Kinetics of the attachment of intrinsic factor-bound cobamides to ileal receptors.

To determine whether the molecular configuration of vitamin B(12) influences the attachment of intrinsic factor-vitamin B(12) complex to ileal microvillous membrane receptor sites, we have examined the kinetics of uptake of intrinsic factor-bound cyanocobalamin by brush borders and microvillous membranes isolated from guinea pig ileum, and have compared this uptake with that of intrinsic factor alone and with that of intrinsic factor complexed with various analogs of cyanocobalamin. We first studied the kinetics of binding of cyanocobalamin and other cobamides to human gastric intrinsic factor. The binding of cyanocobalamin showed saturation kinetics and, at relatively high concentrations of cyanocobalamin, a Scatchard plot of binding was linear. The dissociation constant for the intrinsic factor-cyanocobalamin complex was 0.066 nM. When the binding of various vitamin B(12) analogs to intrinsic factor was determined by competition experiments, the analogs could be separated into two categories: those with affinities similar to that of cyanocobalamin and those with affinities much lower than that of cyanocobalamin. The affinity of cyanocobalamin for intrinsic factor was not altered by various substitutions at the -CN position, while removal of a single amido group on the corrin ring of substitution of the dimethylbenzimidazole base greatly reduced affinity. Removal of the base totally abolished binding. These findings, confirming those reported by others, are consistent with the concept that the cyanocobalamin molecule fits into a "pocket" in the intrinsic factor molecule, with the nucleotide base facing inward and the -CN side of the planar corrin ring facing outward. We then investigated the attachment of intrinsic factor-bound cyanocobalamin to ileal receptor. Attachment to microvillous membranes showed saturation kinetics with a dissociation constant of 0.25 nM. Attachment was rapid and was 70% complete within 5 min; the second-order rate constant for attachment was 1.3 x 10(6) M(-1) s(-1). The half-time for dissociation of intrinsic factor-bound cyanocobalamin from the ileal receptor was approximately 35 min. Free intrinsic factor inhibited the attachment of intrinsic factor-bound cyanocobalamin, but the rate of attachment of free intrinsic factor was slower than that of intrinsic factor bound to cyanocobalamin. When intrinsic factor was complexed with various analogs of cyanocobalamin, the affinities of these complexes for ileal microvillous membranes were similar to that of intrinsic factor-bound cyanocobalamin. These findings suggest that the molecular configuration of vitamin B(12) is not a major determinant in the interaction between intrinsic factor-bound vitamin B(12) and its ileal receptor site.

Bacterial lipopolysaccharide-induced intestinal microvascular lesions leading to acute diarrhea.

Subcutaneous challenge of mice with lipopolysaccharide (LPS) from gram negative bacteria, produced an intestinal microvascular lesion causing fluid exudation into the lumen of the intestine and diarrhea. The microvascular lesion was characterized by endothelial cell damage and microthrombi in the venules and capillaries of the intestinal lamina propria. Marker organisms, given orally to challenged mice, grew in the exuded fluid and could invade the mucosa. Intravenous transfer of postchallenge plasma produced the lesion in normal mice and absorption of such plasma by Sepharose coupled to LPS-antibody abolished this effect. Instillation of large quantities of LPS into the lumen of the intestine produced scattered microvascular lesions, although none of these animals developed diarrhea. Since a similar microvascular lesion has been described in the rectal mucosal lamina propria of adults with acute diarrhea, it is suggested that LPS-induced vascular damage may be a novel mechanism in the pathogenesis of acute diarrhea.

An unidentified inhibitor of lipid peroxidation in intestinal mucosa.

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.

Interaction of melittin with endotoxic lipid A.

Several amphipathic and cationic substances are known to bind lipid A, the toxic component of bacterial lipopolysaccharides. In this report, we have characterized, by fluorescence methods, the interaction of melittin, an amphipathic and basic 26-residue polypeptide isolated from bee venom, with lipid A. The stoichiometry of the complex appears to be two molecules of melittin to one of lipid A with a dissociation constant of 2.5 x 10(-6) M. The binding of melittin not only modifies the endotoxic properties of lipid A in a number of biological assays, but also results in abrogation of the hemolytic activity of melittin. A model of the complex is proposed based on the known structures of lipid A and melittin, and the observed stoichiometry of binding.

Analysis of the binding of polymyxin B to endotoxic lipid A and core glycolipid using a fluorescent displacement probe.

