Effect of system variables involved in packed column supercritical fluid chromatography of stavudine taken as model analyte using response surface methodology along with study of thermodynamic parameters.

A multifactor optimization technique is successfully applied to study the effect of simultaneously varying the system variables on feasibility of stavudine analysis by packed column supercritical fluid chromatography (PC-SFC). The effect of simultaneously varying the pressure, temperature and modifier concentration was studied to optimize the method in order to obtain excellent chromatographic figures of merit. The method is based on isocratic elution using methanol-modified supercritical carbon dioxide as the mobile phase at the flow rate of 3.0 ml/min through a JASCO Finepak SIL-5, ODS [C(18) (5 microm, 25 cm x 4.6 mm, i.d.)] column support using photodiode array detection. The optimal conditions were determined with the aid of the response surface methodology using 3(3) factorial designs. From the response surface graphs optimum regions were selected to be +1, -1, and +1 for temperature (60 degrees C), pressure (20 MPa) and percent modifier concentration (17.81%, v/v), respectively. Linearity dynamic range was found to be in the range of 2.0-150.0 microg/ml with significantly high value of correlation coefficient. The method was validated for precision, robustness and recovery to assess the viability of the established method. The chromatographic limit of detection and quantitation were 0.80 and 1.50 microg/ml respectively. The method has been successfully used to analyze commercial dosage form to assess the chromatographic performance of SFC system which was found to be 99.91%+/-1.62. The present work briefs the thermodynamic applications of PC-SFC with an emphasis on the results of stavudine. The foremost of such applications is the determination of solute diffusion coefficient in supercritical mobile phase by Taylor-Aris peak broadening technique.

Activation of NF kappa B by the respiratory burst of macrophages.

H2O2 and other reduced oxygen species have been proposed as activators of the transcription factor, NF Kappa B. Stimulated macrophages produce superoxide and H2O2 (the respiratory burst). We tested the hypothesis that production of these species could serve as part of the NF Kappa B activation pathway in rat alveolar macrophages and the J774A.1 mouse monocyte/macrophage cell line. Phorbol myristate acetate (PMA) and ADP, which stimulate the respiratory burst, caused NF Kappa B activation in both cells. Catalase abolished NF kappa B activation, while superoxide dismutase produced little inhibition. Thus, H2O2 was the principal agent of respiratory burst-associated NF kappa B activation. Abolition of NF kappa B activation by catalase also suggested that intermediate signaling pathways, such as protein kinase C activation or intracellular free calcium elevation must not be involved. Exogenous H2O2 added as a bolus > or = 50 microM (> or = 50 nmol/10(6) macrophages) also activated NF kappa B in macrophages. Nevertheless, the maximum endogenous production of H2O2 by stimulated alveolar macrophages during a 30-min incubation was < or = 1.3 nmol H2O2/10(6) cells for PMA stimulation and < or = 0.2 nmol H2O2/10(6) cells for ADP stimulation. Thus, relatively little endogenous H2O2 generation was required to produce NF kappa B activation compared to the required amount of exogenous H2O2. As H2O2 rapidly diffuses and is consumed, these results suggest that the site of action for endogenously generated H2O2 is probably close to its origin, the plasma membrane.

Transmembrane redox signaling activates NF-kappaB in macrophages.

In macrophages, NF-kappaB can be activated by H2O2 generated by the respiratory burst or added exogenously. The mechanism of H2O2 signaling may involve changes in the cellular redox state or a redox reaction at the plasma membrane; however, the site of H2O2 action cannot be readily ascertained because of its membrane permeability. Ferricyanide, a nonpermeable redox active anion, activated NF-kappaB in the macrophage cell line, J774A.1. In contrast with exogenous H2O2, activation by ferricyanide did not correlate with net oxidation of NAD(P)H or glutathione, suggesting that a transplasma membrane redox reaction itself was the first signaling process in NF-kappaB activation.

Serologic markers of gluten-sensitive enteropathy in bullous diseases.

