High-performance liquid chromatography (HPLC) determination of inosine, a potential biomarker for initial cardiac ischaemia, using isolated mouse hearts

Each year in the USA approximately 7-8 million patients with non-traumatic chest pain come to hospital emergency rooms. It is estimated that approximately 2-5% of these patients are experiencing cardiac ischaemia, but due to the shortcomings of the available testing methods they are incorrectly diagnosed and discharged without appropriate therapy having been provided. Preliminary data with a globally ischaemic mouse heart model has demonstrated that endogenous inosine might be a potential biomarker of initial cardiac ischaemia before cardiac tissue necrosis. A high-performance liquid chromatographic diode array detection (HPLC-DAD) method was utilized for the detection and quantification of inosine in Krebs-Henseleit (Krebs) buffer solution perfusing from surgically removed and isolated mouse hearts undergoing global cardiac ischaemia. A C₁₈ column at a flow rate of 0.6 ml min^-1 with an aqueous mobile phase of trifluoroacetic acid (0.05% trifluoroacetic acid in deionized water, pH 2.2, v/v) and methanol gradient was used for component separation. The assay detection limit for inosine in Krebs buffer solution was 500 ng ml^-1 using a 100-µl neat injection. The HPLC results were used to determine total cardiac effluxed inosine into the Krebs effluent for each mouse during oxidative stress and compared with the per cent cardiac ventricular functional recovery rate to determine if a relationship exists amongst this cardiovascular parameter during periods of cardiac oxidative stress.     

Effects of salicylic acid on post-ischaemic ventricular function and purine efflux in isolated mouse hearts

Abstract

Acetyl salicylic acid (aspirin) is one of the most widely used drugs in the world. Various plasma concentrations of aspirin and its predominant metabolite, salicylic acid, are required for its antiarthritic (1.5–2.5 mM), anti-inflammatory (0.5–5.0 mM) or antiplatelet (0.18–0.36 mM) actions. A recent study demonstrated the inhibitory effects of both aspirin and salicylic acid on oxidative phosphorylation and ATP synthesis in isolated rat cardiac mitochondria in a dose-dependent manner (0–10 mM concentration range). In this context, the present study was conducted to determine the effects of salicylic acid on inosine efflux (a potential biomarker of acute cardiac ischaemia) as well as cardiac contractile function in the isolated mouse heart following 20 min of zero-flow global ischaemia. Inosine efflux was found at significantly higher concentrations in ischaemic hearts perfused with Krebs buffer fortified with 1.0 mM salicylic acid compared with those without salicylic acid (12575±3319 vs. 1437±348 ng ml^?1 min^?1, mean±SEM, n=6 per group, p<0.01). These results indicate that 1.0 mM salicylic acid potentiates 8.8-fold ATP nucleotide purine catabolism into its metabolites (e.g. inosine, hypoxanthine). Salicylic acid (0.1 or 1.0 mM) did not appreciably inhibit purine nucleoside phosphorylase (the enzyme converts inosine to hypoxanthine) suggesting the augmented inosine efflux was due to the salicylic acid effect on upstream elements of cellular respiration. Whereas post-ischaemic cardiac function was further depressed by 1.0 mM salicylic acid, perfusion with 0.1 mM salicylic acid led to a remarkable functional improvement despite moderately increased inosine efflux (2.7-fold). We conclude that inosine is a sensitive biomarker for detecting cardiac ischaemia and salicylic acid-induced effects on cellular respiration. However, the inosine efflux level appears to be a poor predictor of the individual post-ischaemic cardiac functional recovery in this ex vivo model.     

Effects of salicylic acid on post-ischaemic ventricular function and purine efflux in isolated mouse hearts.

