What makes non-cirrhotic portal hypertension a common disease in India? Analysis for environmental factors

In India, an unexplained enteropathy is present in a majority of non-cirrhotic intrahepatic portal hypertension (NCIPH) patients. Small intestinal bacterial contamination and tropical enteropathy could trigger inflammatory stimuli and activate the endothelium in the portal venous system. Groundwater contaminated with arsenic is an environmental factor of epidemic proportions in large areas of India which has similar consequences. Von Willebrand factor (a sticky protein) expressed by activated endothelium may promote formation of platelet microthrombi and occlusion of intrahepatic portal vein branches leading to NCIPH. Environmental factors linked to suboptimal hygiene and sanitation, which enter through the gastrointestinal (GI) tract, predispose to platelet plugging onto activated endothelium in portal microcirculation. Thus, NCIPH, an example of poverty linked thrombophilia, is a disease mainly affecting the lower socio-economic strata of Indian population. Public health measures to improve sanitation, provide clean drinking water and eliminate arsenic contamination of drinking water are urgently needed. Till such time as these environmental factors are addressed, NCIPH is likely to remain 'an Indian disease'.

Determinants of infant mortality in a developing region in rural Andhra Pradesh.

Introduction: Background & Objectives: Despite several interventions implemented for the improvement of infant survival, the pace of decline in infant mortality in the State of Andhra Pradesh during the last two decades has been rather slow. This paper examines determinants of infant mortality in a rural population of about 45000 in Medchal region of Andhra Pradesh, India. Methods: Data obtained through continuous (longitudinal) enumeration of household population and also from the cross-sectional survey of households carried out during 2008-09 in the study area were analyzed. A univariate analysis was carried out and followed by an application of binary logistic regression model to identify significant factors associated with infant mortality. Results: Despite a substantial increase in institutional deliveries in the study area, infant mortality rate remained relatively constant at 43±1. Socioeconomic variables such as education and household economic status and environmental variables such as access to safe drinking water and sanitation facility and use of clean cooking fuel emerged as significant predictors of infant survival. Interpretation & Conclusions: Promoting institutional deliveries by providing cash assistance to women by itself cannot be expected to bring down infant mortality to a low level in the absence of an improvement in socioeconomic and environmental conditions of the households.

Aetiology and clinical profile of osteomalacia in adolescent girls in northern India.

BACKGROUND: The adolescent age group is particularly prone to nutritional rickets/osteomalacia due to an increased demand for nutrients, especially calcium and vitamin D. Osteomalacia presents with non-specific signs and symptoms because of which diagnosis may be delayed. Vitamin D deficiency is unexpected in India, which is a tropical country with abundant sunshine. METHODS: We prospectively studied the clinical presentation, aetiology and social factors contributing to adolescent rickets/ osteomalacia in our region. RESULTS: We saw 21 symptomatic adolescents with osteomalacia during the study period (November 2000-July 2002). All were girls. Only 1 practised purda and 4 belonged to a low socioeconomic class. The mean (SD) duration of illness before correct diagnosis was 2.8 (2.1) years. Bone pains and muscular weakness were universally present. Non-specific complaints (especially limb pains being mistaken for joint involvement) led to a delay in diagnosis with consequent morbidity. All but 1 patient had low serum 25-hydroxyvitamin D levels (<10 ng/ml), with the mean (SD) being 4.9 (2.7) ng/ml. Their mean dietary calcium intake was low 1265 (199) mg/day, range 40-810 mg/day]. Restricted outdoor activities (n = 19) and the traditional dress code (n = 21) were contributory factors, as they led to poor exposure to sunshine. CONCLUSIONS: Nutritional osteomalacia among adolescents is a poorly recognized entity. Even in non-purda practising communities in the tropics, poor exposure to sunshine due to social factors, compounded by low dietary calcium intake, can lead to osteomalacia in adolescents.

Enterocyte mitochondrial dysfunction due to oxidative stress.

The endogenous production of H2O2 in isolated rat intestinal mitochondria and oxidant induced damage to mitochondria were examined. There was an appreciable amount of H2O2 production in presence of succinate, glutamate and pyruvate, while the presence of rotenone with succinate further increased production. Superoxide generated by the X-XO system induced membrane permeability transition (MPT), calcium influx, lipid peroxidation and changes in membrane fluidity in mitochondria. A decreased mitochondrial ATPase activity and uncoupling of respiration was also observed. Spermine inhibited swelling induced by X-XO and also blocked the calcium influx and reversed the membrane fluidity changes.

Decreased phospholipase A2 activity in butyrate differentiated HT29 cells.

