Michael R. Borenstein

Dietary vitamin E prophylaxis and diabetic embryopathy: Morphologic and biochemical analysis ☆ ☆☆ ★ ★★

OBJECTIVE In this study we sought to determine whether dietary supplementation with vitamin E, a known antioxidant, would reduce the incidence of diabetic embryopathy in an in vivo rat model. STUDY DESIGN Eighty-day-old Sprague-Dawley rats were assigned to one of five groups: two control groups (groups 1 and 2) and three diabetic groups (groups 3, 4, and 5). One group of controls (group 2) and one group of diabetic rats (group 4) received dietary supplements of vitamin E (440 mg/day). The other three groups (groups 1, 3, and 5) received a normal diet only. Group 5 received insulin therapy to control glucose levels. On day 6 of gestation diabetes was induced in groups 3, 4, and 5 with streptozotocin (65 mg/kg). Animals were killed on day 12; embryos were examined for size, protein content, evidence of malformations, and superoxide dismutase activity. RESULTS In both groups (groups 3 and 4) of diabetic rats the mean blood glucose level than was significantly higher in controls. Insulin-treated animals (group 5) had glucose levels that were comparable to those of controls. The unsupplemented diabetic group had a neural tube defect rate of 21.48% +/- 9.6% (percentage of neural tube defects per rat) and a resorption rate of 21.37% +/- 20.39% (percentage of resorptions per rat) as compared with rates in the supplemented diabetic group of 6.92% +/- 4.08% and 2.17% +/- 3.74%, respectively (p < 0.01). Groups 1, 2, and 5 had similar neural tube defect rates (6.63% +/- 5.0%, 5.01% +/- 4.87%, and 3.55% +/- 5.92%, respectively. Vitamin E levels, measured by high-performance liquid chromatography, were significantly higher in maternal serum and embryos in the supplemented groups (p < 0.001) than in controls. Superoxide dismutase activity was reduced in the diabetes groups and was not affected by vitamin E therapy. CONCLUSIONS Supplementation with the antioxidant vitamin E confers a significant protective effect against diabetic embryopathy and may potentially serve as a dietary prophylaxis in the future. We postulate that this protective effect is mediated by a reduction in the oxidative load induced by hyperglycemia.

Dietary polyunsaturated fatty acid prevents malformations in offspring of diabetic rats.

OBJECTIVE The purpose of the current study was to determine whether a dietary source of arachidonic acid could serve as a pharmacologic prophylaxis to obviate the teratogenic effects of hyperglycemia. STUDY DESIGN Eighty-day-old Sprague-Dawley rats were mated, and after conception were randomly allocated to five groups: two groups were nondiabetic normal controls and three groups had diabetes experimentally induced with streptozocin. Of the two control groups, one was fed a normal diet (group 1) and the other group (group 2) received a normal diet and 1.0 ml of safflower oil, a polyunsaturated fatty acid known to increase serum arachidonic acid levels. In the three diabetic groups (groups 3, 4, and 5) glucose levels were allowed to remain > 350 mg/dl by withholding daily insulin therapy. Group 3 received a normal diet without supplementation; group 4 received a normal diet plus normal saline solution sham feedings, whereas group 5 received a normal diet supplemented with 1.0 ml of safflower oil. The oral agents (normal saline solution and polyunsaturated fatty acid) were administered with a tuberculin syringe. RESULTS Diabetic rats not receiving insulin therapy and receiving normal diets produced offspring with malformation rates of 20% compared with control rates of 4.8%. Supplemental normal saline solution or safflower oil given orally to controls did not alter the growth or malformation rates. These rates were similarly unaffected in the diabetic rats receiving oral supplementation of normal saline solution. However, with safflower oil supplementation to diabetic rats the incidence of neural tube defects was decreased from 20.0% to 7.6% (p < 0.0001). An inverse relationship was observed between the malformation rate and the serum arachidonic acid level: 17.83 (SD 5.84 micrograms/ml) in the nondiabetic controls, with a malformation rate of 4.8%, versus 14.18 (SD 2.58 micrograms/ml) in the diabetic rats, with a malformation rate of 20.0% (p < 0.05). With safflower oil supplementation serum levels of arachidonic increased from 14.18 +/- 2.58 micrograms/ml to 19.99 +/- 7.99 micrograms/ml (p < 0.05); this was associated with a concomitant decline in the malformation rate. CONCLUSION These data demonstrate that diabetic embryopathy is associated with a deficiency state in essential fatty acid, corroborating our previous in vitro findings. Furthermore, the use of a dietary polyunsaturated fatty acid that specifically increases arachidonic levels significantly reduced the incidence of diabetic embryopathy. These findings may serve as a basis for developing strategies of pharmacologic prophylaxis against diabetes-induced congenital malformations.