Acetyl salicylic acid (aspirin) is one of the most widely used drugs in the world. Various plasma concentrations of aspirin and its predominant metabolite, salicylic acid, are required for its antiarthritic (1.5-2.5 mM), anti-inflammatory (0.5-5.0 mM) or antiplatelet (0.18-0.36 mM) actions. A recent study demonstrated the inhibitory effects of both aspirin and salicylic acid on oxidative phosphorylation and ATP synthesis in isolated rat cardiac mitochondria in a dose-dependent manner (0-10 mM concentration range). In this context, the present study was conducted to determine the effects of salicylic acid on inosine efflux (a potential biomarker of acute cardiac ischaemia) as well as cardiac contractile function in the isolated mouse heart following 20 min of zero-flow global ischaemia. Inosine efflux was found at significantly higher concentrations in ischaemic hearts perfused with Krebs buffer fortified with 1.0 mM salicylic acid compared with those without salicylic acid (12575+/-3319 vs. 1437+/-348 ng ml(-1) min(-1), mean+/-SEM, n=6 per group, p<0.01). These results indicate that 1.0 mM salicylic acid potentiates 8.8-fold ATP nucleotide purine catabolism into its metabolites (e.g. inosine, hypoxanthine). Salicylic acid (0.1 or 1.0 mM) did not appreciably inhibit purine nucleoside phosphorylase (the enzyme converts inosine to hypoxanthine) suggesting the augmented inosine efflux was due to the salicylic acid effect on upstream elements of cellular respiration. Whereas post-ischaemic cardiac function was further depressed by 1.0 mM salicylic acid, perfusion with 0.1 mM salicylic acid led to a remarkable functional improvement despite moderately increased inosine efflux (2.7-fold). We conclude that inosine is a sensitive biomarker for detecting cardiac ischaemia and salicylic acid-induced effects on cellular respiration. However, the inosine efflux level appears to be a poor predictor of the individual post-ischaemic cardiac functional recovery in this ex vivo model.     

Caffeic acid phenethyl ester changes the indices of oxidative stress in serum of rats with renal ischaemia–reperfusion injury

Oxygen‐derived free radicals have been implicated in the pathogenesis of renal injury after ischaemia–reperfusion. Caffeic acid phenethyl ester (CAPE), an active component of propolis extract, exhibits antioxidant properties. To investigate whether treatment with either CAPE or alpha‐tocopherol modifies the levels of the endogenous indices of oxidant stress, we examined their effects on an in vivo model of renal ischaemia–reperfusion injury in rats. CAPE at 10 μmol kg^?1 or alpha‐tocopherol at 10 mg kg^?1 was administered intraperitoneally before reperfusion. Acute administration of both CAPE and alpha‐tocopherol altered the indices of oxidative stress differently in renal ischaemia–reperfusion injury. Copyright © 2001 John Wiley & Sons, Ltd.     

A rapid and simple chemiluminescence method for screening levels of inosine and hypoxanthine in non‐traumatic chest pain patients

A rapid and simple chemiluminescence method was developed for detection of inosine and hypoxanthine in human plasma. The method utilized a microplate luminometer with direct injectors to automatically dispense reagents during sample analysis. Enzymatic conversions of inosine to hypoxanthine, followed by hypoxanthine to xanthine to uric acid, generated superoxide anion radicals as a useful metabolic by‐product. The free radicals react with Pholasin^®, a sensitive photoprotein used for chemiluminescence detection, to produce measurable blue‐green light. The use of Pholasin^® and a chemiluminescence signal enhancer, Adjuvant‐K?, eliminated the need for plasma clean‐up steps prior to analysis. The method used 20 μL of heparinized plasma, with complete analysis of total hypoxanthine levels (inosine is metabolized to hypoxanthine using purine nucleoside phosphorylase) in approximately 3.7 min. The rapid chemiluminescence method demonstrated the capability of differentiating total hypoxanthine levels between healthy individuals, and patients presenting with non‐traumatic chest pain and potential acute cardiac ischemia. The results support the potential use of chemiluminescence methodology as a diagnostic tool to rapidly screen for elevated levels of inosine and hypoxanthine in human plasma, potential biomarkers of acute cardiac ischemia.Copyright ©2009 John Wiley & Sons, Ltd.     

