A comparative study was conducted to evaluate three different permeabilization methods: FACS Permeabilizing Solution (FPerm), CytoFix/CytoPerm Kit (CFP) and Paraformaldehyde-Tween 20 (PFT) reagents, in cytoplasmic labeling of myeloperoxidase (MPO). Peripheral blood cells from 23 healthy subjects were fixed and permeabilized according to the proposed procedures, prior to direct immunofluorescence staining with CD14, CD45, IgG1, IgG2 and MPO monoclonal antibodies (McAb). Subsequent flow cytometric analysis was performed on FACSCalibur flow cytometer (Becton Dickinson, BD). As far as the antigenic expression of MPO in normal samples is concerned, FPerm and CFP demonstrated better cytoplasmic staining by inducing minor effects on light-scattering properties of the cell populations, whereas PFT-treated samples showed a diminished ability to distinguish the cell types. However, the simple and rapid FPerm method required an earlier processing of samples since the stored whole blood samples (for more than 8 hours) tended to show a significant decrease of fluorescence intensity. We also have demonstrated that P/N ratio possesses added value in evaluation of cell reactivity in immunophenotyping, based upon the apparent nonspecific cytoplasmic staining of MPO in the lymphocyte population.
The onset of 21st century witnessed the awareness among the masses regarding the diet-health linkages. The researchers attempted to explore traditional products/plants were in the domain of pharmacy and nutrition focussing on their health benefits. In the present research intervention, we investigate the role of Nigella sativa fixed oil (NSFO) and essential oil (NSEO) in improving antioxidant status and modulation of enzymes. The National Institute of Health (NIH) provided us 30 Sprague Dawley rats that were equally placed in three groups. The groups were fed on their respective diets (56 days) two experimental diets i.e. D2 (NSFO @ 4.0%) and D3 (NSEO @ 0.30%) and control. The indices pertaining to antioxidant status, antioxidant enzymes, and parameters pertaining to immunity were evaluated at 4 weeks interval. The experimental diets (NSFO@ 4.0% & NSEO@ 0.30%) modulated the activities of antioxidant enzymes i.e., catalase (CAT), superoxide dismutase (SOD), glutathione transferase (GST), glutathione reductase (GR) and glutathione peroxidase (GPx), positively. Indices of antioxidant status like tocopherols and glutathione were in linear relationship with that of GPx, GR and GST (P<0.01). Myeloperoxidase activities were in negative correlation with GST (P<0.01) but positive correlation with some other parameters. In the nutshell, the fixed and essential oil of Nigella sativa are effective in improving the indices pertaining to antioxidant status, however, the immune boosting potential needs further clarification. However, authors are of the view that there is need to explore the molecular targets of Nigella sativa fixed and essential oils. Findings from such studies would be useful to validate this instant study for health promoting potential against diabetes mellitus and cardiovascular disorders.
Nigella sativa is an important component of several traditional herbal preparations in various countries. It finds its applications in improving overall health and boosting immunity. The current study evaluated the role of fixed and essential oil of Nigella sativa against potassium bromate induced oxidative stress with special reference to modulation of glutathione redox enzymes and myeloperoxidase.
A disposable horseradish peroxidase (HRP)-based electrochemical genosensor was developed for chronoamperometric detection of single-stranded asymmetric lolB gene PCR amplicon (118 bp in length) of the food-borne pathogen, Vibrio cholerae. A two-step sandwich-type hybridization strategy using two specific probes was employed for specific detection of the target single-stranded DNA (ssDNA). The analytical performances of the detection platform have been evaluated using a synthetic ssDNA (ST3) which was identical to the target single-stranded amplicon and a total of 19 bacterial strains. Under optimal condition, ST3 was calibrated with a dynamic range of 0.4883-15.6250 nM. By coupling asymmetric PCR amplification, the probe-based electrochemical genosensor was highly specific to the target organism (100% specificity) and able to detect as little as 0.85 ng/μl of V. cholerae genomic DNA.
