A double-blind, randomized, placebo-controlled trial of the short-term effect of vitamin D3 supplementation on insulin sensitivity in apparently healthy, middle-aged, centrally obese men.

AIM: To determine the short-term effect of vitamin D(3) supplementation on insulin sensitivity in apparently healthy, middle-aged, centrally obese men. SUBJECTS AND METHODS: A double-blind randomized controlled trial was conducted at a tertiary care facility in which 100 male volunteers aged > or = 35 years received three doses of vitamin D(3) (120,000 IU each; supplemented group) fortnightly or placebo (control group). Hepatic fasting insulin sensitivity [homeostasis model assessment (HOMA), quantitative insulin-sensitivity check index, HOMA-2], postprandial insulin sensitivity [oral glucose insulin sensitivity (OGIS)], insulin secretion (HOMA%B, HOMA2-%B), lipid profile and blood pressure were measured at baseline and at 6 weeks' follow-up. RESULTS: Seventy-one of the recruited subjects completed the study (35 in supplemented group, 36 in control group). There was an increase in OGIS with supplementation by per protocol analysis (P = 0.038; intention-to-treat analysis P = 0.055). The age- and baseline 25-hydroxyvitamin D level-adjusted difference in change in OGIS was highly significant (mean difference 41.1 +/- 15.5; P = 0.01). No changes in secondary outcome measures (insulin secretion, basal indices of insulin sensitivity, blood pressure or lipid profile) were found with supplementation. CONCLUSION: The trial indicates that vitamin D(3) supplementation improves postprandial insulin sensitivity (OGIS) in apparently healthy men likely to have insulin resistance (centrally obese but non-diabetic).

Plasma membrane calcium pump activity in rat pancreatic islets: an accurate method to measure its calcium-dependent modulation.

The aim of this study was to quantify the glucose modulation of the plasma membrane calcium pump (PMCA) function in rat pancreatic islets. Ca2+-ATPase activity and levels of phosphorylated PMCA intermediates both transiently declined to a minimum in response to stimulation by glucose. Strictly dependent on Ca2+ concentration, this inhibitory effect was fully expressed at physiological concentrations of the cation (less than 0.5 muM), then progressively diminished at higher concentrations. These results, together with those previously reported on the effects of insulin secretagogues and blockers on the activity, expression and cellular distribution of the PMCA, support the concept that the PMCA plays a key role in the regulation of Ca2+ signaling and insulin secretion in pancreatic islets.

Enhanced crystallization of the Cys18 to Ser mutant of bovine gammaB crystallin.

The cysteine residues of the gamma crystallins, a family of ocular lens proteins, are involved in the aggregation and phase separation of these proteins. Both these phenomena are implicated in cataract formation. We have used bovine gammaB crystallin as a model system to study the role of the individual cysteine residues in the aggregation and phase separation of the gamma crystallins. Here, we compare the thermodynamic and kinetic behavior of the recombinant wild-type protein (WT) and the Cys18 to Ser (C18S) mutant. We find that the solubilities of the two proteins are similar. The kinetics of crystallization, however, are different. The WT crystallizes slowly enough for the metastable liquid-liquid coexistence to be easily observed. C18S, on the other hand, crystallizes rapidly; the metastable coexisting liquid phases of the pure mutant do not form. Nevertheless, the coexistence curve of C18S can be determined provided that crystallization is kinetically suppressed. In this way we found that the coexistence curve coincides with that of the WT. Despite the difference in the kinetics of crystallization, the two proteins were found to have the same crystal forms and almost identical X-ray structures. Our results demonstrate that even conservative point mutations can bring about dramatic changes in the kinetics of crystallization. The implications of our findings for cataract formation and protein crystallization are discussed.

Theoretical and experimental basis for the inhibition of cataract.

