Accurate quantitation standards of glutathione via traceable sulfur measurement by inductively coupled plasma optical emission spectrometry and ion chromatography.

The quantitative analysis of glutathione (GSH) is important in different fields like medicine, biology, and biotechnology. Accurate quantitative measurements of this analyte have been hampered by the lack of well characterized reference standards. The proposed procedure is intended to provide an accurate and definitive method for the quantitation of GSH for reference measurements. Measurement of the stoichiometrically existing sulfur content in purified GSH offers an approach for its quantitation and calibration through an appropriate characterized reference material (CRM) for sulfur would provide a methodology for the certification of GSH quantity, that is traceable to SI (International system of units). The inductively coupled plasma optical emission spectrometry (ICP-OES) approach negates the need for any sample digestion. The sulfur content of the purified GSH is quantitatively converted into sulfate ions by microwave-assisted UV digestion in the presence of hydrogen peroxide prior to ion chromatography (IC) measurements. The measurement of sulfur by ICP-OES and IC (as sulfate) using the "high performance" methodology could be useful for characterizing primary calibration standards and certified reference materials with low uncertainties. The relative expanded uncertainties (% U) expressed at 95% confidence interval for ICP-OES analyses varied from 0.1% to 0.3%, while in the case of IC, they were between 0.2% and 1.2%. The described methods are more suitable for characterizing primary calibration standards and certifying reference materials of GSH, than for routine measurements.

DNA quantification via traceable phosphorus measurement through microwave-assisted UV digestion-ion chromatography.

Accurate quantification of deoxyribonucleic acid (DNA) is critical for many analyses in molecular biology and genetic tests. We present a method in which the stoichiometrically existing phosphorus content in purified genomic DNA is quantitatively converted into orthophosphate ions by microwave assisted-UV digestion in the presence of microlitre quantities of dilute reagents (HCl, HNO(3), H(2)O(2)). The tandem use of microwave energy and ultraviolet photons for DNA digestion in pressurized quartz vessels enables a maximum reaction temperature of 240 °C resulting in efficient and fast mineralization of high molecular weight DNA within 30 minutes. Compared to hotplate digestion, the digestion time is reduced by a factor of 32. The MW-UV sample preparation approach coupled with the ion chromatographic measurement of phosphate using a high performance (HP) methodology provides an accurate quantitation of phosphorus mass fractions as low as 0.3 µg g(-1), corresponding to a DNA mass of 25 µg. The relative expanded uncertainties (% U) expressed at 95% confidence for these analyses range from 0.2 to 0.6%. Critically, the matrix of the calibrant solution is also matched with respect to the digested matrix anions (chloride, nitrate), without which significant bias in IC performance is observed. The phosphorus content of the calf thymus DNA was also measured using high-performance inductively coupled plasma optical emission spectroscopy (HP-ICP-OES), which provided independent data for comparison with the MW-UV digestion-IC based approach. Ion chromatography requires smaller volume of materials to perform the analysis and could be useful for characterizing primary calibration standards and certified reference materials with low uncertainties.

Effect of nitric oxide modulators on pylorus-ligation-induced ulcers in the rat.

In the present investigation, the effect of nitric oxide (NO) modulators on pylorus-ligation-induced gastric ulcers in rats was studied. Sodium nitroprusside (SNP, 1 mg kg-1), a NO donor, l-arginine (l-Arg, 300 mg kg-1), the NO precursor, nitro-l-arginine methyl ester (l-NAME), a nitric oxide synthase (NOS) inhibitor and lipopolysaccharide (LPS, 3 mg kg-1), a NOS inducer have been administered prior to pylorus ligation. The effects of these interventions on the gastric mucosal nitrite content, the incidence of ulcers, the ulcer index, the volume of gastric secretions and the free and total acidity 4 h after pylorus ligation were investigated. SNP, l-Arg and LPS pretreatment increased the mucosal nitrite contents and protected the animals against pyloric-ligation-induced increase in acidity and ulcer index. However, inhibition of NOS activity by l-NAME (10 mg kg-1) decreased the nitrite content and augmented the ulcer-induced increase in the gastric acid contents. Coadministration of l-Arg with l-NAME prevented the l-NAME-induced changes. Interventions which increased the mucosal nitrite content were found to be protective against ulcers. However, the NOS inhibitor l-NAME decreased mucosal nitrite levels and was ulcerogenic. Results obtained thus indicate the protective effect of NO on the pyloric-ligation-induced ulcers in the rat.