Biomarkers play a pivotal role in the diagnosis and management of patients with acute coronary syndrome. This study aimed to investigate the differences in level of several biomarkers, i.e. C-reactive protein, myeloperoxidase, soluble CD40 ligand and placental growth factor, between acute coronary syndrome and chronic stable angina patients. The relationship between these biomarkers in the coronary circulation and systemic circulation was also investigated.
This study aimed to work out a simple and high-yield procedure for the immobilization of horseradish peroxidase on silver nanoparticle. Ultraviolet-visible (UV-vis) and Fourier-transform infrared spectroscopy and transmission electron microscopy were used to characterize silver nanoparticles. Horseradish peroxidase was immobilized on β-cyclodextrin-capped silver nanoparticles via glutaraldehyde cross-linking. Single-cell gel electrophoresis (Comet assay) was also performed to confirm the genotoxicity of silver nanoparticles. To decrease toxicity, silver nanoparticles were capped with β-cyclodextrin. A comparative stability study of soluble and immobilized enzyme preparations was investigated against pH, temperature, and chaotropic agent, urea. The results showed that the cross-linked peroxidase was significantly more stable as compared to the soluble counterpart. The immobilized enzyme exhibited stable enzyme activities after repeated uses.
A study was undertaken to evaluate the ability of flow cytometric analysis of intracellular myeloperoxidase (MPO) in differentiating populations of lymphocytes (L), monocytes (M) and granulocytes (G), by means of lysed whole blood method. Anticoagulated blood from 23 normal individuals was lysed with FACS lysing solution and permeabilized with FACS permeabilizing solution before subjected to direct immunofluorescence staining. The geometric means of the fluorescence intensity were measured using FACSCalibur flow cytometer (Becton Dickinson). Populations of L, M and G were gated based on their light scatter characteristics and expression of CD14 and CD45. Then, the fluorescence intensity of MPO expression was studied in these individual cell populations. The results showed that fluorescence intensity of MPO was the strongest in G and weakest in L, whereas M showed intermediate fluorescence intensity. Our findings reveal that discrimination of these three cell types is achievable based upon the sole expression of intracellular MPO.
The specificity and sensitivity of an indirect and two (an 'ordinary' and a 'rapid') double sandwich enzyme-linked immunosorbent assay (ELISA) procedures for the quantitation of Calloselasma rhodostoma (Malayan pit viper) venom were examined. The three assays were equally sensitive and the accuracy of the assays was not substantially affected by individual variation in the venom composition. The specificity of the assays was examined against 26 venoms from snakes of the families Viperidae and Elapidae. While the double sandwich ELISA procedures were sufficiently specific to be used in the clinical immunodiagnosis of C. rhodostoma bite in Malaysia, the indirect ELISA procedure exhibited extensive cross-reactivity with other Malaysian pit viper venoms. Attempts were made to improve the specificity of the indirect ELISA procedure for the quantitation of C. rhodostoma venom. A 'low ELISA cross-reactivity' venom fraction (termed VF52) was isolated from C. rhodostoma venom by repeated Sephadex G-100 gel filtration chromatography. The indirect ELISA procedure using antibodies to VF52 as immunoreagent showed an improvement in specificity. The use of the indirect ELISA procedure for the detection of C. rhodostoma antibodies was also examined and the results show that the assay was sufficiently specific to be used for retrospective diagnosis of C. rhodostoma bite in Malaysia, in particular when VF52 was used as the coating antigen.
Composition, physicochemical properties and enzyme inactivation kinetics of coconut water were compared between immature (IMC), mature (MC) and overly-mature coconuts (OMC). Among the samples studied, pH, turbidity and mineral contents for OMC water was the highest, whereas water volume, titratable acidity, total soluble solids and total phenolics content for OMC water were the lowest. Maturity was found to affect sugar contents. Sucrose content was found to increase with maturity, and the reverse trend was observed for fructose and glucose. Enzyme activity assessment showed that polyphenol oxidase (PPO) in all samples was more heat resistant than peroxidase (POD). Compared to IMC and MC, PPO and POD from OMC water showed the lowest thermal resistance, with D83.3°C=243.9s (z=27.9°C), and D83.3°C=129.9s (z=19.5°C), respectively.