Our earlier work has shown that in butyrate differentiated colonic HT29 cells, there is an alteration in phospholipid composition as compared to control. To know more about these changes, butyrate treated and control cell homogenates were incubated in presence of calcium and phospholipids were analyzed. It was observed that incubation with calcium was associated with increase in lysophosphatidylcholine (lysoPC) and free fatty acids and the increase was much higher in control as compared to butyrate treated cells. There was no alteration in lysoPC content. These products are formed by the action of phospholipase A2 (PLA2) which is activated by calcium and suggests that butyrate-induced differentiation is associated with decrease in PLA2 activity.

Oxidative stress response in Shigella & nonpathogenic gut bacteria.

The effect of oxidative stress in the form of exogenous H2O2 on the survival of four species of Shigella and two nonpathogenic Gram negative gut bacteria and the role of catalase as an antioxidant enzyme, neutralizing the effect of H2O2 were examined. A significant decrease in the number of colony forming units (CFUs) after exposure to exogenous H2O2 (122 +/- 37), compared to control bacteria (218 +/- 63, P < 0.001) was observed. There was an induction of catalase activity after exposure to exogenous H2O2 and the specific activity of catalase in H2O2 exposed bacteria was significantly increased (2.88 +/- 1.25), compared to control bacteria (1.5 +/- 0.44; P < 0.05). A direct correlation was observed between the decrease in bacterial counts and increase in catalase activity after exposure of H2O2 (regression coefficient (0.56). Gut bacteria appear to be susceptible to oxidative stress and inducible catalase activity may form an important part of the antioxidant defence mechanism against oxidative stress.

Microtiter plate assay for superoxide dismutase using MTT reduction by superoxide.

A simple microtiter plate based colorimetric assay for superoxide dismutase is described. The method, involves generation of superoxide by pyrogallol autoxidation and the inhibition of superoxide dependent reduction of the tetrazolium dye MTT [3-(4,5-dimethyl-thiazol-2-yl) 2,5-diphenyl tetrazolium bromide] to its formazan, measured at 570 nm. The reaction was terminated by the addition of dimethyl sulfoxide (DMSO) which also helps to solubilize the formazan formed and the colour evolved was stable for many hours. The method was compared with other known methods to measure the activity of purified erythrocyte Cu,ZnSOD and superoxide dismutase activity from various rat tissues. This procedure involves inexpensive reagents, allows a rapid and sensitive measurement of SOD activity and the microtiter plate assay is suitable for use with large number of samples.

Precios de medicamentos al por menor: la ley de la jungla / Costs of medicaments by units: the jungle law

Un analisis critico del precio al por menor de 21 medicamentos comunmente utilizados en 39 paises alrededor del mundo lleva a concluir que el fijar precios a los medicamentos se asemeja a la ley de la jungla, donde lo que se puede resulta correcto. Los datos presentados sugieren que la guia principal que al parecer la industria de medicamentos adopta, al fijar sus precios, es establecer los limites mas altos de acuerdo con lo que el mercado pueda soportar. La maximalizacion de utilidades parece ser el unico objetivo de la industria

A microtiter plate assay for superoxide using MTT reduction method.

A simple, rapid and sensitive microtiter plate assay for superoxide using the reduction of tetrazolium dye MTT to its coloured formazan has been developed. The colour formed can be measured using a microtiter plate reader and the extent of reduction of MTT indicates the amount of superoxide generation. A comparison of the sensitivities of different procedures for the quantitation of superoxide generated by X-XO system has been made. The MTT reduction due to superoxide was confirmed by inhibiting the reduction using purified superoxide dismutase. Using this method superoxide generation by mitochondria and microsomes was demonstrated and this procedure is suitable for detection of intracellularly generated superoxide. The proposed method is inexpensive and is suitable for a routine analysis of large number of samples.

Effect of enterotoxin on glutathione status in the intestinal mucosa.

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.

Effect of ischemia/reperfusion on intestinal brush border membrane lipid composition, fluidity and enzyme activities.

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.

Lipid composition and membrane fluidity of monkey small intestinal brush border membrane: regional differences.

Brush border membrane vesicles (BBMV) isolated from various regions of monkey small intestine were compared for lipid composition, membrane fluidity and sodium-dependent D-glucose transport. Total phospholipid content was same in all regions whereas cholesterol content was high in duodenum and jejunum as compared to ileum resulting in higher cholesterol/phospholipid molar ratios. Regional differences in individual phospholipid subclasses and fatty acids in total lipids were also observed. Fluidity measurements showed that the membranes of duodenum and jejunum were less fluid than ileum. The change in BBMV fluidity appears to be due to alteration in cholesterol/phospholipid ratio and phospholipid composition. The sodium dependent D-glucose uptake was more in duodenum and jejunum as compared to ileum. These results suggest that there is a regional difference in the lipid composition and fluidity of BBMV in monkey small intestine which may influence their function.

Preliminary study of androgen, thyroid & adrenal status in alcoholic men during deaddiction.