Aggregation of the lens proteins to form high molecular weight clusters is a major contributing factor in age-onset nuclear cataract [Benedek, G. B. (1971) Theory of transparency of the eye. Appl. Optics, 10, 459-473]. This aggregation occurs continually throughout life and contributes to an exponential increase, as a function of age, in the intensity of the light backscattered out of the lens. The time constant deltaT for this exponential increase in human populations is a valuable index, helpful for conducting clinical trials. In-vitro studies have identified reagents capable of inhibiting high molecular weight aggregate formation, as well as the non-covalent interprotein interactions responsible for phase separation. These reagents are also found experimentally to be effective cataract inhibitors in animal model systems in vivo. We believe that the stage is now set for human clinical trials of putative cataract inhibitors. We present rough quantitative estimates of the trial parameters needed to assure an unambiguous determination of efficacy in a trial population. Such a trial simply requires a measurement of the time constant deltaT in the treated population relative to the untreated population. A successful outcome of the trial is indicated if deltaT increases by 20% over that found for the untreated population. Our estimates suggest efficacy could be determined in a two year trial involving about 300 subjects in the treated group.

Aggregation in aqueous solutions of bovine lens gamma-crystallins: special role of gamma(s).

PURPOSE: To study the aggregation of bovine gamma-crystallins in aqueous solutions and the effect of gamma(s)-crystallin on the aggregation of other gamma-crystallins. METHODS: Aggregation in aqueous solutions of gamma(s)-, gammaII-, and gammaIVa-crystallin was monitored by quasielastic light scattering. Aggregation in mixtures of gamma(s)- and gammaII-crystallin, and gamma(s)- and gammaIVa-crystallin, was also monitored by light scattering. RESULTS: All of the gamma-crystallins studied formed large aggregates (or "megamers") in aqueous solutions. However, each protein differed in the relative rates of formation of megamers. Gamma(s)-crystallin formed megamers much more slowly than gammaII- and gammaIVa-crystallin. In solutions containing mixtures of gammaII and gamma(s), and gammaIVa and gamma(s), gamma(s)-crystallin significantly suppressed the aggregation of gammaII- and gammaIVa-crystallin. Megamerization seemed to be associated with thiol oxidation in these proteins. CONCLUSIONS: Gamma-crystallins undergo aggregation in which a small fraction of the proteins form a few large aggregates, whereas the larger proportion of the proteins remain monomeric. This suggests that the megamerization is preceded by an initial modification of the protein. The modification is associated with the thiol groups, and only such modified protein species participate in megamerization. The presence of gamma(s) significantly slows the megamerization of gammaII- and gammaIVa-crystallin. This fact, together with the previous finding that gamma(s) strongly reduces the phase separation temperatures of the gamma-crystallins, suggests that gamma(s)-crystallin plays an important role in suppressing the formation of light-scattering elements and helps maintain lens transparency.

Glucocorticoid receptors in bronchoalveolar lavage fluid in sarcoidosis. A preliminary report.

Glucocorticoid receptor (GR) content was measured in bronchoalveolar lavage fluid (BALF) cells of 20 untreated patients with sarcoidosis. A significantly higher (p less than 0.001) lymphocyte count and GR content of BALF cells was noted in patients with sarcoidosis as compared to normal volunteers. The GR content correlated positively with lymphocytes (%) (r = 0.6; p less than 0.01) and was significantly higher (p less than 0.001) in patients who showed roentgenographic improvement, physiologic improvement, and improvement in both parameters compared to those who did not show improvement. Above findings suggest that the GR content of BALF cells is increased in sarcoidosis and a major contribution to this may be made by the lymphocytes.

Bronchoalveolar lavage in pulmonary tuberculosis: a decision analysis approach.

We assessed the utility of bronchoalveolar lavage (BAL) in the diagnosis of pulmonary tuberculosis (PTB) in 50 consecutive HIV-negative patients with clinical and radiographic findings suggestive of PTB, but with negative microscopy for acid-fast bacilli (AFB) on sputum smear. Patients were grouped, using a scoring system, into relative likelihoods of having PTB (I-IV, in descending probability). Patients were started on anti-tuberculosis treatment according to the BAL results. Bacteriological diagnosis of PTB was confirmed in 22/50 BAL; 11 (91.6%), seven (37%) and four (40%) of groups I-III, respectively. In 13 cases, an early diagnosis of PTB was made by positive microscopy for AFB on BAL; an alternative diagnosis was made in six cases (bacterial pneumonia 4, carcinoma 2). A decision analysis model was created to assess the overall utility of BAL. This suggested that in a region of high PTB prevalence, and when the clinical diagnosis of PTB is likely, empirical treatment is the best course of action, with BAL being reserved for further investigation of non-responders. Early BAL should be considered when the diagnosis of PTB is uncertain.

