Glucose-6-phosphate dehydrogenase inhibition attenuates acute lung injury through reduction in NADPH oxidase-derived reactive oxygen species.

Acute lung injury (ALI) is a heterogeneous disease with the hallmarks of alveolar capillary membrane injury, increased pulmonary oedema and pulmonary inflammation. The most common direct aetiological factor for ALI is usually parenchymal lung infection or haemorrhage. Reactive oxygen species (ROS) generated by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX2) are thought to play an important role in the pathophysiology of ALI. Glucose-6-phosphate dehydrogenase (G6PD) plays an important role both in production of ROS as well as their removal through the supply of NADPH. However, how G6PD modulation affects NOX2-mediated ROS in the airway epithelial cells (AECs) during acute lung injury has not been explored previously. Therefore, we investigated the effect of G6PD inhibitor, 6-aminonicotinamide on G6PD activity, NOX2 expression, ROS production and enzymatic anti-oxidants in AECs in a mouse model of ALI induced by lipopolysaccharide (LPS). ALI led to increased G6PD activity in the AECs with concomitant elevation of NOX2, ROS, SOD1 and nitrotyrosine. G6PD inhibitor led to reduction of LPS-induced airway inflammation, bronchoalveolar lavage fluid protein concentration as well as NOX2-derived ROS and subsequent oxidative stress. Conversely, ALI led to decreased glutathione reductase activity in AECs, which was normalized by G6PD inhibitor. These data show that activation of G6PD is associated with enhancement of oxidative inflammation in during ALI. Therefore, inhibition of G6PD might be a beneficial strategy during ALI to limit oxidative damage and ameliorate airway inflammation.

A new spectrophotometric method for the determination of methyldopa.

A new, simple and low cost spectrophotometric method for the determination of methyldopa in pharmaceutical preparations was developed. The method was based on the coupling of methyldopa with 2,6-dichloroquinone-4-chlorimide (DCQ). The absorbance maximum (λ max) of the resulted colored product was at 400 nm. Different buffers were used to determine the optimal pH for the reaction. About 1% w/v acetate buffer with pH 8.0 gave the optimal pH required for the reaction. Of the different solvents tried, water and ethanol were found to be the most suitable solvents. Beer's law was obeyed in concentration range of 4-20 µg/ml methyldopa. The correlation coefficient was found to be (r = 0.9975). The limit of detection and limit of quantification were 1.1 µg/ml and 3.21 µg/ml, respectively. The reaction ratio between methyldopa and DCQ was studied and found to be 1:3. The work included the study of the possible interference of hydrochlorothiazide found in combination with methyldopa tablets. The method was validated and results obtained for the assay of two different brands of methyldopa tablets were compared with the BP method (colorimetric). The repeatability and reproducibility of the developed method were evaluated and the obtained results quoted. The derivative formed as a result of the reaction of methyldopa with DCQ was isolated and its possible mechanistic pathway was suggested.

Unusual manifestation of a concurrent demodectic and sarcoptic mange in a Zebu-Friesian cross-bred heifer.

We describe a rare case of a concurrent demodectic and sarcoptic mange in a 2-year-old heifer in Khartoum, Sudan. The lesions were massive lumps of granulomatous tumour-like dermatitis with thick, nodular folds mainly covering the head, neck and shoulders. Histopathological examination of the lesions revealed the presence of both Demodex bovis and Sarcoptes scabiei. The animal died regardless of the anti-parasitic treatment it received.

Antischistosomal effects of praziquantel, its alkaline hydrolysis and sun decomposed products on experimentally S. mansoni infected albino mice. (A) Efficacy assessment based on clinicopathological findings.

The antischistosomal activity of praziquantel (PZQ), its alkaline hydrolysis product (HP) and its sun-decomposed (SD) products was investigated in S. mansoni experimentally infected mice. The evaluation was made depending on the degree of clinico-pathological changes. The results obtained revealed that, PZQ, HP and the SD have induced partial suppression of worm fecundity as judged by the significant reduction in eggs per gram of faeces in comparison with the infected untreated control. The effect on tissue egg deposition in the treated groups was comparable to the infection of untrea ted control. Worm recovery showed large reduction in the number of worms for SD (47.6%) and HP (28.6%) compared to PZQ (16.6%) treated groups. So, the former two compounds have the superior antischistosomal activities. Glutamate pyruvate transominase (GPT) and glutamate oxaloacetate transaminase (GOT) concentrations were measured. The GOT values for all treated groups were significantly higher than those for the healthy control group (p=0.01). The SD group enzyme concentration was even higher than the infected untreated control. The GPT values of all groups were greater than the uninfected control and the difference was significant for the infected untreated, PZQ and the SD treated groups (p=0.05).