To evaluate the status of the testes, thyroid, and adrenals in male alcoholics during the period of voluntary abstinence and therapy, chronic male drinkers undergoing a 4 wk inpatient deaddiction programme in a social hospital were recruited. Levels of a few serum and urinary hormones/metabolites viz., serum testosterone, total triiodothyronine (T3) and thyroxine (T4) and urinary total 17-ketosteroids (17-KS), estrone, estradiol, and 17-hydroxy corticosteroids (17-OHCS) were assessed in alcoholics thrice during the treatment programme at hospital i.e., on the zero (day of admission), 10th, and 20th day and compared to those of non-alcoholic controls. Alcoholics registered elevated serum total T3, and reduced total T4 and testosterone levels at admission, which persisted even after 20 days of the rehabilitative programme. Markedly high urinary levels of total 17-KS, estrone, and 17-OHCS were observed on zero day of admission. Urinary estrone and 17-OHCS, unlike total 17-KS, showed a trend to return to the normal range during the 20 days period. Urinary estradiol levels, however, recorded no significant alteration. The results of this preliminary study are suggestive of alcohol-induced perturbations on the functional integrity of the testes, thyroid, and adrenal in male alcohol addicts, wherein 20 days period of total alcohol abstinence and rehabilitative programme failed to reverse alcohol-induced hypoandrogenization and altered thyroidal status, but only partially restored certain biochemical events associated with the excretion of steroid metabolites.

Lipid peroxidation of colonocyte membranes.

Effects of various oxidants on the colonic membrane lipid peroxidation have been studied in rats. 2,2-Azobis (2-amidinopropane) dihydrochloride (ABAP), which generates free radicals by thermal decomposition, induced peroxidation as judged by the formation of conjugated diene, malondialdehyde (MDA), and depletion of arachidonic acid. Exposure to other oxidants which require free iron for peroxidation was ineffective. Alpha tocopherol level was not altered on exposure to various oxidants except with ABAP which depleted its level in these membranes. Exposure of the membranes to both ABAP and xanthine-xanthine oxidase (X-XO) decreased total protein thiols, whereas other oxidants had no significant effect. Isolated colonocyte membranes were found to contain considerable amount of nonesterified fatty acids as part of the total lipids and removal of free fatty acids from the membrane using fatty acid-free albumin made the membranes susceptible to iron-induced free radical generation and lipid peroxidation. These studies suggest that colonocytes are possibly protected from lipid peroxidation by the free fatty acids associated with the membrane.

Studies on nonesterified fatty acids in rat intestinal cell membranes.

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.

Studies on iron binding by free fatty acids.

The effect of various saturated and unsaturated fatty acids on iron binding and the translocation of this complex into the organic phase were studied. The amount of iron translocated into the organic phase depended on the affinity of the respective fatty acid to iron. It was seen that saturated and monounsaturated fatty acids translocated more iron compared to polyunsaturated fatty acids. Positional isomers of oleic acid were as effective as oleic acid in translocating iron. It appears that the free carboxyl group on the fatty acid is essential, as the methyl ester of the fatty acid failed to translocate iron. Effect of varying the concentration of fatty acid and iron on iron translocation suggested the requirement of 3 to 4 moles of fatty acid for 1 mole of iron binding. Divalent cations such as Ca2+ and Mg2+ or lipids such as cholesterol or triolein did not compete with fatty acids but there was reduced translocation when phosphate buffer or phospholipid was included in the incubation medium, suggesting that phosphate interferes with the formation of fatty acid-iron complex. Thus it seems that free fatty acids are capable of forming a complex with iron.

Specific activities of seminal vesicular phosphomonoesterases and Mg2+-,Ca2+- and Na+/K(+)-adenosine triphosphatases in hypo- and hyperthyroid albino rats.

Hypothyroidism (surgical thyroidectomy) inhibited the activities of acid phosphatase and Mg(2+)-ATPase in seminal vesicular tissue and fluid and that of Ca(2+)- and Na+/K(+)-ATPases in fluid alone, and T4 supplementation restored normalcy in all, except acid phosphatase. Hyperthyroidism (T4 25 micrograms/100g body weight/day for 60 days, im) enhanced the activities of alkaline phosphatase and ATPases in seminal vesicular tissue and fluid, and decreased acid phosphatase activity in tissue alone. Withdrawal of T4 treatment from hyperthyroid rats (after 30 days) augmented the activity of ATPases in tissue and impaired the same in fluid, while phosphomonoesterases remained at hyperthyroid level. The results suggest specific responses of various seminal vesicular phosphatases to altered thyroid hormone status. Modification in the specific threshold of androgen/estrogen action on different phosphatases in seminal vesicles appears to be the plausible mechanism underlying these changes in hypo- and hyperthyroid conditions.

Modulation of monkey small intestinal brush border membrane D-glucose transport by nonesterified fatty acids.

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.