Clinical profile, laboratory characteristics and outcome in miliary tuberculosis.

We report a retrospective series of 100 non-HIV adult patients with miliary tuberculosis (MTB) treated in a tertiary care centre. There were 51 males. Their mean age was 35 years. Predisposing conditions existed in 34. Twelve patients had larger-than-miliary (> 2 mm) shadows in their chest roentgenograms. Five presented with acute respiratory failure, and early treatment cured four of them. Hyponatraemia occurred in 42/60 patients (70%) for whom values were available. Twelve patients (12%) died of MTB. Temperature > or = 39.3 degrees C (p < 0.01), hypoalbuminaemia (p < 0.01), hyponatraemia (p < 0.001), history of vomiting (p < 0.001) and presence of crepitations on auscultation (p < 0.001) were independent predictors of mortality. Diagnosis of MTB is difficult even in an endemic area, as the clinical symptoms are non-specific and the chest roentgenograms do not always reveal classical miliary changes. A high index of clinical suspicion and diligent efforts in confirming the diagnosis are needed, as early therapy yields good results.

Oligomerization and phase separation in globular protein solutions.

We have chemically crosslinked a globular protein, gamma IIIb-crystallin, to produce a system of well-defined oligomers: monomers, dimers, trimers and a mixture of higher n-mers. Gel electrophoresis, size exclusion chromatography, quasielastic light scattering spectroscopy, and electrospray ionization mass spectrometry were used to characterize the oligomers formed. The liquid-liquid phase separation boundaries of the various oligomers were measured. We find that at a given concentration the phase separation temperature strongly increases with the molecular weight of the oligomers. This phase behavior is very similar to previous findings for gamma II-crystallin, for which oxidation-induced oligomerization is accompanied by an increase in the phase separation temperature. These findings imply that for phase separation, the detailed changes of the surface properties of the proteins are less important than the purely steric effects of oligomerization.

Long-term effect of a continuous low dose of megestrol acetate on the genital organs and fertility of female rats.

PIP: Micro doses (1.5 mcg/rat/day) of megestrol acetate (6-methyl-17beta-acetoxypregna-4,6-diene-3,20 dione) were administered to 16 rats for 1 year to determine the effect on the genital organs and female fertility. No noteworthy ponderal or histologic effect of the genital organs or the pituitary was observed. However, uterus glycogen concentration and alkaline phosphatase activity were greatly reduced (versus controls, p.05 and p.01, respectively) while glucose-6-phosphate and lactic dehydrogenase activities increased significantly (versus controls, p.01). In the fertility performance test, 86% of the 14 controls showed positive mating compared with 50% for the treated group. By Day 10 of pregnancy many of the fetuses in the treated group were in the process of resorption. The factors contributing to pregnancy failure were inhibition of mating, implantation failure and fetal resorption.^ieng

Pantethine inhibits the formation of high-Tc protein aggregates in gamma B crystallin solutions.

PURPOSE: Solutions of the bovine lens protein gamma B (or gamma II) crystallin at neutral pH in the absence of reducing agents, undergo a slow, partial conversion to a new protein species, gamma IIH. This species is an aggregate composed of an intermolecular, disulfide-crosslinked dimer (approximately equal to 32% of total protein by weight) and loosely associated dimers (approximately equal to 66%). gamma IIH has a phase separation temperature (Tph), at least 40 degrees C higher than that of native gamma II crystallin at any given protein concentration. In this paper we demonstrate that pantethine, a derivative of coenzyme A, inhibits the formation of gamma IIH. METHODS: gamma II crystallin solutions were incubated at pH 7.1 and room temperature with increasing amounts of pantethine. The Tph of the solutions was monitored as a function of incubation time. Corresponding to each Tph measurement, aliquots of each solution were analyzed by cation-exchange HPLC to determine the amount of gamma IIH formed. RESULTS: Incubation of gamma II crystallin with increasing amounts of pantethine lowers Tph and suppresses the formation of gamma IIH. With pantethine to protein mole ratios of 0.66, 1 and 2, the Tph of gamma II crystallin is lowered from 8 degrees C in the native protein, to 2 degrees C, -3 degrees C respectively, at a protein concentration of approximately equal to 200 mg/ml. The amount of gamma IIH accumulated decreases from approximately 25% in the native protein to 10%, 1% and 0% respectively in these pantethine-treated protein solutions. For complete suppression of the rise in Tph and inhibition of gamma IIH formation, a 2:1 mole ratio of pantethine to protein is required. CONCLUSIONS: We suggest that pantethine reacts with two cysteine residues of gamma IIH crystallin by forming a mixed disulfide, and effectively suppress protein aggregation and lowers Tph. This is due to the strong polar character of pantethine which reduces the net attractive interactions between the protein molecules.