Effects of alkali and simulated gastric and intestinal fluids on danazol stability.

The degradation kinetics of methanolic solution of danazol (0.020% w/v) in aqueous buffers and sodium hydroxide was investigated using stability-indicating HPLC method. The drug degrades in alkaline medium through a base-catalysed proton abstraction rather than via an oxidative mechanism involving oxygen species. The degradation followed pseudo-first-order kinetics. The rates pH-profile exhibited specific base catalysis. The stability of the drug was found to be dependent on pH, buffer concentration, buffer species (acetate, borate, phosphate) and temperature. The ionic strength did not affect the stability of the drug. The energy of activation according to Arrhenius plot was estimated to be 22.62 kcal mol(-1) at pH 12 and temperatures between 30 and 60 degrees C. The effect of simulated gastric and intestinal fluids on the drug stability was also investigated. Two major hydrolytic degradation products were separated and identified by IR, NMR and mass spectrometry and the degradative pathway suggested.

Voltammetric determination of josamycin (a macrolide antibiotic) in dosage forms and spiked human urine.

The voltammetric behaviour of josamycin (a macrolide antibiotic) has been studied using direct current (DC(t)) alternating current (AC(t)) and differential pulse polarography (DPP). In Britton-Robinson buffers, josamycin developed cathodic waves over the pH range 7-12. At pH 10, a well-defined cathodic wave with diffusion current constant of 1.06 +/- 0.19 (n = 5) was obtained. The wave was characterized as being diffusion-controlled; and partially affected by adsorption phenomenon. The current-concentrations plots are rectilinear over the range 10-60 and 6-50 microg/ml using DC(t) mode and DPP mode, respectively. The minimum detectability limit was 1.2 microg/ml (1.9 x 10(-6) M) adopting the DPP mode. A method was proposed for the determination of josamycin in its tablets adopting both DC(t) and DPP modes. The results obtained were in good agreement with those given by the manufacturer. The method was extended to the in-vitro determination of the drug in spiked human urine; the % recovery was 98.06 +/- 1.76% (n = 5). The number of electrons involved in the reduction process was accomplished and a proposal of the electrode reaction was presented.

Sequential injection technique for automated titration: spectrophotometric assay of vitamin C in pharmaceutical products using cerium(IV) in sulfuric acid.

For the first time sequential injection analysis (SIA) technique has been employed for titrimetry. A new SI titrimetric spectrophotometric method for the assay of vitamin C in drug formulations was explored. The method is based on the oxidation reaction of vitamin C with cerium(IV) in sulfuric acid media using a spectrophotometer as a detector with the wavelength monitored at 410 nm. A 2(3) factorial design chemometric approach was employed to study the interaction effect of the chemical and system variables, mainly cerium(IV), sulfuric acid concentrations and the flow rate. The results of the chemometric optimization revealed that the optimum operating conditions for the SI titrimetric analysis of vitamin C were 7.0 x 10(-3) M cerium(IV), 0.455 M sulfuric acid and 28.9 microL s-1 flow rate. A linear calibration plot for the determination of vitamin C was obtained in the concentration range between 30 to 200 ppm. The method was applied to the determination of vitamin C in pharmaceutical preparations and no excipient was found to pose any interference, thus rendering the method suitable for the determination of the drug in pharmaceutical preparations. The SIA method is found to be accurate when the results were statistically compared with the results obtained by the BP standard method. The SIA method is superior when compared to the conventional titration method, the BP standard method and previous methods with respect to precision and automation in solution handling.

Sequential injection spectrophotometric assay of bromazepam complexed with iron(II) in hydrochloric acid with chemometric optimization.