Dietary intake of myo-inositol and neural tube defects in offspring of diabetic rats

OBJECTIVE Embryopathy in diabetic mothers occurs at a rate four to five times higher than that observed in the general population. The current investigation was undertaken to assess the use of dietary myo-inositol supplementation as a pharmacologic prophylaxis to obviate the teratogenic effects of hyperglycemia in an in vivo study. STUDY DESIGN Seventy Sprague-Dawley rats were mated and after conception were randomly divided into five groups: one group was nondiabetic normal controls and four groups had diabetes experimentally induced with streptozotocin. Of the diabetic groups, one received the usual diet, whereas the others received, respectively, 0.08, 0.16, and 0.5 mg/day supplemental myo-inositol orally. RESULTS With the myo-inositol supplementation (0.08 mg/day), the incidence of neural tube defects was significantly reduced from 20.4% to 9.5% (p < 0.01). The most effective dosage of myo-inositol was 0.08 mg/day. Increasing the dose of myo-inositol beyond that level did not significantly reduce the rate of neural tube defects. However, the resorption rate was increased to 29.8%. CONCLUSION These data demonstrate that myo-inositol supplementation reduces the incidence of diabetic embryopathy and may serve as a pharmacologic prophylaxis against diabetes-induced congenital malformations.

Effects of topical corticosteroids on inflammatory mediator–induced eicosanoid release by human airway epithelial cells

BACKGROUND Airway epithelial cells are among the first cells to come in contact with aerosolized corticosteroids. However, the relative potencies and time course of action of the several commonly used aerosolized corticosteroids on eicosanoid production by airway epithelial cells are unknown. OBJECTIVES This study compared the effects of fluticasone, budesonide, and triamcinolone on eicosanoid output by human airway epithelial cells in vitro. We also determined the spectrum of eicosanoids affected and the mechanism for corticosteroid action. METHODS Cultured BEAS-2B airway epithelial cells (a transformed cell line) were exposed to corticosteroids (1 nmol/L to 1 micromol/L) for 2 to 48 hours and then assayed for basal- and bradykinin (BK)-stimulated eicosanoid output. The eicosanoid profile was identified by HPLC in tritiated arachidonic acid prelabelled cells, and PGE2, the major eicosanoid product, was quantitated by RIA. The effect of corticosteroids on the immunoreactivity of key proteins involved in eicosanoid metabolism (ie, cyclooxygenase [COX], phospholipase A2 [PLA2], and Clara cell protein, a PLA2 inhibitor) was determined by Western blotting. RESULTS Eicosanoid output was largely confined to prostaglandins with values of 5 +/- 2 and 82 +/- 35 ng PGE2/10(6) cells for basal- and BK stimulation, respectively (n = 8). All 3 corticosteroids inhibited basal- and BK-induced PGE2 output in a dose- and time-dependent manner. Fluticasone and budesonide completely eliminated PGE2 output in nanomolar concentrations in contrast to triamcinolone, which required micromolar concentration. The rank order of potency was: fluticasone = budesonide > triamcinolone. The time course of action for PGE2 inhibition also differed, with budesonide acting more slowly than the other 2 corticosteroids (P = .04). All 3 corticosteroids markedly reduced COX2 with little effect on COX1, cPLA2 (Type IV), or iPLA2 (Type VI) immunoreactivity or their relative distribution in cytosol versus membrane fractions. Clara cell protein immunoreactivity was undetectable in control and corticosteroid-treated cell lysates. CONCLUSION These results show that in a human airway epithelial cell line, the 3 inhaled corticosteroids commonly used to treat asthma differ in onsets of action as inhibitors of prostaglandin synthesis and vary considerably in potency. All 3 corticosteroids act mechanistically in similar fashion by inhibiting COX2 synthesis.