Inosine ameliorates the effects of hemin-induced oxidative stress in broilers

The objective of these studies was to determine whether inosine, a precursor of the antioxidant uric acid, can ameliorate hemin-induced oxidative stress. Dietary inclusion of inosine was begun either before or after hemin-induced oxidative stress. Broilers (4 weeks) were divided into four treatment groups (Control, Hemin, Inosine, Hemin/Inosine). Throughout the study control birds (n = 10) were injected daily with a buffer solution, while hemin birds (n = 10) were injected daily (i.p.) with a 20 mg/kg body weight hemin buffer solution. Leukocyte oxidative activity (LOA) and concentrations of plasma uric acid (PUA) were measured. Results from the first study showed that hemin birds had increased levels of LOA (P = 0.0333) and lower PUA (P = 0.1174). On day 10, control and hemin birds were subdivided into inosine birds (n = 5) and hemin/inosine birds (n = 5). These birds were given 0.6 M/kg of feed/day of dry inosine. Plasma concentrations of uric acid and LOA were then measured on day 15. Results showed that inosine raised concentrations of PUA (P = 0.0001) and lowered LOA (P = 0.0044) as induced by hemin. In the second study pretreatment of broilers with hemin prevented the increase in LOA induced by hemin (P = 0.0001). These results show that modulating the concentrations of uric acid can markedly affect oxidative stress.     

Plasma low-molecular-weight thiol/disulphide homeostasis as an early indicator of global and focal cerebral ischaemia

Objective: Recent studies have shown that cerebral ischaemia causes not only local, but also systemic oxidative stress. This leads to oxidation of thiol-containing compounds, including low-molecular-weight thiols (cysteine, glutathione, homocysteine and others). Therefore, the aim of this work was to verify the hypothesis that the thiol/disulphide homeostasis of low-molecular-weight thiols is disturbed in the early stages of cerebral ischaemia.

Methods: Two experimental rat models of ischaemia were used: a global model of vascular ischaemia (clamping the common carotid arteries?+?haemorrhage) and focal ischaemia (middle cerebral artery occlusion). The total levels of thiols and their reduced forms were measured before surgery and after 40 minutes of reperfusion (global) or 3?hours (focal) ischaemia.

Results: The global ischaemia model caused a marked (2.5–4 times, P?P?Discussion: These results suggest that plasma low-molecular-weight thiols are actively involved in oxidation reactions at early stages of cerebral ischaemia; therefore, their reduced forms or redox state may serve as a sensitive indicator of acute cerebrovascular insufficiency.     

Cordycepin ameliorates cardiac hypertrophy via activating the AMPKα pathway

Increase of myocardial oxidative stress is closely related to the occurrence and development of cardiac hypertrophy. Cordycepin, also known as 3'‐deoxyadenosine, is a natural bioactive substance extracted from Cordyceps militaris (which is widely cultivated for commercial use in functional foods and medicine). Since cordycepin suppresses oxidative stress both in vitro and in vivo, we hypothesized that cordycepin would inhibit cardiac hypertrophy by blocking oxidative stress‐dependent related signalling. In our study, a mouse model of cardiac hypertrophy was induced by aortic banding (AB) surgery. Mice were intraperitoneally injected with cordycepin (20 mg/kg/d) or the same volume of vehicle 3 days after‐surgery for 4 weeks. Our data demonstrated that cordycepin prevented cardiac hypertrophy induced by AB, as assessed by haemodynamic parameters analysis and echocardiographic, histological and molecular analyses. Oxidative stress was estimated by detecting superoxide generation, superoxide dismutase (SOD) activity and malondialdehyde levels, and by detecting the protein levels of gp91^phox and SOD. Mechanistically, we found that cordycepin activated activated protein kinase α (AMPKα) signalling and attenuated oxidative stress both in vivo in cordycepin‐treated mice and in vitro in cordycepin treated cardiomyocytes. Taken together, the results suggest that cordycepin protects against post‐AB cardiac hypertrophy through activation of the AMPKα pathway, which subsequently attenuates oxidative stress.     

An HPLC method for determination of inosine and hypoxanthine in human plasma from healthy volunteers and patients presenting with potential acute cardiac ischemia

A simple and sensitive high-performance liquid chromatography (HPLC) method utilizing ultraviolet (UV) detection was developed for the determination of inosine and hypoxanthine in human plasma. For component separation, a monolithic C(18) column at a flow rate of 1.0 mL/min with an aqueous mobile phase of trifluoroacetic acid (0.1% TFA in deionized water pH 2.2, v/v) and methanol gradient was used. The method employed a one-step sample preparation utilizing centrifugal filtration with high component recoveries (approximately 98%) from plasma, which eliminated the need of an internal standard. The method demonstrated excellent linearity (0.25-5 microg/mL, R>0.9990) for both inosine and hypoxanthine with detection limits of 100 ng/mL. This simple and cost effective method was utilized to evaluate potential endogenous plasma biomarker(s), which may aid hospital emergency personnel in the early detection of acute cardiac ischemia in patients presenting with non-traumatic chest pain.     