BACKGROUND AND DESIGN: Dermatitis herpetiformis (DH) is characterized immunologically by the presence of IgA immune deposits in the skin and by the presence of various serum antibodies. Of these, antibodies to gliadin, reticulin, and endomysium have been found to be significant. There are, however, conflicting reports as to the exact specificity and sensitivity of these serologic markers in diagnosing DH. We examined the disease specificity of these three antibody markers in 14 patients with DH, in 98 patients with pemphigus and pemphigoid, and in 26 normal subjects. Reticulin and endomysium antibodies were detected by indirect immunofluorescence and gliadin antibodies by means of the enzyme-linked immunosorbent assay method. RESULTS: Among the various bullous diseases, endomysial and reticulin antibodies were found to be disease specific for DH. Endomysial antibodies occurred in twice the number of DH patients (72%) compared with the occurrence of reticulin antibodies (36%). Antigliadin antibodies were detected in two thirds of DH patients and were not disease specific since increased frequencies of these antibodies were also detected in patients with pemphigus and pemphigoid. CONCLUSION: These studies support the earlier findings of the high degree of specificity of endomysial antibodies for DH and, thus, help to differentiate DH from other bullous disorders.

Alpha-tocopherol and atherosclerosis.

Cardiovascular disease is the leading cause of morbidity and mortality in the Western world. There is compelling evidence incriminating oxidative stress in the pathogenesis of the atherosclerotic lesion. Several lines of evidence suggest that antioxidants, especially alpha-tocopherol, have potential beneficial effects with regard to cardiovascular disease. In vitro, alpha-tocopherol has been shown to inhibit platelet adhesion and aggregation and smooth muscle cell proliferation, exert anti-inflammatory effects on monocytes, and improve endothelial function. Also, supplementation with alpha-tocopherol has been shown to decrease lipid peroxidation, platelet aggregation, and pro-inflammatory activity of monocytes. However, clinical trials with alpha-tocopherol supplementation to date have been equivocal. Thus, although mounting in vitro evidence and animal models provide a sound scientific basis for alpha-tocopherol supplementation, further clinical trials are required before a definitive recommendation can be made with respect to the primary and secondary prevention of heart disease.

Free radicals and the heart.

Because of the molecular configuration, most free radicals are highly reactive and can cause cell injury. Protective mechanisms have evolved to provide defense against free-radical injury. Any time these defense systems are overwhelmed, such as during disease states, cell dysfunction may occur. In this review we discuss cellular sources as well as the significance of free radicals, oxidative stress, and antioxidants. A probable role of oxidative stress in various cardiac pathologies has been also analyzed. Although some methods for the detection of free radicals as well as oxidative stress have been cited, better methods to study the quantity as well as subcellular distribution of free radicals are needed in order to understand fully the role of free radicals in both health and disease.

Vitamin E and carotene status in pre-eclamptic pregnant women from India.

BACKGROUND: The imbalance between oxidative stress and the protective antioxidant system of the body enhanced the free radical mediated membrane lipid peroxidation and possibly the vascular endothelial damage due to peroxidation plays a major role in the aetiology of pre-eclampsia. With present day awareness on micronutrient antioxidants, we did investigate vitamin E and carotene status in Indian pre-eclamptic pregnant and full term normotensive pregnant women. Fresh vegetables and oils are considered to be good sources of vitamin E and carotene. The subjects were used to have good intake of fresh vegetable and oil as per Indian standard prescribed by Indian council of Medical research (ICMR) for this sub-continent. METHODS: The blood samples were processed for RBC vitamin E, serum carotene and serum cholesterol analysis. Routine laboratory tests like hemogram, serum urea, urate, malonyldialdehyde, urine sugar and albumin were performed. RESULTS: All pregnant subjects, both cases and control were maternal and gestational age matched. Routine check up showed no significant differences in means of white blood cell count, Hb/hematocrit and platelets. Serum urate and malonyldialdehyde were significantly raised in pre-eclamptic cases. The severely affected pre-eclamptic cases (diastolic BP >100 mmHg with proteinuria 2+ and more) showed markedly low levels of vitamin E and carotene whereas their levels were comparable between mild cases (diastolic BP <100 mmHg with+/-trace albuminuria) and normotensive pregnant control. CONCLUSIONS: The study concluded that the levels of vitamin E and carotene were markedly lowered in severe pre-eclamptic pregnant women from India. The mild pre-eclamptic cases did not show noticeable changes from that of control pregnant women. Further studies are needed to verify their therapeutic and prophylatic roles against pre-eclamptic complication suring pregnancy.

Probucol improves antioxidant activity and modulates development of diabetic cardiomyopathy.