Free radicals and antioxidant status following pylorus ligation induced gastric mucosal injury in rats.

The present investigation was undertaken to suggest the involvement of free radicals in the pathogenesis of pyloric ligation induced ulcers in rats. Lipid peroxidation product (MDA), antioxidant contents and secretory activity have been studied in the rat stomach at different time intervals after pylorus ligation induced ulcers. A time-dependent increase in the peptic activity, free and total acid content in the gastric juice was observed. MDA level, myeloperoxidase (a neutrophil maker) and catalase activity in the rat stomach homogenate were augmented 2, 4 and 19 h after pylorus ligation. While a significant and time-dependent decrease in the glutathione content, superoxide dismutase and glutathione peroxidase activity was observed after pyloric ligation. An increase in the acid and pepsin content in the gastric juice in N-ethyl maleamide (GSH depletor) or aminotriazole (catalase inhibitor) pretreated animals, further suggests that depletion in the antioxidant levels enhance ulceration. Thus the results obtained have shown alterations in the antioxidant status following ulceration, indicating that free radicals seems to be associated with the pylorus ligation induced ulceration in rats.

Interferon-inducer polyriboinosinic-polyribocytidylic acid: a potent anti-gastric ulcer agent and inhibitor of the gastric proton pump in rats.

1. Polyriboinosinic-polyribocytidylic acid (Poly I:Poly C), an interferon inducer was studied for its effect on gastric ulceration in rats. Polyriboinosinic-polyribocytidylic acid (1, 2 and 4 mg/kg, i.m.) showed a dose-dependent inhibition of gastric ulcers induced by aspirin, cold restraint stress and pylorus ligation (Shay's model). Protective dose (PD50) +/- SEM values of Poly I:Poly C on these models of ulcers were 1.9 +/- 0.2, 2.3 +/- 0.4 and 2.8 +/- 0.4 (mg/kg, i.m.) respectively. 2. Polyriboinosinic-polyribocytidylic acid (10-60 micrograms) produced dose-dependent inhibition of gastric proton pump (H+/K(+)-ATPase) activity in the gastric parietal microsomal fraction. The concentration of Poly I:Poly C causing a 50% inhibition (IC50) +/- SEM was found to be 17.6 +/- 1.2 micrograms. 3. Polyriboinosinic-polyribocytidylic acid caused a significant decrease in free and total acid and pepsin and an increase in mucin content in Shay (pylorus-ligated) rat. 4. Polyriboinosinic-polyribocytidylic acid did not exert a significant influence on isolated tissue preparations for anti-cholinergic (acetylcholine-induced contraction of guinea-pig ileum) and H2-anti-histaminic (histamine-induced contraction of rat uterus and guinea-pig auricle) activities. 5. Thus, the present study indicates that Poly I:Poly C may possess anti-gastric ulcer activity as a result of inhibition of the gastric proton pump.

Prevention of ischaemia-induced biochemical changes by curcumin & quinidine in the cat heart.

Effect of myocardial ischaemia on the bioantioxidants levels in the cat heart was evaluated. In addition, effect of curcumin, an anti-inflammatory and anti-thrombotic drug, and quinidine, a standard antiarrhythmic drug, was also studied in the cat. Myocardial ischaemia was induced by the ligation of left descending coronary artery. Quinidine (1 mg/kg, iv) was administered 15 min prior to while curcumin (100 mg/kg, ip) was given 30 min before ligation. Hearts were removed 4 h post coronary artery ligation. Levels of glutathione (GSH), malonaldelhyde (MDA), myeloperoxidase (MPO), superoxide dismutase (SOD), catalase (CAT) and lactate dehydrogenase (LDH) were estimated in the ischaemic and non-ischaemic zones. Both the drugs protected the animals against decrease in the heart rate and blood pressure following ischaemia. In the ischaemic zone, after 4 h of ligation, an increase in the level of MDA and activities of MPO and SOD (cytosolic fraction) were observed. Quinidine and curcumin pretreatment prevented the ischaemia-induced elevation in MDA contents and LDH release. Curcumin pretreatment did not prevent the increase in MPO activity while quinidine did. Results obtained indicate alterations in the bioantioxidants following ischaemia and both curcumin and quinidine prevented ischaemia induced changes in the cat heart.