Dyspnoea, lung function & respiratory muscle pressures in patients with Graves' disease.

To understand the pathophysiology of dyspnoea in patients with hyperthyroidism, lung function, maximum inspiratory, expiratory respiratory muscle pressures (MIP and MEP) and intensity of dyspnoea (after six minutes walking test) were recorded in 12 consecutive patients with active Graves' disease. Reassessment was done after achieving euthyroidism with 8-12 wk of carbimazole therapy. Patients covered similar distance during 6 min walking before and after carbimazole therapy. However, there was a significant reduction in dyspnoea following euthyroidism. This was accompanied by significant decrease in respiratory rate, minute ventilation, forced expiratory volume in one second (FEV1%) and improvement in the forced vital capacity (FVC). No significant changes in tidal volume (TV) and maximum-midexpiratory flow rates (MMEFR), MIP and MEP were observed. Lung function parameters, MIP and MEP did not correlate with the severity of dyspnoea. Serum T4 levels correlated inversely with the distance covered during 6 min walking test, MIP and MEP. To conclude, increased breathing effort in presence of reduced FVC may lead to dyspnoea during hyperthyroid phase in patients with active Graves' disease. Lack of correlation between the severity of dyspnoea and abnormalities in lung function suggests that other mechanisms of dyspnoea may also operate in these patients.

Effect of di-norgestrel (13beta-ethyl-17alpha-ethynyl-19-nortestosterone, Wy 3707) on the reproductive organs of rabbit.

PIP: The effect of dl-norgestrel (13 beta-ethyl-17 alpha-ethynyl-19-nortestosterone, Wy 3707) on the biochemical makeup of rabbit ovary and uterus was investigated. 20 adult, healthy virgin female rabbits either received olive oil only or olive oil plus 12 mcg of norgestrel/rabbit/day for 30 days. The animals were sacrificed 24 hours after the last treatment. In the ovary protein concentration and activities of glucose-6-phosphate dehydrogenase (G6PD), lactate dehydrogenase (LDH), and malate dehydrogenase (MDH) remained unaffected. However, in the uterus the level of protein was significantly elevated (p less than .01), the activities of G6PD and acid phosphatase were enhanced (p less than .05), and those of adenosine triphosphatase (ATPase) and alkaline phosphatase were diminished (p less than .05). LDH and MDH in the uterus remained unchanged. The effect of norgestrel at this antiovulatory dose in rabbits consisted of a disturbance in the biochemical constituents of the uterus leading to a lowering of the ATPase activity and an impairment of the anaerobic mechanism of the organ.^ieng

Postoperative pulmonary complications in patients with preoperative lung disease.

A prospective study was carried out on the incidence of pulmonary complications in 728 patients who underwent major surgical procedures at a reputed hospital in New Delhi. These patients were preoperatively assessed on the basis of history, clinical examination and bedside pulmonary function tests including PEFR, VC, FVC and FEV1. On the basis of these criteria, 212 patients had significant preoperative pulmonary disease, while 516 had normal lung functions. Postoperative pulmonary complications developed in 7.69 per cent of the patients. They were more frequent in smokers (P < 0.001) and in those with preoperative pulmonary dysfunction (P < 0.001). Their incidence was greater following thoraco-abdominal and musculoskeletal and miscellaneous operations compared to lower abdominal surgery (P < 0.001). The incidence was also higher following exposure to general anaesthesia compared to regional anaesthesia (P < 0.05). Pneumonia and atelectasis were the most common postoperative pulmonary complications. It was observed that the recovery of pulmonary functions was delayed in patients who developed postoperative pulmonary complications.