To examine the role of free radicals in diabetic cardiomyopathy, myocardial antioxidants as well as lipid peroxide content were examined in rats made diabetic with a single injection of streptozotocin (65 mg/kg i.v). At 4 wk, the left ventricular peak systolic (LVSP) as well as aortic pressures were depressed in the diabetic group. Hearts from diabetic animals showed about a 100% increase in thiobarbituric acid reactive substances (TBARS), indicating increased lipid peroxidation. This was accompanied by about a 50% decrease in superoxide dismutase (SOD) and 60% decrease in glutathione peroxidase (GSHPx) enzyme activities. Catalase activity in these hearts showed a small but significant increase. Treatment with probucol (10 mg/kg i.p., on alternate days), a known lipid-lowering drug with strong antioxidant properties, was initiated 1 d after the induction of diabetes and was continued for 4 wk. In probucol-treated diabetic animals, LVSP was not different from controls. Probucol treatment caused a small but significant improvement in serum insulin and decrease in glucose levels as well as increased myocardial SOD, GSHPx, and catalase activities with a concomitant decrease in TBARS in the diabetic animals. These data provide evidence that diabetic cardiomyopathy is associated with an antioxidant deficit, and a better cardiac function due to treatment with probucol may be related to the improved insulin levels as well as maintenance of the antioxidant status of the heart.

Neutrophil oxygen free radical production proportionates with the degree of myocardial ischemia.

OBJECTIVE: To assess the production of oxygen free radicals by chemiluminescence and to assess leukocyte aggregation, in patients with acute myocardial infarction and angina pectoris, and to compare these to creatine kinase-MB levels. DESIGN: Prospective study with serial estimation at presentation and 72 h later. SETTING: Referral, tertiary care hospital. PATIENTS: Group 1, acute myocardial infarction (n = 18); group 2, stable angina pectoris (n = 8); and age-and sex-matched normal healthy persons (n = 12). All patients included had pain of less than 24 h duration with typical electrocardiographic and laboratory abnormalities. Patients or controls who had any inflammatory disease in the preceding two weeks or who were on anti-inflammatory drugs, calcium channel or beta-adrenoceptor blockers, were excluded. TESTS: Venous blood samples taken at presentation and 72 h later were analyzed for creatine kinase-MB using a standard kit, neutrophilic chemiluminescence and leukocyte aggregation. MAIN RESULTS: In group 1 there were significant rises in both creatine kinase-MB and chemiluminescence, which subsequently regressed (P less than 0.02). There was, however, no statistical correlation between the two. The qualitative pattern of the rise and fall of chemiluminescence was similar in group 2. Changes in leukergy in both groups were not significant. CONCLUSIONS: Oxygen free radical generation occurs early in myocardial ischemia with regression by 72 h. Neutrophilic chemiluminescence may provide an alternative method for assessment of myocardial ischemia.

Effect of nifedipine on Leishmania donovani infection in-vivo and in-vitro: chemiluminescence responses of peritoneal macrophages and neutrophils.

After peritoneal macrophages had been exposed to different concentrations of nifedipine (10-120 ng mL-1) there was a significant increase (P less than 0.001) in the percentage of Leishmania donovani infected macrophages compared with controls. Parasite load was also significantly increased (P less than 0.001) in nifedipine-treated, L. donovani infected, BALB/c mice, compared with untreated, infected mice, post-inoculation. Peak chemiluminescence responses were significantly depressed (P less than 0.001) in nifedipine-treated infected mice compared with untreated mice post-inoculation. It is suggested that availability of intracellular calcium is a factor in the defense mechanism of inflammatory cells in L. donovani infections.

Patent ductus arteriosus device embolization.

Nonsurgical closure of patent ductus arteriosus (PDA) using a duct occluder placed percutaneously is currently the first line of therapy and the success rate is quite high. Several devices are currently available. An eight year child underwent device closure of the ductus. However after deployment of the device it, became dislodged into the left pulmonary artery. Several attempts at catheter retrieval failed. The child underwent successful surgical removal of the device without cardiopulmonary bypass.

Clinical significance of glycosylated haemoglobin (HbA1C) over fasting blood sugar for monitoring metabolic control in diabetic patients with or without complications.