Oxidative stress is associated with decreased heart rate variability in patients with chronic kidney disease

Objectives: Elevated oxidative stress and reduced heart rate variability (HRV) is prevalent in patients with chronic kidney disease (CKD) and is associated with increased morbidity and mortality. Previous studies have identified a positive association between elevated oxidative stress and autonomic dysfunction, however this relationship has not yet been investigated in the CKD population.

Methods: Plasma was collected from 78 patients with stage 3–4 CKD (estimated glomerular filtration rate 25–60?ml/min/1.73?m²) for the assessment of oxidative stress, including plasma total F2-isoprostanes, glutathione peroxidase activity and total antioxidant capacity. Time and frequency HRV parameters were measured from a three lead electrocardiogram.

Results: Participants with elevated F2-isoprostanes had reduced HRV compared to patients with normal levels of F2-isoprostanes. A number of HRV parameters were found to be inversely correlated with F2-isoprostanes in all CKD patients, including SDNN (r?=??0.337; P?r?=??0.281, P?=?0.01), LF (r?=??0.315, P?r?=??0.288, P?=?0.01). Multiple linear regression found F2-isoprostanes to be an independent predictor of SDNN (r²?=?0.287, β?=??0.272, P?=?0.01).

Discussion: Oxidative stress is significantly and independently associated with HRV in patients with CKD. Identifying oxidative stress in the pathogenesis of autonomic dysfunction may help target therapeutic strategies.     

Regulation of and intervention into the oxidative pentose phosphate pathway and adenine nucleotide metabolism in the heart

The capacity of the oxidative pentose phosphate pathway (PPP) in the heart is limited, since the activity of glucose-6-phosphate dehydrogenase (G-6-PD), the first and regulating enzyme of this pathway, is very low. Two mechanisms are involved in the regulation of this pathway. Under normal conditions, G-6-PD is inhibited by NADPH. This can be overcome in the isolated perfused rat heart by increasing the oxidized glutathione and by elevating the NADP⁺/NADPH ratio. Besides this rapid control mechanism, there is a long-term regulation which involves the synthesis of G-6-PD. The activity of G-6-PD was elevated in the rat heart during the development of cardiac hypertrophy due to constriction of the abdominal aorta and in the non-ischemic part of the rat heart subsequent to myocardial infarction. The catecholamines isoproterenol and norepinephrine stimulated the activity of myocardial G-6-PD in a time- and dose-dependent manner. The isoproterenol-induced stimulation was cAMP-dependent and due to increased new synthesis of enzyme protein. The G-6-PD mRNA was elevated by norepinephrine. As a consequence of the stimulation of the oxidative PPP, the available pool of 5-phosphoribosyl-l-pyrophosphate (PRPP) was expanded. PRPP is an important precursor substrate for purine and pyrimidine nucleotide synthesis. The limiting step in the oxidative PPP, the G-6-PD reaction, can be bypassed with ribose. This leads to an elevation of the cardiac PRPP pool. The decline in ATP that is induced in many pathophysiological conditions was attenuated or even entirely prevented by i.v. infusion of ribose. In two in vivo rat models, the overloaded and catecholamine-stimulated heart and the infarcted heart, the normalization of the cardiac adenine nucleotide pool by ribose was accompanied by an improvement of global heart function. Combination of ribose with adenine or inosine in isoproterenol-treated rats was more effective to restore completely the cardiac ATP level within a short period of time than either intervention alone. (Mol Cell Biochem 160/161: 101–109, 1996)     

Hesperidin regresses cardiac hypertrophy by virtue of PPAR‐γ agonistic,anti‐inflammatory,antiapoptotic, and antioxidant properties