Immunologic & pulmonary function abnormalities in tropical pulmonary eosinophilia.

Twenty six patients (24 males and 2 females) with tropical pulmonary eosinophilia (TPE) were studied. Arterial blood gas analysis, pulmonary functions, peripheral blood examination and bronchoalveolar lavage (BAL) were performed. Peripheral blood and BAL fluid analyses were performed in healthy volunteers. Pulmonary functions revealed a mild restrictive ventilatory defect with airways obstruction. Mild hypoxaemia was observed on arterial blood gas analysis. Serum immunoglobulins IgG (P < 0.01), IgA (P < 0.001) and IgM (P < 0.001) were significantly raised as compared to normal controls. Serum complement (C3) level was higher, however, it was not significantly different as compared to normal controls. Serum haemolytic component of the complement (CH50) was significantly higher (P < 0.001) in patients with TPE compared to normal control subjects. The immunoglobulins IgG, IgA and IgM in the BAL fluid were significantly (P < 0.001) increased as compared to normal controls. The fibronectin (FN) level was also significantly increased (P < 0.001) in the BAL fluid. It is concluded that patients with TPE have mild restrictive ventilatory defect with airways obstruction and mild hypoxaemia. They have eosinophilic alveolitis with increased levels of immunoglobulins in the peripheral blood and BAL fluid. The significance of elevated FN in the BAL fluid is not clear and serial estimations may have to be done in order to clarify its role in the pathogenesis of fibrosis in chronic TPE.

Effect of inhalation of salbutamol, beclomethasone dipropionate & ipratropium bromide on mucociliary clearance in some patients with chronic stable bronchial asthma.

BACKGROUND & OBJECTIVES: Asthma is now regarded as an inflammatory disease and bronchial inflammation may disrupt mucociliary function. Inhaled drugs may act by improving mucociliary function. The aim of the study was to investigate the effect of salbutamol, ipratropium bromide and beclomethasone on mucociliary clearance in patients with chronic stable asthma and to compare the efficacy of these drugs on mucociliary clearance. METHODS: Ten patients with chronic stable asthma were enrolled in the study, but two patients did not complete the study. Patients with bronchial asthma were chosen on clinical grounds. (99m)Tc phytate radioaerosol generated through a nebulizer, was given to each patient on four days. After each administration the radioactivity over the thorax was constantly measured in sequential frame mode for 120 min. Radioactivity in the thorax was also measured after 24 h. A base-line pulmonary function test with reversibility was obtained. Salbutamol, ipratropium bromide, beclomethasone dipropionate and placebo inhalation were given randomly to each patient on four days. RESULTS: The mean age of patients (n = 8) was 36 +/- 9.3 yr and mean duration of symptoms was 5 +/- 6.6 yr. There was no visual impression that mucociliary clearance was enhanced with any of the drugs. The time activity curves did not show any visually recognisable change in slope. In only one patient the curve tended to show a steeper slope with ipratropium inhalation. In the rest of the patients the curves showed no difference at all with medication when compared with placebo. All the quantitative indices analyzed by two-way ANOVA at the end of one and two hours were comparable for the three test drugs and placebo. None of the three test drugs demonstrated statistically significant mucociliary clearance effect compared with placebo. However, the temporal difference in airways clearance efficiency (ACE) was significant with beclomethasone and ipratropium bromide. INTERPRETATION & CONCLUSION: Inhalation of any of the three drugs tested did not produce any immediate improvement in mucociliary clearance as compared to placebo in patients with stable bronchial asthma suggesting the need for further studies using higher doses of drugs for longer duration in a large sample.

Effect of corticosteroid treatment on various serological & bronchoalveolar lavage abnormalities in patients with sarcoidosis.

