid, 27.6% arachidonic acid, 18.0%. Mixtures of authentic fatty acids of the same relative concentration showed inhibition of peroxidation, comparable with the purified inhibitor from intestinal mucosa. A study of the inhibitory activity of the components of the mixture using malonaldehyde estimation, diene conjugation and arachidonic acid estimation showed that the inhibitory activity was due to palmitoleic and oleic acids only, the latter being the major component.

Lipid peroxidation in vitro was tested by malonaldehyde production in gastrointestinal mucosa and compared with other tissues. It was observed that gastrointestinal mucosa was resistant to both non-enzymatic and enzymatic lipid peroxidation. This was due to the presence of an inhibitor of lipid peroxidation in the membranous fractions of intestinal mucosa. This inhibitor was capable of inhibiting other recognised peroxidation systems, such as liver mitochondria. This effect was confirmed by measurement of diene conjugation and utilisation of arachidonic acid as other markers of peroxidation, in addition to malonaldehyde production. Preliminary characterisation of this inhibitor revealed that it is resistant to proteolysis, non-diffusable and extractable from membranes by organic solvents. It was partially purified by methanol extraction of the mucosa and by three successive preparative thin-layer chromatography steps. The purified material gave a single spot on thin-layer chromatography, using a number of different solvent systems. Mobility of the inhibitor on thin-layer chromatography was different from that of authentic tocopherol, and it was present in the intestine of vitamin-E-deficient animals. These results suggest that the resistance of intestinal mucosa to lipid peroxidation is due to the presence of a novel inhibitor which is lipidic in nature.