Hesperidin (HES), a flavanone glycoside, predominant in citrus fruits, has an agonistic activity on peroxisome proliferator‐activated receptor gamma (PPAR‐γ). PPAR‐γ is an inhibitor of cardiac hypertrophy (CH) signaling pathways. In this study, we investigated the cardioprotective effect of HES in isoproterenol (ISO)‐induced CH through PPAR‐γ agonistic activity. For this, male albino Wistar rats were divided into six groups (n = 6), that is, normal, ISO‐control, HES treatment group (200 mg kg^?1; p.o.), HES per se (200 mg kg^?1; p.o.), enalapril treatment group (30 mg kg^?1; p.o.), and combination group (HES 200 mg kg^?1; p.o.+enalapril 30 mg kg^?1; p.o.). ISO (3 mg kg^?1; s.c.) was administered to all groups except normal and per se to induce CH. HES or enalapril treatment of 28 days significantly attenuated pathological changes, improved cardiac hemodynamics, suppressed oxidative stress, and apoptosis along with an increased PPAR‐γ expression. The combination of enalapril with HES exhibited an effect similar to that of HES or enalapril alone on all the aforementioned parameters. Therefore, HES may be further evaluated as a promising molecule for the alleviation of CH.     

CD38 promotes angiotensin II‐induced cardiac hypertrophy

Cardiac hypertrophy is an early hallmark during the clinical course of heart failure and regulated by various signalling pathways. Recently, we observed that mouse embryonic fibroblasts from CD38 knockout mice were significantly resistant to oxidative stress such as H₂O₂‐induced injury and hypoxia/reoxygenation‐induced injury. In addition, we also found that CD38 knockout mice protected heart from ischaemia reperfusion injury through activating SIRT1/FOXOs‐mediated antioxidative stress pathway. However, the role of CD38 in cardiac hypertrophy is not explored. Here, we investigated the roles and mechanisms of CD38 in angiotensin II (Ang‐II)‐induced cardiac hypertrophy. Following 14 days of Ang‐II infusion with osmotic mini‐pumps, a comparable hypertension was generated in both of CD38 knockout and wild‐type mice. However, the cardiac hypertrophy and fibrosis were much more severe in wild‐type mice compared with CD38 knockout mice. Consistently, RNAi‐induced knockdown of CD38 decreased the gene expressions of atrial natriuretic factor (ANF) and brain natriuretic peptide (BNP) and reactive oxygen species generation in Ang‐II‐stimulated H9c2 cells. In addition, the expression of SIRT3 was elevated in CD38 knockdown H9c2 cells, in which SIRT3 may further activate the FOXO3 antioxidant pathway. The intracellular Ca²⁺ release induced by Ang‐II markedly decreased in CD38 knockdown H9c2 cells, which might be associated with the decrease of nuclear translocation of NFATc4 and inhibition of ERK/AKT phosphorylation. We concluded that CD38 plays an essential role in cardiac hypertrophy probably via inhibition of SIRT3 expression and activation of Ca²⁺‐NFAT signalling pathway. Thus, CD38 may be a novel target for treating cardiac hypertrophy.     

Flux balance analysis of Chlorella sp. FC2 IITG under photoautotrophic and heterotrophic growth conditions

Quantification of carbon flux distribution in the metabolic network of microalgae remains important to understand the complex interplay between energy metabolism, carbon fixation, and assimilation pathways. This is even more relevant with respect to cyclic metabolism of microalgae under light–dark cycle. In the present study, flux balance analysis (FBA) was carried out for an indigenous isolate Chlorella sp. FC2 IITG under photoautotrophic and heterotrophic growth conditions. A shift in intracellular flux distribution was predicted during transition from nutrient sufficient phase to nutrient starvation phase of growth. Further, dynamic flux analysis (dFBA) was carried out to capture light–dark metabolism over discretized pseudo steady state time intervals. Our key findings include the following: (i) unlike heterotrophic condition, oxidative pentose phosphate (PP) pathway, and Krebs cycle were relatively inactive under photoautotrophic growth; (ii) in both growth conditions, while transhydrogenation reaction was highly active, glyoxalate shunt was found to be nonoperative; (iii) flux distribution during transition period was marked with up regulation of carbon flux toward nongrowth associated (NGA) maintenance energy, oxidative phosphorylation, and photophosphorylation; (iv) redirection of carbon flux from polysaccharide and neutral lipid resulted in up regulation of Krebs cycle flux in the dark phase; (v) elevated glycolytic and acetyl-CoA flux were coupled with induction of neutral lipid during light cycle of the growth; (vi) significantly active photophosphorylation in the light phase was able to satisfy cellular energy requirement without need of oxidative PP pathway; and (vi) unlike static FBA, dFBA predicted an unaltered NGA maintenance energy of 1.5 mmol g^?1 DCW h^?1.     