Dansylcadaverine, a cationic fluorescent probe binds to bacterial lipopolysaccharide and lipid A, and is displaced competitively by other compounds which possess affinity toward endotoxins. The binding parameters of dansylcadaverine for lipid A were determined by Scatchard analysis to be two apparently equivalent sites with apparent dissociation constants (Kd) ranging between 16 microM to 26 microM, while that obtained for core glycolipid from Salmonella minnesota Re595 yielded a Kd of 22 microM to 28 microM with three binding sites. The Kd of polymyxin B for lipid A was computed from dansylcadaverine displacement by the method of Horovitz and Levitzki (Horovitz, A., and Levitzki, A. (1987) Proc. Natl. Acad. Sci. USA 84, 6654-6658). The applicability of this method for analyzing fluorescence data was validated by comparing the Kds of melittin for lipid A obtained by direct Scatchard analysis, and by the Horovitz-Levitzki method. The displacement of dansylcadaverine from lipid A by polymyxin B was distinctly biphasic with Kds for polymyxin B-lipid A interactions corresponding to 0.4 microM and 1.5 microM, probably resulting as a consequence of lipid A being a mixture of mono- and di-phosphoryl species. This was not observed with core glycolipid, for which the Kd for polymyxin was estimated to range from 1.1 microM to 5.8 microM. The use of dansylcadaverine as a displacement probe offers a novel and convenient method of quantitating the interactions of a wide variety of substances with lipid A.

Interaction of cationic amphiphilic drugs with lipid A: implications for development of endotoxin antagonists.

This report presents evidence for the interactions of several classes of cationic amphiphilic drugs including the phenothiazines, aminoquinolines, biguanides, and aromatic diamidines, with lipid A, the endotoxic principle of lipopolysaccharides. The interactions of the drugs were quantitatively assessed by fluorescence methods. The affinities of the drugs for lipid A parallel their endotoxin-antagonistic effects in the Limulus gelation assay. Dicationic compounds bind lipid A with greater affinity; the affinity of such molecules increases exponentially as a function of the distance between the basic moieties. The bis-amidine drug--pentamidine--examined in greater detail, binds lipid A with high affinity (apparent Kd: 0.12 microM), and LPS, probably due to simultaneous interactions of the terminal amidine groups with the anionic phosphates on lipid A. The sequestration of endotoxin by pentamidine reduces its propensity to bind to cells, and the complex exhibits attenuated toxicity in biological assays. These results have implications in the development of therapeutic strategies against endotoxin-related disease states.

Purification and chemical characterisation of the inhibitor of lipid peroxidation from intestinal mucosa.

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.

Evidence regarding the minimal daily requirement of dietary vitamin B12.

The minimal daily requirement of dietary vitamin B12 is defined as that amount which must be ingested in the diet to prevent the occurrence of megaloblastosis, anemia, or other manifestations of vitamin B12 deficiency. An attempt has been made to define this requirement by studying hematological responses to varying amounts of the vitamin in five south Indian subjects with vitamin B12 deficiency anemia (one due to gastric atrophy and four due to dietary deficiency). Amounts ranging from 0.07 to 0.25 micrograms of dietary vitamin B12 were shown to be inadequate but amounts ranging from 0.3 to 0.65 microgram were adequate or possibly more than adequate. It is concluded that the minimal daily requirement of dietary vitamin B12 for the average south Indian adult is in the region of 0.5 microgram/day and that an intake of 1.0 microgram/day would cover the needs of the vast majority of the population and allow a wide margin of safety.

Absorption of nutrient energy in southern Indian control subjects and patients with tropical sprue.

Fecal energy excretion was measured by bomb calorimetry, in a group of 30 healthy volunteers and 15 patients with tropical sprue, to determine the absorption of nutrient energy. The mean energy absorption in the healthy volunteers (91.6%) was less than in similar subjects in temperate climates. The reduction of energy absorption in the volunteers, who all have tropical enteropathy, suggests that this condition leads to wastage of 5% of energy intake. In patients with tropical sprue the energy absorption was significantly lower. A strict metabolic balance study was not essential to detect energy malabsorbers.

Plasma lipids in tropical sprue.

Previous studies indicated that many patients with tropical sprue in southern India have triglyceride accumulation within the cells of the intestinal mucosa. This could be due to essential fatty acid deficiency as a result of steatorrhea in subjects on a diet normally low in linoleic acid. Plasma lipids have, therefore, been studied in patients with tropical sprue and the results compared to values observed in healthy controls. The low intake of linoleic acid was found to be adequate to maintain normal levels of arachidonic acid and to repress the formation of 5,8,11-eicosatrienoic acid in rural controls. Patients with tropical sprue had decreased levels of plasma linoleic acid, raised monoenoic fatty acids and the appearance of 5,8,11-eicosatrienoic acid in the lecithin fraction. All of these observations indicate essential fatty acid depletion although unequivocal evidence of essential fatty acid deficiency was not present in any of the patients. The degree of essential fatty acid depletion observed is unlikely to be the cause of the mucosal accumulation of triglyceride in tropical sprue.