A sequential injection spectrophotometric method for the assay of bromazepam anxiolytic drug has been reported. The method is based on the complexation reaction of bromazepam with iron(II) in hydrochloric acid media and spectrophotometrically measuring the product at lambda(max)=585 nm. A comprehensive chemometrical optimization treatment was successfully utilized for determining the proper optimum operating conditions for both the system and the chemical variables. The experimental design approach was employed and a 2(k) factorial design was run for studying the interaction effects of four factors namely, hydrochloric acid concentration, iron(II) concentration, delay time and flow rate. The super modified simplex algorithm was utilized for optimizing the three highly interacting factors which were, hydrochloric acid, iron(II), and delay time. The conditions obtained were 150 microl 0.110 mol l(-3) hydrochloric acid, 75 microl 0.328 mol l(-3) iron(II), 1200 s delay time and 40 microl s(-1) flow rate. The method was found to be suitable for the determination of Bromazepam in pharmaceutical preparations and the results obtained for the assay of the compound in proprietary drugs indicate that the method suffers no interference from excipients.

Photodegradation studies on chloroquine phosphate by high-performance liquid chromatography.

A method based on high-performance liquid chromatography (HPLC) was developed to study degraded chloroquine samples produced after exposure to sunlight in the Sudan. The method was also used to investigate chloroquine photodegradation after irradiation by UV and sunlight at ambient temperature. The study showed that the photodecomposition of chloroquine was pH and solvent dependent. Moreover, the extent of reaction was found to increase in the absence of oxygen. At pH 8, where the reaction rate was high, the photodecomposition was found to follow pseudo-first-order reaction kinetics. The HPLC method developed was also employed to analyse chloroquine and its degradation products in two commercially available brands of chloroquine injections which had been stored under local conditions in the Sudan. A number of degradation products were separated and examined by photodiode array spectroscopy and preparative TLC.

Determination of chloroquine and its decomposition products in various brands of different dosage forms by liquid chromatography.

Various commercial preparations of chloroquine dosage forms have been examined both by thin-layer chromatography (TLC) and liquid chromatography (LC). TLC showed that all these preparations yielded more than one spot, indicating possible degradation. An LC method has been adapted for the determination of chloroquine in these drug formulations. The standard calibration curve was linear over the concentration range 1-6 micrograms ml-1. Chloroquine was assayed in various brands of different forms (ampoules, tablets and syrups). However, it was observed, for some samples, that the bands obtained were rather broad, showing shoulders or peak splitting, indicating the presence of other compounds coeluting with chloroquine. The utility of this method for the quality control of this major drug is assessed in the context of the need to carefully monitor drug purity in a tropical climate, particularly in situations where there may be doubt about the quality of the primary manufacturer.

A study of chloroquine and desethylchloroquine plasma levels in patients infected with sensitive and resistant malaria parasites.

This study was carried out on 63 patients in the town of Gadaref in eastern Sudan; each patient was given the standard therapeutic dose of chloroquine (CQ). Plasma levels of chloroquine and its major metabolite desethylchloroquine (DCQ) were measured by means of a high-performance liquid chromatographic method (HPLC) in patients infected with sensitive (sensitive group) and resistant (resistant groups) strains of Plasmodium falciparum. The ratios of chloroquine to desethylchloroquine (CQ/DCQ) in different groups were calculated and the results obtained were compared and correlated with the degree of parasitaemia. The statistical analysis of the results showed that the plasma content of CQ and the CQ/DCQ ratio in the majority of the patients fall within the normal mode of distribution. A small group of patients showed a deviation from the normal mode by having a rather high CQ plasma level and a high ratio of CQ/DCQ. The mean plasma levels of CQ and the CQ/DCQ ratio in the sensitive group was found to be higher than that in the resistant groups. However, these differences were found to be not significant. Correlation tests showed that the levels of CQ and the CQ/DCQ ratios increase with the increase of the degree of parasitaemia in the sensitive group but decrease with the increase of parasitaemia in resistant groups.

Plasma chloroquine measurement in the evaluation of Plasmodium falciparum sensitivity.

In-vivo assessment of the sensitivity of Plasmodium falciparum to chloroquine was carried out in 63 patients in eastern Sudan. Standard triple-dose therapy with chloroquine (25 mg base kg-1 body wt) failed in curing 30 patients. All grades of resistance were demonstrated in the study, confirming that the phenomenon of chloroquine resistance is well established in this area. Factors which may have accelerated the spread of resistant strains include: a drop in the immunity of the local people caused by previous drought, introduction of non-immune refugees, increased transmission following heavy rains, and massive drug pressure. Plasma levels of chloroquine attained in our patients exceeded the therapeutic level and thus ruled out ineffective levels as a possible cause of treatment failure. Moreover, there was no significant difference between chloroquine levels in patients infected with sensitive or resistant strains. As some patients were cured with an additional dose of chloroquine, it is proposed that chloroquine measurement be carried out in patients treated for falciparum malaria with a view to defining new effective levels for semi-immune populations.