Human diploid fibroblasts (HDFs) undergo a limited number of cellular divisions in culture and progressively reach a state of irreversible growth arrest, a process termed cellular ageing. Even though beneficial effects of Piper betle, Chlorella vulgaris and tocotrienol-rich fraction (TRF) have been reported, ongoing studies in relation to ageing is of interest to determine possible protective effects that may reverse the effect of ageing. The aim of this study was to evaluate the effect of P. betle, C. vulgaris and TRF in preventing cellular ageing of HDFs by determining the activity of antioxidant enzymes viz.; catalase, superoxide dismutase (SOD) and glutathione peroxidase.
Exercise is beneficial to health, but during exercise the body generates reactive oxygen species (ROS) which are known to result in oxidative stress. The present study analysed the effects of vitamin E (Tri E®) on antioxidant enzymes; superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (Cat) activity and DNA damage in rats undergoing eight weeks exercise.
The efficiency of advanced oxidation processes (AOPs), enzymatic treatment and combined enzymatic/AOP sequences for the colour remediation of soda and chemimechanical pulp and paper mill effluent was investigated. The results indicated that under all circumstances, the AOP using ultraviolet irradiation (photo-Fenton) was more efficient in the degradation of effluent components in comparison with the dark reaction. It was found that both versatile peroxidase (VP) from Bjerkandera adusta and laccase from Trametes versicolor, as pure enzymes, decolorize the deep brown effluent to a clear light-yellow solution. In addition, it was found that in the laccase treatment, the decolorization rates of both effluents were enhanced in the presence of 2, 2'-azinobis (3-ethylbenzthiazoline-6-sulfonate), while in the case of VP, Mn(+2) decreased the efficiency of the decolorization treatment. The concomitant use of enzymes and AOPs imposes a considerable effect on the colour remediation of effluent samples.
Hyperglycemia-induced increase in oxidative stress is implicated in diabetic complications. This study investigated the effect of metformin and/or glibenclamide in combination with honey on antioxidant enzymes and oxidative stress markers in the kidneys of streptozotocin (60 mg/kg; intraperitoneal)-induced diabetic rats. Diabetic rats were randomized into eight groups of five to seven rats and received distilled water (0.5 mL); honey (1.0 g/kg); metformin (100 mg/kg); metformin (100 mg/kg) and honey (1.0 g/kg); glibenclamide (0.6 mg/kg); glibenclamide (0.6 mg/kg) and honey (1.0 g/kg); metformin (100 mg/kg) and glibenclamide (0.6 mg/kg); or metformin (100 mg/kg), glibenclamide (0.6 mg/kg) and honey (1.0 g/kg) orally once daily for four weeks. Malondialdehyde (MDA) levels, glutathione peroxidase (GPx) and superoxide dismutase (SOD) activities were significantly elevated while catalase (CAT) activity, total antioxidant status (TAS), reduced glutathione (GSH), and GSH:oxidized glutathione (GSSG) ratio was significantly reduced in the diabetic kidneys. CAT, glutathione reductase (GR), TAS, and GSH remained significantly reduced in the diabetic rats treated with metformin and/or glibenclamide. In contrast, metformin or glibenclamide combined with honey significantly increased CAT, GR, TAS, and GSH. These results suggest that combination of honey with metformin or glibenclamide might offer additional antioxidant effect to these drugs. This might reduce oxidative stress-mediated damage in diabetic kidneys.