Antioxidant metallothionein alleviates endoplasmic reticulum stress-induced myocardial apoptosis and contractile dysfunction

Abstract

Aims. Endoplasmic reticulum (ER) stress exerts myocardial oxidative stress, apoptosis, and contractile anomalies, although the precise interplay between ER stress and apoptosis remains elusive. This study was designed to examine the impact of the cysteine-rich free radical scavenger metallothionein on ER stress-induced myocardial contractile defect and underlying mechanisms. Methods and results. Wild-type friendly virus B and transgenic mice with cardiac-specific overexpression of metallothionein were challenged with the ER stress inducer tunicamycin (1 mg/kg, intraperitoneal, 48 h) prior to the assessment of myocardial function, oxidative stress, and apoptosis. Our results revealed that tunicamycin promoted cardiac remodeling (enlarged left ventricular end systolic/diastolic diameters with little changes in left ventricular wall thickness), suppressed fractional shortening and cardiomyocyte contractile function, elevated resting Ca²⁺, decreased stimulated Ca²⁺ release, prolonged intracellular Ca²⁺ clearance, and downregulated sarco(endo)plasmic reticulum Ca²⁺-ATPase levels, the effects of which were negated by metallothionein. Treatment with tunicamycin caused cardiomyocyte mitochondrial injury, as evidenced by decreased mitochondrial membrane potential (??m, assessed by JC-1 staining), the effect of which was negated by the antioxidant. Moreover, tunicamycin challenge dramatically facilitated myocardial apoptosis as manifested by increased Bax, caspase 9, and caspase 12 protein levels, as well as elevated caspase 3 activity. Interestingly, metallothionein transgene significantly alleviated tunicamycin-induced myocardial apoptosis. Conclusion. Taken together, our data favor a beneficial effect of metallothionein against ER stress-induced cardiac dysfunction possibly associated with attenuation of myocardial apoptosis.     

Demethylation of theophylline (1,3-dimethylxanthine) to 1-methylxanthine: the first step of an antioxidising cascade

Abstract

The reaction of theophylline with HO^? radical, produced by photolytic methods at pH 7, was studied in aqueous solution and the products characterised by HPLC and GC-MS. In addition to the expected 1,3-dimethyluric acid, the formation of 1-methylxanthine and, to a lesser extent, of 3-methylxanthine was observed. Theoretical calculations confirmed the preferred formation of the former compound. Both demethylated products were also observed upon reaction of theophylline with O^?– radical anion at pH～13, and 1-methylxanthine was consumed faster than 3-methylxanthine after its formation. Molecular oxygen had no significant effect on the formation of the mono-methylxanthine derivatives. A reaction mechanism for the demethylation of theophylline by oxidising radicals is proposed. This demethylation reaction can play an important role in the protection of biological targets against oxidative stress as the first step of an antioxidising cascade.     

Prolongation of oxidative stress by long-lived reactive protein species induced by X-ray radiation and their genotoxic action

Abstract

The formation of long-lived reactive protein species of bovine serum albumin (BSA), ovalbumin, casein and casein hydrolyzate with a half-life of 3–5 hours was shown using chemiluminescence induced by X-ray radiation. It was found that long-lived reactive protein species are capable of generating reactive oxygen species (ROS) (H₂O₂, OH^?, HO₂^{?, 1}O₂) in the aquatic environment over a long period of time in vitro. The interaction of X-ray-irradiated BSA with DNA in vitro led to the formation of 8-oxoguanine (8-oxo-7,8-dihydroguanine), a biomarker of oxidative damage to DNA. Some natural antioxidants are effective scavengers of ROS (inosine, tryptophan, methionine and ascorbate). They protect DNA from the action of long-lived reactive protein species leading to ROS generation and the formation of 8-oxoguanine. The intravenous injection of X-ray radiation-induced, long-lived reactive protein species to rats, as well as the peroral and intraperitoneal administration of these products to mice, gave rise to cytogenetic injuries in the cells of their red bone marrow through the formation of micronuclei in polychromatophilic erythrocytes. The administration of the same natural antioxidants used for in vitro experiments soon after irradiation made it possible to effectively eliminate the genotoxic action of oxidative stress caused by radiation-induced, long-lived reactive protein species. Our data represent clear evidence that the oxidative damage to proteins induced by X-rays is directly involved in the induction of a response to DNA damage in rodents.     