Cutaneous habronemiasis in horses and domestic donkeys (Equus asinus asinus).

Cutaneous habronemiasis in 15 horses and 5 donkeys is described. The lesions were distributed in many parts of the body involving the medial canthus, shoulder and pectoral regions, knee and fetlock joints, abdominal wall and prepuce. Some animals had more than one lesion. The lesions were ulcerative and filled with soft light red granulation tissue. When curretted, the deeper layers revealed a dense fibrous tissue with calcified foci. Close examination of the lesions showed that the superficial layer of this dense fibrous tissue contained small caseated and necrotic foci. The same features prevailed in lesions involving the muscular areas, but the deeper layers consisted of a dense granuloma with no evidence of cicatrization. Curretted material digested in potassium hydroxide revealed fragments of larvae of the nematode suggestive of Draschia or Habronema. The histopathological changes were severe and comprised: necrotic foci in a dense fibrous stroma infiltrated with eosinophils, macrophages and few giant cells. Sections of the larvae surrounded by eosinophils were encountered in lesions of the muscular areas. Curretting and excision of the lesion were effective and produced complete healing of the wound by scar tissue formation.

Studies on gangrenous mastitis in goats.

One hundred and fifty goats were diagnosed as having proven gangrenous mastitis. The disease was categorized into early, intermediate and late stages. Gangrenous mastitis in goats is typified by a sudden onset, dark hyperemia, and edema with progressive discoloration of the distal part of the udder. The disease affected lactating goats but not the dry ones. Coagulase positive Staphylococcus aureus was isolated from 60% of composite and half milk samples obtained from the diseased goats. The histopathological changes mainly comprised proliferation of connective tissue, thrombosis and necrosis involving a group of lobules. Treatment of the early and intermediate stages of the disease was successful through the administration of systemic and intramammary terramycin together with diuretics and topical antiseptic cream. The late stage of the disease was successfully treated only through surgery.

Experimental transmission of a goat strain of Sarcoptes scabiei to desert sheep and its treatment with ivermectin.

Desert sheep were experimentally infected with a goat strain of Sarcoptes scabiei, proving that this mite is not completely host specific. More severe lesions were produced on sheep when the mites were applied to lacerated than when applied to scarified or non-scarified areas. Lesions were more pronounced on moistened areas than dry areas and lesions produced on dry scarified areas resembled those produced on non-scarified moistened ones. Skin scrapings from the experimental lesions in the sheep contained numerous mites of all stages, proving the goat mites had become well established and were reproducing actively in the experimental lesions. Severe histopathological changes were observed in skin sections, comprising mainly thick crust formation, mites beneath the keratin and severe degenerative and necrotic changes. Treatment of sheep with ivermectin at a dose rate of 200 micrograms kg-1 body weight produced rapid and excellent cure.

Experimental infection of the domestic donkey (Equus asinus asinus) with a goat strain of Sarcoptes scabiei, and treatment with ivermectin.

Donkeys were experimentally infected with Sarcoptes scabiei from goats, proving that this mite is not completely host specific. The lesions produced on the donkeys were characterized by extensive areas of alopecia, covered with thin crusts and bran-like scales. The skin had many excoriations but showed no cracks or fissures. Skin scrapings from the experimental lesions in donkeys contained numerous mites of all stages and skin sections showed severe histopathological changes, proving that the goat mites had become well established and were reproducing actively in experimental lesions. Treatment of the donkeys with ivermectin produced excellent and rapid cure.

Effect of 6-hydroxydopamine on the metabolism of endogenous octopamines and catecholamines.

The daily excretion in rat urine of o-(OHMA) (0.2), m-(MHMA) (0.9), p-(PHMA) hydroxymandelic acid (78), vanillylmandelic acid (VMA) (3.5) and homovanillic acid (30 micrograms day-1), terminal metabolites of the isomeric octopamines and catecholamines, has been determined by gas chromatography-mass spectrometry. Chemical sympathectomy with 6-hydroxydopamine (i.p.) caused the daily output of each acid (except OHMA) to drop by 50% and that of PHMA was restored to normal the most rapidly after cessation of treatment. These results indicate that m- and p- (but not o-) octopamine coexist with catecholamines in sympathetic nerve terminals, are subject to the same compensatory biosynthetic mechanism following chemical sympathectomy and that p-octopamine has the highest turnover rate of these biogenic amines in the rat.