Dietary myo‐inositol therapy in hyperglycemia‐induced embryopathy

Dysmorphogenesis in diabetic mothers occurs more frequently than in the general population. This phenomenon is believed to be caused by the teratogenic effects of metabolic fuel mixtures with associated membrane injury and aberrations in the biochemical constituents. The present experiment was designed to determine: 1) if hyperglycemia-induced membrane injury is associated with intracellular and/or extracellular lipid disturbances; 2) if supplemental myo-inositol therapy prevents hyperglycemia-induced embryopathy; 3) if a correlation exists between dietary myo-inositol, serum and tissue levels of myo-inositol, and conceptus development; and 4) the cellular content of arachidonic acid following myo-inositol supplementation. Sixty-five female Sprague-Dawley rats were mated, and divided into three groups. One group was nondiabetic normal controls, and two groups had diabetes experimentally induced with streptozotocin. Of the diabetic groups, one received a normal diet, while the other received a myo-inositol-supplemented diet during the period of organogenesis. Blood samples were collected on days 0 and 12 of pregnancy. Embryos and yolk sacs were analyzed for myo-inositol and arachidonic acid levels, using mass spectrochromatography. Dietary myo-inositol supplementation of diabetic mothers resulted in a significant decrease in the incidence of neural tube defects when compared with diabetics not receiving supplements (9.5 vs. 20.4%; P < 0.05). This protective effect was incomplete, based on the incidence observed in the nondiabetic controls (9.5 vs. 3.8%; P < 0.05). The myo-inositol embryonic tissue levels in the diabetic group which had been fed a regular diet without supplementation were significantly lower than in the nondiabetic group. Dietary therapy successfully restored myo-inositol levels in the yolk sacs, as suggested by similar tissue levels in diabetics receiving myo-inositol supplementation and normal controls (18.7 +/- 1.3 vs. 19.1 +/- 2.0 ng/mg; P = ns). Dietary therapy, however, failed to restore myo-inositol levels in the embryos, suggesting hyperglycemia-induced faulty transport of nutrients from the yolk sac to the embryo. No correlation was noted between maternal blood levels of myo-inositol, with or without supplementation, and the clinical outcome. Tissue arachidonic acid levels were markedly reduced in the conceptuses of diabetic mothers with (0.4 +/- 0.1 micrograms/mg) or without (0.25 +/- 0.08 micrograms/mg) myo-inositol supplementation when compared to the nondiabetic controls (3.33 +/- 0.24 micrograms/mg). These data demonstrate that diabetes-induced embryopathy is associated with a deficiency state in both myo-inositol and arachidonic acid. The myo-inositol deficiency is not demonstrated at the serum level, but rather at the tissue level, suggesting a paracrine action. Dietary supplementation of myo-inositol is associated with an increase in tissue myo-inositol levels and a decrease in malformations. This therapy holds promise for use as a dietary prophylaxis against diabetic embryopathy.

Gas chromatographic method using nitrogen-phosphorus detection for the measurement of tramadol and its O-desmethyl metabolite in plasma and brain tissue of mice and rats

A method that allows the measurement of plasma and brain levels of the centrally-acting analgesic tramadol and its major metabolite (O-desmethyl tramadol) in mice and rats was developed using gas chromatography equipped with nitrogen-phosphorus detection (GC-NPD). Plasma samples were extracted with methyl tert.-butyl ether (MTBE) and were injected directly into the GC system. Brain tissue homogenates were precipitated with methanol, the resulting supernatant was dried then acidified with hydrochloric acid. The aqueous solution was washed with MTBE twice, alkalinized, and extracted with MTBE. The MTBE layer was dried, reconstituted and injected into the GC system. The GC assay used a DB-1 capillary column with an oven temperature ramp (135 to 179 degrees C at 4 degrees C/min). Dextromethorphan was used as the internal standard. The calibration curves for tramadol and O-desmethyl tramadol in plasma and brain tissue were linear in the range of 10 to 10000 ng/ml (plasma) and ng/g (brain). Assay accuracy and precision of back calculated standards were within +/- 15%.

