Role of active nitrogen molecules in progression of septic shock.

INTRODUCTION: Active nitrogen molecules are formed as a result of cell metabolism. They are essential for cell metabolism, but when produced in excess, they contribute to the pathogenesis of several disease processes. These nitrogen molecules play an important role in vascular instability of septic shock. This study was planned to detect the role of active nitrogen molecules in the progression of septic shock. MATERIALS AND METHODS: Blood samples were collected from 118 critically ill patients admitted in ICU and from 95 healthy relatives accompanying the patients. Patients were categorized into three groups: systemic inflammatory response syndrome (n = 54), sepsis (n = 35) and septic shock (n = 29). Plasma total nitrite (nitrites and nitrates), cytokines like tumour necrosis factor-α (TNF-α) and plasma lactate were measured to assess inflammatory activity and severity of septic shock. RESULTS: High plasma levels of nitrite and nitrate (No2-/No3-) were observed in critically ill patients (mean level 78.92 µmol/l in sepsis and 97.20 µmol/l in septic shock). Mean plasma TNF-α level in sepsis was 213.50 pg/ml and septic shock was 227.38 pg/ml. CONCLUSION: Plasma No2-/No3- and TNF-α levels were high in patients with sepsis and septic shock, which increased with severity of sepsis.

Allium sativum constituents: effect on free radical generation from rat neutrophils.

Reactive oxygen species formation or respiratory burst by the neutrophils helps to remove the invaded pathogens and thus constitute a major defense against pathogenic microorganisms. Production of these radicals by activated neutrophils at the site of inflammation however inflicts damage to the host tissue. Modulation of the neutrophil respiratory burst is therefore important in determining the balance between immune defense and host tissue injury during inflammatory conditions. Garlic extracts and various compounds isolated from garlic have been found to possess various activities, however no report is available on their effect on neutrophil free radical generation. The present study was therefore undertaken to evaluate the effect of garlic aqueous extract, alcoholic extract and various fractions on the free radical generation from neutrophils. Among the tested fractions, chloroform fraction of garlic seems to be very potent in attenuating the free radical generation from rat neutrophils, which could be beneficial in the inflammation associated pathological conditions.

Biochemical and molecular evaluation of neutrophil NOS in spontaneously hypertensive rats.

Resting neutrophils generate NO, while activation leads to the production of reactive oxygen and nitrogen species. Nowadays cardiovascular pathological conditions such as hypertension, cardiac ischemia, reperfusion and heart failure are associated with inflammation. This project explores the respiratory burst potential and NO generation status in the neutrophils, plasma, aorta, and kidneys from normotensive Wistar and spontaneously hypertensive rats (SHR). Total and protein associated nitrite content was quantitated using Griess reagent following cadmium reduction and mercuric chloride treatment respectively. NO and superoxide generation evaluated by Flowcytometry and peroxynitrite by spectrofluorimetric method. Expression of NOS isoforms was analyzed by RT-PCR. NO generation from SHR neutrophils was significantly augmented in comparison to normotensive counterparts. Neutrophils activated in response to arachidonic acid, PMA, fMLP or E. coli generated more superoxide radicals among SHR, and consequentially peroxynitrite. Expression of iNOS was significantly more in the SHR neutrophils, while that of nNOS remained unaffected. Results suggest that NO generated in SHR is utilized in scavenging superoxide radicals thereby limiting its bioavailability. Thus induction of NOS in neutrophils combined with augmented oxidative stress might influence its association with endothelium and contribute to inflammatory responses under hypertensive condition.

Nitric oxide synthase localization in the rat neutrophils: immunocytochemical, molecular, and biochemical studies.

Nitric oxide (NO) modulates diverse functions of polymorphonuclear neutrophils (PMNs), but localization of NO synthase (NOS) and identification of its interacting proteins remain the least defined. The present study discerns subcellular distribution of NOS and caveolin-1, a prominent NOS-interacting protein in rat PMNs. Localization of NOS was explored by confocal and immunogold electron microscopy, and its activity was assessed by L-[3H] arginine and 4,5-diaminofluorescein diacetate (DAF-2DA). Reverse transcriptase-polymerase chain reaction using NOS primers and Western blotting demonstrated the presence of neuronal NOS (nNOS) and inducible NOS (iNOS) in PMNs. Immunocytochemical studies exhibited distribution of nNOS and iNOS in cytoplasm and nucleus, and L-[3H] citrulline formation and DAF fluorescence confirmed NOS activity in both fractions. NOS activity correlated positively with calmodulin concentration in both of the fractions. nNOS and iNOS colocalized with caveolin-1, as evidenced by immunocytochemical and immunoprecipitation studies. The results thus provide first evidence of nNOS and iNOS in the nuclear compartment and suggest NOS interaction with caveolin-1 in rat PMNs.

