Inhibitory effect of docosahexaenoic acid (DHA) on the intestinal metabolism of midazolam: in vitro and in vivo studies in rats.

The aim of this study was to evaluate the effects of docosahexaenoic acid (DHA) on the intestinal cytochrome P450 isoenzyme (CYP3A) and P-glycoprotein (P-gp) functions using midazolam and rhodamine-123 as specific substrates of CYP3A and P-gp, respectively. Perfused everted intestinal segments from rats were employed to determine the effects of DHA on midazolam metabolism and rhodamine-123 transport. In addition, the effects of DHA on in vitro midazolam metabolism in rat intestinal microsomes and on midazolam bioavailability in rats were examined. The intestinal extraction ratio (ER G) of midazolam was determined to be 0.43 and decreased significantly to 0.12, 0.07, and 0.06 in the presence of 50, 100, and 200 microM DHA, respectively, in a concentration-dependent manner. The results from an in vitro study using rat intestinal microsomes demonstrated that DHA competitively inhibited the intestinal CYP3A activity with Ki of 15.7 and 27.1 microM for the formations of 1'-OH midazolam and 4-OH midazolam, respectively. Moreover, the oral administration of DHA (100mg/kg) increased the AUC infinity, Cmax, and oral bioavailability (F) of midazolam by about 50% in rats, without affecting the T 1/2, V dss/F, or CL tot/F. In contrast, DHA did not change the serosal-to-mucosal transport of rhodamine-123 in the perfused everted intestine and oral administration of DHA (100mg/kg) had no influence on the pharmacokinetics of intravenously administered midazolam in rats, thus suggesting that DHA has little effect on the intestinal P-gp activity and hepatic clearance of midazolam. This study provided the first direct evidence to show that DHA has an inhibitory effect on the intestinal pre-systemic metabolism of a CYP3A substrate and that DHA has little, if any, effect on the P-gp activity in the gut.

The use of PCR in detecting toxoplasma parasites in the blood and brains of mice experimentally infected with Toxoplasma gondii.

Polymerase chain reaction (PCR) has been extensively used for diagnosis recently because of its very high sensitivity and specificity. We studied the applicability of PCR to the early diagnosis of toxoplasmosis in a murine model orally infected with Toxoplasma gondii (S-273). PCR was performed using EH24 and HE27 primers synthesized by the phosphoramidite method. Mice blood and brains collected on various post infection days (PID) were analysed by PCR (35 cycles). A portion of the brain tissue from each mouse was examined microscopically for the presence of parasite cysts. Blood and brain PCR were positive on the 9th and 12th day post-infection (DPI). Toxoplasma cysts in brain tissue appeared only on the 18th PID. The results showed that Toxoplasma parasites can be detected earlier in the blood than in the brain during primary infection, indicating that blood PCR is the more useful procedure.

Seroprevalence of hepatitis B and C viruses in eastern Nepal.

The seroprevalence of hepatitis B virus (HBV) and hepatitis C virus (HCV) in 303 serum samples collected from that apparently healthy population inhabitating different areas in eastern Nepal was studied. Samples were collected at Dharan Municipality, Sunsari85), Pancha Kanya Village Development Committee, Ilam86), Dhankuta Hile, Dhankuta82) and Basantapur Village Development Committee, Tehrathm50). HBsAg and anti-HBsAg antibody was screened by reverse passive haemagglutination (RPHA) and passive haemagglutination (PHA) respectively and positivity was confirmed by enzyme linked immunosorbent assay (ELISA). Anti-HCV antibody was detected by ELISA. None of the samples were positive for HBsAg. Anti-HBsAg antibody was positive in 1.9% (6/303). The positive rate increased with age reaching 25% positivity among the elderly. The anti-HBsAg antibody positivity was 2.35, 2.32, 1.22 and 2.00 in Dharan, Ilam, Dhankuta and Tehrathum respectively Anti-HCV antibody was detected only in one sample (15-year-old boy) collected in Dharan. These findings indicate that the HBV and HCV infections are not active in eastern Nepal.

Maintenance of pancreatic endocrine cells of neonatal rats: effect of 2-deoxyglucose on insulin biosynthesis.