Lentinula edodes (Berk) Pegler, commonly known as Shiitake mushroom has been used as medicinal food in Asian countries, especially in China and Japan and is believed to possess strong immunomodulatory property. In the present study, the methanolic extract of the fruit bodies of L. edodes was investigated for cytoprotective effect against H2O2-induced cytotoxicity in human peripheral blood mononuclear cells (PBMCs) by measuring the activities of xanthine oxidase (XO) and glutathione peroxidase (GPx) . H2O2 at a concentration of 5 microM caused 50% inhibition of PBMCs viability. The extract improved the PBMC viability and exerted a dose-dependent protection against H2O2-induced cytotoxicity. At 100 microg/ml of extract concentration, the cell viability increased by 60% compared with the PBMCs incubated with H2O2 alone. The extract also inhibited XO activity in PBMC, while showing moderate stimulatory effect on GPx. However, in the presence of H2O2 alone, both the enzyme activities were increased significantly. The GPx activity increased, possibly in response to the increased availability of H2O2 in the cell. When the cells were pretreated with the extract and washed (to remove the extract) prior to the addition of H2O2, the GPx and XO activities as well as the cell viability were comparable to those when incubated with the extract alone. Thus, it is suggested that one of the possible mechanisms via which L. edodes methanolic extract confers protection against H2O2-induced oxidative stress in PBMC is by inhibiting the superoxide-producing XO and increasing GPx activity which could rapidly inactivate H2O2.
The trace elements copper, zinc, and selenium are important immune modulators and essential cofactors of the antioxidant enzymes. In the present study, the proliferative effect of human peripheral mononuclear cells (PBMCs) that have been exposed to copper, zinc, and selenium and the corresponding activities of antioxidant enzymes, namely superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase, were determined. Zinc and copper stimulated the PBMC proliferation in a dose-dependent manner within the dose range 25-200 micromol/L. SOD and GPx activities in PBMCs exposed to zinc were inhibited, whereas catalase activity was unaffected. All the three antioxidant enzymes in the cells exposed to copper were inhibited. Selenium exerted more potent inhibition of the cell proliferation while causing stimulation of the antioxidant enzymes at the lowest dose (25 micromol/L) than at the highest dose (200 micromol/L) tested. A significant negative correlation was observed between proliferation and antioxidant enzyme (SOD and GPx) activities in trace-element-exposed PBMC. The present findings substantiate the importance of trace elements as immune modulators and the involvement of enzymatic antioxidant system in the immune cell regulation.
The hepatic and pulmonary effects of nitrofurantoin (40 mg/kg, intraperitoneally) were determined at 4 and 24 hr following its administration in mice fed for 10 weeks with a vitamin E sufficient, deficient or enriched diet. Liver glutathione (GSH) was reduced by nitrofurantoin at 4 hr but was unchanged 20 hr later. Nitrofurantoin did not affect liver glutathione peroxidase, glutathione reductase or superoxide dismutase activities. Liver catalase activities were decreased by nitrofurantoin at 4 hr. Lung GSH levels were increased whilst glutathione peroxidase activity was decreased at 4 and 24 hr. Lung glutathione reductase activity was reduced in certain groups. Nitrofurantoin did not affect lung superoxide dismutase, but catalase was decreased at 24 hr. Liver malondialdehyde levels were increased by nitrofurantoin in the vitamin E deficient group whilst lung malondialdehyde levels remained unchanged. Both liver and lung malondialdehyde levels were unaffected by vitamin E supplementation when compared to the vitamin E-sufficient group. These results suggest that nitrofurantoin (40 mg/kg) was deleterious to the liver and lung. Nitrofurantoin-induced lipid peroxidation was seen in vitamin E deficiency but an increase in dietary vitamin E content did not provide additional protection compared to the recommended daily allowance. The antioxidant activities of alpha-tocopherol and gamma-enriched tocotrienol were similar.
1. The mean levels of lipid peroxidation products, namely conjugated diene and malonaldehyde, were increased in the soleus muscles of hyperthyroid cats, while the mean glutathione peroxidase activity was decreased. No corresponding similar changes were noted in the fast extensor digitorum longus muscles and serum. 2. Propranolol administration prevented the increase in conjugated diene level in the soleus muscles of hyperthyroid cat but not the malonaldehyde level. It also prevented the reduction in glutathione peroxidase activity in the slow oxidative soleus muscles of hyperthyroid cats. 3. Maximal twitch tension, subtetanic tension and maximum tetanic tension of soleus and EDL muscles were reduced in hyperthyroid cats. Propranolol administration for 5 weeks to hyperthyroid cats did not prevent the reduction in tension of contractions of these muscles. 4. It is suggested that lipid peroxidation might not be responsible for the myopathy in hyperthyroidism and propranolol administration does not improve skeletal muscle function in hyperthyroid animals.