Effect of daily oral prednisolone treatment was studied in 29 patients with pulmonary sarcoidosis. Twenty normal control subjects were also studied. Pretreatment absolute lymphocyte counts and proportion of lymphocytes in peripheral blood were significantly lower in patients with sarcoidosis as compared to normal controls. Total cell count and the proportion of lymphocytes in bronchoalveolar lavage (BAL) fluid were significantly higher in sarcoidosis. The proportion of CD3+ and CD4+ was significantly lower in peripheral blood and higher in BAL fluid in patients with sarcoidosis. Immunoglobulins (IgG, A and M) and complements (C3, C4 and CH50) were significantly higher both in peripheral blood and BAL fluid. Patients with sarcoidosis were treated with daily oral prednisolone (30 mg/day). Repeat studies were performed after an interval of 4-6 months in 20 patients with sarcoidosis. A significant increase in absolute lymphocyte counts in peripheral blood and decrease in the proportion of lymphocytes in BAL fluid occurred with prednisolone treatment. Proportion of CD3+, CD4+ and B cells increased in peripheral blood and decreased in BAL fluid. Complement and immunoglobulin levels revealed a significant reduction in peripheral blood and BAL fluid. It is concluded that patients with sarcoidosis have peripheral blood lymphopaenia and lymphocytic alveolitis. They have increased levels of complement and immunoglobulins both in the peripheral blood and BAL fluid. All these abnormalities show significant improvement with prednisolone treatment.

Preparation & standardization of the goat lung surfactant extract.

Exogenous goat lung surfactant extract (GLSE) was obtained by chloroform-methanol extraction of the centrifuged lung lavage. Four lungs provided around 250 mg of surfactant. GLSE has a phospholipid content of 50 percent, cholesterol of 1.5 percent and protein content under 1 percent. The preparation was bacteriologically sterile and had a shelf life of at least one month at 4 degrees C. Endotracheal administration of surfactant (50 mg/kg) in a rat lung model rendered surfactant deficient by saline lavage, restored the compliance characteristics to normal, thus documenting biological activity of the preparation. The cost of a course of 2 doses of surfactant replacement therapy was estimated to be as low as Rs 500/-. The study opens up the possibility of surfactant replacement therapy for the management of neonatal respiratory distress syndrome in India by an indigenous product at an affordable cost.

The use of diagnostic bronchoscopy in lung cancer.

BACKGROUND: The role of fibreoptic bronchoscopy in the diagnosis of bronchogenic carcinoma is well established. However, in developing countries, where the burden of illness is large and diagnostic facilities are limited, only a small number of patients are diagnosed at a stage when they might benefit from operation. We felt it would be desirable to identify subsets of patients suspected of harbouring lung cancers, in whom bronchoscopy would have not only a high diagnostic yield but also provide useful information which might influence treatment, the patient's quality of life and duration of survival. METHOD: We analysed the records of 588 patients, who had been bronchoscoped for suspected lung cancer, over a period of 8 years at a tertiary referral centre in north India. The patients were divided into different clinical subgroups on the basis of their clinical and radiological presentation, and the diagnostic yield from bronchoscopy in each group was calculated. A decision analysis model was constructed and the expected value of clinical information was determined for each group. RESULTS: A tissue diagnosis was established by bronchoscopy in 177 (30%) patients and by additional investigations in 43 (7.3%) patients. A positive tissue diagnosis was obtained most often in patients with clinical and radiological evidence of pulmonary collapse (50%) and mass lesions (38-42%). Only 12% of patients with malignancy underwent resection and 70% of them belonged to the above two groups. The expected value of clinical information was greatest in patients with collapse (0.077) or mass lesions (0.067-0.065). CONCLUSIONS: A diagnostic work-up including fibreoptic bronchoscopy is indicated early in patients with collapse or mass lesions of the lung. Patients with non-resolving pneumonia, pleural effusion, metastatic disease and non-specific lesions on chest X-ray should receive a low priority for bronchoscopy.

Acetylator status, drug metabolism and disease.

Acetylation polymorphism, although discovered 40 years ago, still holds interest not only because many drugs and carcinogens are metabolized by acetylation in the liver but also because advances have been made in the understanding of the molecular genetics of acetylation. It is this genetic variation of drug metabolism that is one of the causes of inter-individual variation of the effect of a drug. Acetylation polymorphism relates to the metabolism of a number of arylamine and hydrazine drugs and carcinogens by cytosolic N-acetyltransferase--NAT2. In humans, 2 genes--NAT1 and NAT2--are responsible for the N-acetyltransferase activity. Studies have revealed several allelic variants of both NAT1 and NAT2. It has been suggested that some of these variants modify the individual susceptibility to disease.