The relationship between insulin biosynthesis and proinsulin mRNA activity was examined in pancreatic monolayer cultures of the neonatal rat. Monolayer cultures were maintained in TCM 199 medium containing 16.7 mM of either glucose or 2-deoxyglucose or in a basal medium not containing glucose for 24 hr. The fractions containing mRNA extracted from these cultures were translated in a cell-free protein synthesizing system of rabbit reticulocyte lysate. The proinsulin mRNA activity was determined by the radioactivity of the translation product immunoprecipitated using anti-insulin serum, and identified as preproinsulin with a molecular weight of 11,500 on gel electrophoresis. The amount of insulin biosynthesis was determined by the incorporation of [3H]-leucine into (pro)insulin. In islet B cells cultured in a medium with 16.7 mM glucose, insulin biosynthesis induced by 16.7 mM glucose was enhanced by 158% when cultured in basal medium without glucose. This increment corresponded nicely with a 185% increase in the proinsulin mRNA activity. However, the addition of 16.7 mM 2-deoxyglucose to the basal medium resulted in a 293% increase in glucose-induced insulin biosynthesis despite a 16% drop in the proinsulin mRNA activity, and primed a dose-dependent increase of insulin biosynthesis over the concentration range of 0 to 16.7 mM glucose. These results suggest first that in neonatal B cells insulin biosynthesis may be regulated at the transcriptional level, and second, that 2-deoxyglucose may cause the transition of the neonatal B cell to an adult-type response.

Effect of 2-deoxy-D-glucose on maintenance in culture of neonatal B cell of rat.

The effect of 2-deoxy-D-glucose on maintenance in culture of B cells of the neonatal rat was examined by supplementation of Medium 199 containing 5.5 mM glucose with 1 mM 2-deoxy-D-glucose. Islets maintained in medium with 5.5 mM glucose (basal medium) for 7 d underwent remarkable decreases in glucose sensitivity, and the levels of insulin in the medium dropped. By contrast, addition of 2-deoxy-D-glucose promoted a higher insulin content in medium and an increase in the glucose-induced insulin release and biosynthesis. Moreover, the addition of the deoxysugar caused a selective deletion of fibroblasts and prevented the deterioration of islet cells in basal medium, yielding clusters mostly consisting of islet cells at the end of culture.

Maintenance of neonatal endocrine cells of the rat. I. Effects of 2-deoxyglucose and 2-deoxy-2-fluoroglucose.

Pancreatic endocrine cells of the neonatal rat cultured in medium with 5.5 mM glucose for 7 days showed no response to glucose. By contrast, the supplementation of the medium with 1.0 mM 2-deoxyglucose or with 0.1-5.0 mM 2-deoxy-2-fluoroglucose maintained the capacity of glucose-induced insulin release and biosynthesis, and the recovery of insulin in cells at day 7 at levels significantly higher than in basal medium; the highest responses were recorded for 1.0 mM deoxysugars. Moreover, the addition of 1.0 mM deoxysugars caused a selective deletion of fibroblasts and yielded monolayers mostly consisted of endocrine cells at the end of the culture study period. In these monolayer cells, the stimulating level of c-AMP release was significantly higher than the basal. On the other hand, the in vitro function of A cells in culture was also better preserved in media with 1.0 mM deoxysugars.

Effect of D-glucosamine on the functional maturation of cultured B cells of the neonatal rat.

The present study demonstrates the effect of glucosamine on the functional maturation of cultured B cells of the neonatal rat. When B cells had been maintained at a physiological concentration (5.5 mM) of glucose for 7 days, a drop in the stimulatory effect of 16.7 mM glucose on insulin release and biosynthesis was observed together with a reduced insulin content. By contrast, the sensitivity of glucose-induced insulin release was increased after one week of culture with 5.5 mM glucose and 5 mM glucosamine. And both the insulin content and glucose-induced insulin biosynthesis also remained at the same level as observed at the first day of culture with 5.5 mM glucose alone. In summary, it was suggested that glucosamine-supplemented culture may result in the transition of B cells of neonatal rat from a poor glucose sensitivity to adult-type response of insulin release.

Insulin receptors in rabbit erythrocyte.

The existence of insulin receptors in rabbit erythrocytes was studied by evaluating the specific binding of 125I-insulin to erythrocyte membranes. The binding of 125I-insulin was pH, time and temperature dependent. Maximal binding was achieved by incubation for 20 hr at 0 degrees C. The optimum pH was 7.4. Treatment with cations and enzymes enhanced the specific binding except for with trypsin, the treatment which greatly reduced the binding. Unlabeled insulin over a wide range of concentrations competitively inhibited the binding of 125I-insulin, while the binding was little affected by structurally unrelated hormones. Scatchard plot was represented as a concave curve. Binding sites of relatively high affinity (K1 = 0.9 X 10(9) M-1) and low capacity (8.0 X 10(13)/g protein) could be distinguished from those of lower affinity (K2 = 0.8 X 10(7) M-1) and higher capacity (1.8 X 10(15)/g protein). Hill's analysis and dissociation of 125I-insulin from membranes demonstrated the characteristics of negative cooperation between receptor sites. Both incorporation of H3(32)PO4 to erythrocyte membranes and uptake of 45Ca were significantly reduced by the addition of unlabeled insulin. Unlabeled insulin produced no effect on uptake of 45Ca into trypsin-treated erythrocytes. On the basis of these results, it was suggested that rabbit erythrocytes might possess biologically significant insulin receptors located on the cell membranes.