Differential tramadol and O-desmethyl metabolite levels in brain vs. plasma of mice and rats administered tramadol hydrochloride orally.

Objective: To investigate a possible differential brain uptake of tramadol vs. its major metabolite (O‐desmethyl tramadol; M1) in mice and rats.

Determination of the adsorption of tramadol hydrochloride by activated charcoal in vitro and in vivo.

Although tramadol is one of the most widely used centrally acting analgesics worldwide, no literature is available regarding adsorption of tramadol HCl powder or tablets (Ultram; 50 mg tramadol HCl per tablet) by activated charcoal (AC) for use as potential adjunct treatment of overdose. The present study incorporated a novel combination of in vitro and in vivo methods to investigate this question. Based on a binding curve of tramadol UV absorbance (UV(a); 225 nm) plotted against the amount of AC, the ratio of amount of tramadol completely adsorbed by AC was 0.05 mg/mg. Also based on UV(a), no tramadol was detected in filtrate of slurries in which up to 62 tablets of Ultram were mixed with 50 g AC; 4.6% of unbound tramadol was detected when 100 tablets of Ultram were mixed with AC. The ratio of amount of tramadol completely adsorbed by AC in this test was 0.10. In vivo, co-administration of 0.1 g/ml of AC produced a 13- to 14-fold rightward shift in tramadols antinociceptive dose-response curve and a 1.6-fold rightward shift in tramadols lethality dose-response curve.

Manufacture of Porous Cross-Linked HEMA Spheres for Size Exclusion Packing Material

Abstract2-Hydroxyethyl methacrylate (HEMA), methyl methacrylate (MMA) monomers and ethyleneglycol dimethacrylate (EDMA) as a cross-linker, were reacted with the initiator 2,2,azobis-2-methylpropionitrile (AIBN) in the presence of dodecanol. The effect of increasing concentrations of dodecanol, as a porogen agent, was evaluated. The radical polymerization was carried out using an in situ prepared Mg(OH)2 gel, at 70°C for 5 h. Following polymerization, the suspension was eliminated by neutralization for recovery of beads. The spherical particles obtained were filtered, washed with methanol using simple extraction and using a Soxhlet thimble, and dried at 60°C under vacuum. Scanning Electron Microscopy (SEM) revealed regularly shaped spherical beads containing macropores. Nitrogen Sorption Analysis (NSA) and Mercury Intrusion Porosimetry (MIP) were performed for evaluation of surface area, pore size and pore volume.

Hydrophilic Porous Hydroxypropyl Methacrylate–Ethyleneglycol Dimethacrylate Packing Materials Suitable for Use in Size Exclusion Chromatography

Packing materials suitable for use in size exclusion chromatography (SEC) were produced by radical polymerization at 70°C for 1 hour. Hydroxypropyl methacrylate (HPMA) monomer, ethyleneglycol dimethacrylate (EDMA) cross-linker, 2, 2, azobis-2-methylpropionitrile (AIBN) initiator and the porogen dodecanol (DOD) were mixed and added to a polyvinyl alcohol (PVA) solution suspending medium. Elimination of the suspending medium by centrifugation was followed by filtering, rinsing with methanol, Soxhlet extraction and vacuum drying (60°C, 12 h) to yield polymeric spherical particles. The effect of increasing the concentrations of the porogen agent dodecanol was evaluated. Scanning electron microscopy (SEM) revealed regularly shaped spherical porous beads ranging in size from 10–20 μm. Nitrogen Sorption Analysis (NSA) was employed to determine the specific surface area and pore size.