Antenatal and postnatal environments are hypothesised to influence the development of hypertension. This study investigates the synergistic effect of cross-fostering and melatonin supplementation on the development of hypertension and renal glutathione system in spontaneously hypertensive rats (SHR). In one experiment, 1-day-old male SHR pups were fostered to either SHR (shr-SHR) or Wistar-Kyoto rats, (shr-WKY). In a concurrent experiment, SHR dams were given melatonin in drinking water (10 mg/kg body weight) from day 1 of pregnancy. Immediately following delivery, 1-day-old male pups were fostered either to SHR (Mel-shr-SHR) or WKY (Mel-shr-WKY) dams receiving melatonin supplementation until weaning on day 21. Upon weaning, melatonin supplementation was continued to these pups until the age of 16 weeks. Systolic blood pressures (SBP) were recorded at the age of 4, 6, 8, 12 and 16 weeks. Renal antioxidant activities were measured. Mean SBP of shr-WKY, Mel-shr-SHR and Mel-shr-WKY was significantly lower than that in shr-SHR until the age of 8 weeks. At 12 and 16 weeks of age, mean SBP of Mel-shr-WKY was lower than those in non-treated shr-SHR and shr-WKY pups but was not significantly different from that in Mel-shr-SHR. Renal glutathione peroxidase (GPx) and glutathione S-transferase (GST) activities were significantly higher in Mel-shr-SHR and Mel-shr-WKY at 16 weeks of age. It appears that combination of cross-fostering and melatonin supplementation exerts no synergistic effect on delaying the rise in blood pressure in SHR. The elevated GPx and GST activities are likely to be due to the effect of melatonin supplementation.
1. The hypotensive effect of cross-fostering in spontaneously hypertensive rats (SHR) is thought to involve adjustments in renal function. However, its association with renal anti-oxidant/oxidant balance during cross-fostering is not known. 2. The present study examined the effect of cross-fostering and in-fostering of 1-day-old offspring between SHR and Wistar-Kyoto (WKY) dams on renal anti-oxidant/oxidant status and systolic blood pressure (SBP). Renal anti-oxidant/oxidant status and SBP were determined in the offspring from 4-16 weeks of age. 3. Cross-fostered SHR had significantly lower SBP than in-fostered SHR at 6, 8 and 12 weeks, but not at 16 weeks (127 ± 1 vs 144 ± 2, 138 ± 1 vs 160 ± 1, 174 ± 2 vs 184 ± 2 and 199 ± 2 vs 194 ± 3 mmHg at 6, 8, 12 and 16 weeks, respectively). No differences in SBP were evident between cross-fostered and in-fostered WKY rats. There were no significant differences in levels of thiobarbituric acid-reactive substances (TBARS), protein carbonyl and total anti-oxidant status (TAS) or superoxide dismutase, catalase, glutathione peroxidase (GPx), glutathione S-transferase and glutathione reductase activity between cross-fostered and in-fostered SHR or WKY offspring. However, compared with WKY rats, catalase activity was higher at 6 and 16 weeks, TAS was higher at 16 weeks and GPx activity and TBARS were lower at 16 weeks in SHR. 4. It appears that cross-fostering of SHR offspring to WKY dams during the early postnatal period causes a transient delay in the rise in blood pressure in SHR and that this does not involve the renal anti-oxidant/oxidant system.
The effect of tocotrienol on the activities of glutathione S-transferases (GSTs), glutathione reductase (GR) and glutathione peroxidase (GPx) in rats given 2-acetylaminofluorene (AAF) was investigated over a 20 week period. Liver and kidney GST and liver GR activities were significantly increased after AAF administration. Kidney GPx activities were significantly affected; activity assayed with cumene hydroperoxide (cu-OOH) was increased but activity assayed with H2O2 was reduced. Supplementation of the diet with tocotrienol in the AAF-treated rats reduced the increase in enzyme activities. Tocotrienol on its own had no effect on the enzyme activities.