Oxidative stress and antioxidant deficiencies in asthma: potential modification by diet

Abstract

The lungs of asthmatic patients are exposed to oxidative stress due to the generation of reactive oxygen and nitrogen species as a consequence of chronic airway inflammation. Increased concentrations of NO^?, H₂O₂ and 8-isoprostane have been measured in exhaled breath and induced sputum of asthmatic patients. O₂^??, NO^?, and halides interact to form highly reactive species such as peroxynitrite and HOBr, which in turn cause nitration and bromination of protein tyrosine residues. Oxidative stress may also reduce glutathione levels and cause inactivation of antioxidant enzymes such as superoxide dismutase, with a consequent increase in apoptosis, shedding of airway epithelial cells and airway remodelling. The oxidant/antioxidant equilibrium in asthmatic patients may be further perturbed by low dietary intakes of the antioxidant vitamins C and E, selenium and flavonoids, with a consequent lowering of the concentrations of these and other non-dietary antioxidants such as bilirubin and albumin in plasma and airway epithelial lining fluid. Although supplementation with vitamins C and E appears to offer protection against the adverse effects of ozone, recent randomised, placebo-controlled trials of vitamin C or E supplements for patients with mild asthma have not shown significant benefits over standard therapy. However, genetic variation in glutathione S-transferase may influence the susceptibility of asthmatic individuals to oxidative stress and the extent to which they are likely to benefit from antioxidant supplementation. Long-term prospective trials are required to determine whether modification of dietary intake will benefit asthma patients and reduce the socio-economic burden of asthma in the community.     

A novel chemiluminescent method for determination of phloroglucinol

It was found that the inhibition and enhancement by phloroglucinol of the chemiluminescence from the luminol–K₃Fe(CN)₆ system were dependent on the pH of luminol solution and the concentration of phloroglucinol. In Na₂CO₃–NaHCO₃ buffer, phloroglucinol exhibited strong chemiluminescent enhancement at pH 9.4. On this basis, a flow injection method was developed for the determination of phloroglucinol. The method was simple, rapid, convenient and sensitive, with a detection limit of 2.0 × 10^?9 mol/L. It is effective for determining phloroglucinol in the range of 1.0 × 10^?5–5.0 × 10^?9 mol/L. The relative standard deviation is 1.3% within one day and 3.2% between days for the determination of 5.0 × 10^?7 mol/L phloroglucinol. The method has been successfully used to determine phloroglucinol in environmental water, with satisfactory results. Copyright © 2003 John Wiley & Sons, Ltd.     

Some kinetic studies on ribonucleosides degradation by extracts of penicillium citrinum

Cell-free extracts of 3–4 days old mats of nitrate-grown Penicillium citrinum catalyze the hydrolytic cleavage of the N-glycosidic bonds of inosine, guanosine and adenosine optimally at pH 4, 0.1 M citrate buffer. The same extracts catalyze the hydrolytic deamination of cytidine at a maximum rate in 0.08 M Tris-acetate buffer pH 6.5, 40°C and 50°C were the most suitable degrees for purine nucleoside hydrolysis and cytidine deamination, respectively. The incubation of the extracts at 60°C, in the absence of cytidine caused a loss in the deaminating activity, while freezing and thawing had no effect on both activities. The deaminating activity seems to be cytidine specific as neither cytosine, adenine, adenosine nor guanosine could be deaminated. Uridine competively inhibited this activity, while ammonia had no effect. The apparent K_m value of this enzyme for cytidine was 1.57×10^?3M and its K_i value for uridine was 7.8×10^?3M. The apparent K_m values of the N-glycosidic bond cleaving enzyme for inosine, guanosine and adenosine were 13.3, 14.2 and 20×10^?3 M, respectively.