Enzyme-linked immunosorbent assay for quantification of anti-lipid A antibody. Enhancement of sensitivity by antigen immobilization at low pH.

An enzyme-linked immunosorbent assay (ELISA) to quantitate anti-lipid A antibodies in sera has been developed. The sensitivity of the ELISA was improved when the antigen (lipid A) was immobilized at pH 2.0, presumably by enhanced solid-phase adsorption of lipid A which is presumed to be aggregated at low pH. This was also verified by solid-phase immunoradiometry in which a four-fold improvement in the signal-to-noise ratio was observed when antigen coating was performed at pH 2.0 as compared to coating at pH 9.6. The enhanced sensitivity permitted the use of low concentrations of lipid A (10 micrograms/ml) for antigen coating of microtiter plates. The assay was able to clearly detect differences in IgG anti-lipid A levels between patients with chronic liver disease and normal controls.

One-hour serum xylose as an absorption test in the tropics.

The 1-hour serum xylose (surface area corrected) as an indication of xylose absorption after 5 g oral D-xylose has been compared with the 5-hour urine excretion test in a tropical population. The study confirmed that the peak serum xylose concentration occurs at 1 hour and that correction to a constant body surface are improves the discrimination between subjects with normal and impaired xylose absorption. The significantly lower reference range for the 1-hour surface area corrected serum xylose (0.55-1.11 mmol/l) compared to the UK figure reflects the reduced absorptive capacity of the jejunum, a result of tropical enteropathy. In view of the difficulties in obtaining accurate urine collections in tropical countries, especially in field studies, it is recommended that the 1-hour serum xylose (surface area corrected) should be adopted as the standard test of xylose absorption.

Salicylate inhibits fimbriae mediated HEp-2 cell adherence of and haemagglutination by enteroaggregative Escherichia coli.

Enteroaggregative Escherichia coli (EAggEC) are associated with both acute and persistent diarrhoea in children. Bowel colonisation due to fimbrial adherence factors appears to play a major role in the disease process. In this study, we investigated the effect of sodium salicylate and 5-aminosalicylic acid on adherence of a type strain and 40 clinical isolates of EAggEC to HEp-2 cells and erythrocytes from different species. Growth in the presence of 10 mM salicylate resulted in markedly decreased adherence to tissue culture cells with 33/40 (82.5%) isolates, and was also associated with inhibition of haemagglutination in 20/33 (60.6%) isolates. Complete or partial inhibition of adherence was also seen in two of five isolates showing localised adherence and three of five isolates with diffuse adherence. Decrease in adherence was associated with decreased or absent expression of fimbriae in 28/40 (70%) of the EAggEC isolates, although production of outer membrane proteins was not affected. Salicylates appear to inhibit adherence mediated by fimbrial adhesins.

The prevalence of bacterial intestinal pathogens in a healthy rural population in southern India.

In a one-year prospective survey bacterial intestinal pathogens unassociated with diarrhoeal episodes were isolated from 20.5% of stool samples from 48.5% of a stratified random sample of the population of a village in southern India. Campylobacter jejuni was the pathogen most frequently isolated, followed by enteropathogenic serotypes of Escherichia coli. The incidence of diarrhoea in the study population was lower than the frequency of isolation of bacterial intestinal pathogens. It is necessary to understand the prevalence of intestinal pathogens in this ecosystem to know the dynamics of intestinal infection and the pathogenesis of diarrhoea.

Bacterial flora of the jejunum: a comparison of luminal aspirate and mucosal biopsy.

The aerobic and anaerobic bacterial flora grown from 40 human jejunal aspirates were compared with the flora grown from an intestinal mucosal biopsy obtained simultaneously from the same level. In four paired samples the flora was identical; in nine the flora differed by only one organism. In six pairs the flora differed by two organisms, and in 11 pairs by three or more organisms. In nine pairs the jejunal aspirate grew only one organism or was sterile, but the biopsy showed considerable numbers of organisms. In one pair the jejunal aspirate grew organisms, but there was no growth from the biopsy. It is apparent that for adequate bacteriological study of the intestine, the flora at both sites should be investigated.

A comparison of the susceptibility of three human gut tumour-derived differentiated epithelial cell lines, primary monkey kidney cells and human rhabdomyosarcoma cell line to 66-prototype strains of human enteroviruses.