Glycosylated haemoglobin was studied in 30 cases of mild to severe diabetes in the age group 12-60 years. Ten patients were keto-acidotic. Glycosylated haemoglobin and fasting blood sugar levels were studied in patients with various complications of diabetes like neuropathy, nephropathy, retinopathy, keto-acidosis, cardiac and respiratory complications. There was a significant correlation between fasting blood sugar and glycosylated haemoglobin in normal subjects as well as in diabetic patients. There was a significant correlation between levels of glycosylated haemoglobin and blood sugar over preceding 4-6 weeks. Most frequent complication being retinopathy and keto-acidosis was associated with maximum glycosylated haemoglobin with poor metabolic control.

The effect of alpha-tocopherol on monocyte proatherogenic activity.

Atherosclerosis is the leading cause of morbidity and mortality in Westernized populations. The monocyte is a crucial cell in the genesis of the atherosclerotic lesion and is present during all stages of atherosclerosis. alpha-Tocopherol (AT) is the most active component of the vitamin E family and is the principal and most potent lipid-soluble antioxidant in plasma and LDL. With regard to monocyte function, AT supplementation (1200 IU/d) has been shown to decrease release of reactive oxygen species, lipid oxidation, release of cytokines such as interleukin-1ss (IL-1ss) and tumor necrosis factor-alpha (TNF-alpha) and decrease adhesion of monocytes to human endothelium. The mechanism of inhibition of superoxide and lipid oxidation by monocytes appears to be via inhibition of protein kinase C (PKC), the decrease in IL-1ss and TNF-alpha release by inhibition of 5-lipoxygenase and the inhibition of monocyte-endothelial cell adhesion via decrease in adhesion molecules on monocytes, CD11b and VLA-4 and by decreasing DNA-binding activity of nuclear transcription factor kappaB. Thus, in addition to the decrease in oxidative stress resulting from AT supplementation, as evidenced by decreased F(2)-isoprostanes and LDL oxidizability, AT is anti-inflammatory and exerts beneficial antiatherogenic effects on cells crucial in atherogenesis such as monocytes.

Oxygen free radicals and protective effect of captopril on myocardial infarct size.

Captopril (0.25 mg/kg and 0.5 mg/kg, p.o.) decreased the myocardial infarct size and prevented the progressive decrease in voltage of the R wave in rats. It had no marked effect on systolic blood pressure at these dose levels but higher doses (1 mg/kg, p.o.) reduced systolic blood pressure. It also produced a concentration-dependent (50-700 ng/10(6) cells) decrease of chemiluminescence response from rat neutrophils and markedly reduced serum malonyldialdehyde levels, elevated as a consequence of left coronary artery ligation. It is suggested that the protective effect of captopril may be mediated through a decreased formation or scavenging of reactive oxygen species.

Probucol promotes endogenous antioxidants and provides protection against adriamycin-induced cardiomyopathy in rats.

BACKGROUND: The potential usefulness of adriamycin (ADR) is restricted because of its cardiotoxic side effects. Since free radicals and lipid peroxidation are suggested to be involved in ADR cardiomyopathy, we examined the beneficial effects of probucol, a lipid-lowering drug with strong antioxidant properties. METHODS AND RESULTS: ADR was administered to rats in six equal intraperitoneal injections over a period of 2 weeks (cumulative dose of 15 mg/kg). After a 3-week posttreatment period, cardiomyopathy and congestive heart failure were characterized by ascites, congested liver, depressed cardiac function, elevated left ventricular end-diastolic pressure, and myocardial cell damage. Myocardial glutathione peroxidase (GSHPx) activity was decreased, and lipid peroxidation was increased. Probucol (cumulative dose, 60 mg/kg IP) was administered in six equal injections over a 2-week period on days alternating with ADR treatment. Probucol significantly attenuated the myocardial effects of ADR, improved left ventricular function, and lowered mortality as well as the amount of ascites. Treatment with probucol was also accompanied by an increase in myocardial GSHPx and superoxide dismutase activities, with a concomitant decrease in lipid peroxidation. CONCLUSIONS: These data provide evidence that ADR cardiomyopathy is associated with an antioxidant deficit. Improved cardiac function resulting from treatment with probucol may be related to the maintenance of the antioxidant status of the heart. The study suggests potential usefulness of antioxidant (probucol) therapy in ADR cardiomyopathy.