The growth of prototype strains of 31 serotypes of ECHO, 3 polio, 6 Coxsackie B, 24 Coxsackie A and enterovirus serotypes 70 and 71 were tested in parallel in primary monkey kidney cells (PMK), RD cells and three gut tumour-derived differentiated epithelial cell lines (HRT-18 HT-29 in SKCO-1). All 31 serotypes of ECHO viruses grew in HT-29, 27 and SKCO-1, 5 in HRT-18, 29 in PMK and 29 in RD. There was good growth of poliovirus serotypes in all five cell types. Coxsackie B viruses grew well in all the cell lines except RD. Fifteen of the Coxsackie A viruses grew in SKCO-1, 4 in HT-29, 3 in HRT-18 and 7 in RD. Enterovirus serotypes 70 and 71 grew only in RD cells after 3 serial passages. These results showed that 2 of the gut tumour-derived cell lines, HT-29 and SKCO-1 had a markedly wider susceptibility, with comparable or wider sensitivity, for enteroviruses, than PMK and RD. While their use for field isolation from clinical samples is not yet fully established HT-29 and SKCO-1 would appear to be ideal for a variety of laboratory manipulations of the majority of enteroviruses.

Tropical sprue in southern India.

Tropical sprue, a primary malabsorption syndrome affecting residents and visitors to several tropical regions, occurs in southern India in endemic and epidemic forms. The stomach, the small intestine and colon are affected and malabsorption results in nutrient deficiency. Enterocyte damage, the primary lesion in southern Indian tropical sprue, is the result of a persistent lesion of the stem cell compartment. This lesion occurs on a background of tropical enteropathy and the available evidence suggests that an immunity conferring agent may be responsible for initiating the damage.

Jejunal surface pH measurements in tropical sprue.

The mucosal surface pH of jejunal biopsy samples incubated in vitro in Krebs-phosphate buffer was measured. Biopsies from 7 healthy individuals or tropical sprue subjects in complete remission had a mean surface pH of 5.8 +/- 0.09, similar to values for normal Caucasian subjects living in the UK. The mean surface pH of biopsies from 20 sprue patients, 6.0 +/- 0.08, was significantly higher (P less than 0.05) than that of the control subjects. Sprue patients could be sub-divided into those with 2 or more abnormal results in 3 intestinal function tests, and those with one or no abnormal test. The 9 low scorers showed a mean surface pH of 5.75 +/- 0.06 resembling the control mean, whereas the 11 high scorers had a higher (P less than 0.01) mean surface pH of 6.17 +/- 0.08. Mucosal surface pH correlated directly with 3 d mean faecal fat excretion and inversely with xylose and vitamin B12 absorption values but not with the nutritional indicators serum albumen, folate or blood haemoglobin levels. As in coeliac disease, mucosal surface pH in the jejunum is elevated above normal in tropical sprue and may reflect the extent to which normal ion transport processes are affected.

Food-based solutions are a viable alternative to glucose-electrolyte solutions for oral hydration in acute diarrhoea--studies in a rat model of secretory diarrhoea.

A survey of acute diarrhoea and its treatment, in 3 groups of villages in south India, revealed that use of the World Health Organization oral rehydration solution (WHO-ORS) was poor or virtually non-existent and that several liquid foods were given to children during acute diarrhoea. The effects of the most commonly used, boiled and cooled supernatants of these liquid foods [rice (Oryza sativa)-water, ragi (Eleusine coracana)-water, arrowroot (Maranta arundinacea)-water], and tender coconut-water, and of the bicarbonate- and citrate-WHO-ORS on intestinal water transport were evaluated using a rat model of secretory diarrhoea. All solutions either decreased cholera toxin-induced net water secretion (arrowroot-water) or reversed it to net absorption. Ragi-water produced maximum net water absorption, significantly greater than the WHO oral rehydration solutions. WHO-ORS utilization is poor in some developing countries, and locally used food-based solutions could be used for maintaining hydration or correcting the dehydration due to acute diarrhoea once their effectiveness has been proved by clinical trials.

The relative merits of faecal and duodenal juice microscopy in the diagnosis of giardiasis.

We compared the diagnostic accuracy of microscopical examination of multiple faecal specimens with duodenal juice examination in the diagnosis of giardiasis. Of 292 patients who had stool microscopy and duodenal aspirate, Giardia were identified in either stools or duodenal fluid from 73 patients (25%). Giardiasis was diagnosed in 62 (73%) with the first faecal specimen, but examination of 3 specimens increased the diagnostic yield to 85%. Giardia, however, were found in only 32 of 73 duodenal aspirates examined (44%). This finding is contrary to the widely held belief that duodenal fluid examination is superior to stool microscopy for the diagnosis of giardiasis. The 2 approaches are complementary, however, since Giardia was found in duodenal fluid, only, from 15% of patients.