A preliminary investigation into maximal expiratory pressures in some village children.

A clinical mercury sphygmomanometer was used to measure Maximal Expiratory Pressure (MEP) in 29 boys (mean age 8 +/- 1.4 yr) and 21 girls (mean age 7.6 +/- 1.5 yr) of a village in interior Maharashtra. The values of 70.6 +/- 13.4 mmHg SD for the boys and 61.9 +/- 18.9 mmHg for the girls were quite comparable to the respiratory pressures reported elsewhere in literature, even though the subjects were apparently poorly nourished. There was no statistical difference between the MEPs of boys and girls. The MEP was positively and significantly (P<0.01) correlated to height (r=0.51) and weight (r=0.05) in the boys. The MEP denoting respiratory muscle strength also correlated positively with handgrip power used to represent non-respiratory muscle strength (r=0.34) (P>0.05). The simple, reproducible method of measuring MEP as described may be useful for measuring this important physiological parameter at the bedside in children whose respiratory muscle function needs to be evaluated.

Contribution of genomics and proteomics in understanding the role of modifying factors in Parkinson's disease.

Parkinson's disease (PD) is a complex neurological disorder, characterized by selective degeneration of nigrostriatal dopaminergic neurons. It is a multi-factorial disease, contributed by a combination of age, genetic and environmental factors. Etiology of sporadic PD and mechanism underlying selective loss of dopaminergic neurons has not yet been clearly understood. Recent developments in genomics and proteomics have revolutionized the research on PD at genetic level. Differential gene expression patterns (DNA biochip technology), age-dependent complex genetic patterns (SNP genotyping), and protein expression profiles (proteomics) of PD patients have started providing the specific and rigorous molecular explanation and role of modifying factors in PD. Genomics and proteomics are further expected to help in developing biomarkers for diagnosis of early onset PD and also to develop valuable and potential therapeutic strategies for its treatment. In this review, we have discussed the progress made by genomics and proteomics, in understanding the role of modifying factors in PD.

Lung functions with spirometry: an Indian perspective--II: on the vital capacity of Indians.

Spirometry has been used in India since 1929 to evaluate vital capacity. The mean value for this parameter has changed slightly for the better over about eight decades. It is currently recorded at about 21.8 ml/cm height for males and about 18 ml/cm height for females, the difference between the two sexes being statistically significant throughout the period studied. The vital capacity reaches its peak at about 30 years of age in both Indian men and women and declines there after. There is no significant statistical difference in the vital capacities of subjects from different regions of India. Composite regressions have been generated for use as reference equations for estimating. Vital capacity of Indians is lower than that of Caucasians, but the age related decline is much greater for Caucasians.

Lung functions with spirometry: an Indian perspective--I. Peak expiratory flow rates.

Peak expiratory flow rate is an effective measure of effort dependent airflow. It is relatively a simple procedure, and may be carried out in the field using portable instruments. The average PEFR of healthy young Indian males and females is around 500 and 350 lpm respectively. The PEFR reaches a peak at about 18-20 years, maintains this level up to about 30 years in males, and about 40 years in females, and then declines with age. Common regression equations for Indians enveloping major studies from various parts of the country have been formulated. Indian PEFR values compare favourably with other ethnic groups such as Americans and Europeans.

Alterations in the bioantioxidants following thrombosis.

The role of free radicals in rat thrombosis has been demonstrated by studying its scavenging enzyme system. Changes in the bio-antioxidants (GSH-redox cycle, total thiol groups, vitamins E and C) that also offer protection against the free radicals, have, however, not been studied so far. This investigation was undertaken to understand the involvement of these antioxidants during thrombosis. The tissues investigated following thrombosis were blood, platelets, polymorphonuclear leukocytes (PMNLs), heart, and lung. Glutathione (GSH) content in the platelets was observed to be depleted. However, oxidized glutathione (GSSG) contents in the platelets, PMNLs, and blood remain unaltered. In addition, in the whole blood GSH levels were increased significantly, whereas there was no change in the GSSG level. Activity of glutathione reductase (GR) was decreased significantly in platelets and lungs with an increase in the total thiol groups in the lung homogenate. Activity of Glutathione peroxidase (Gpx) remained unaltered in all the tissues studied. In addition, 24% and 15% decrease in the alpha-tocopherol concentration was observed in thrombocytes and PMNLs, respectively, with no change in the ascorbate levels in these cells. Results of this investigation suggest alterations in the GSH-redox cycle in blood, platelets, and lung after thrombosis in the rat.

Alterations in free radical scavenging mechanisms following blood-brain barrier disruption.

It has been reported earlier that rat microvessels which constitute the blood-brain barrier (BBB) are rich in free radical scavenging enzymes. In the present investigation, BBB of rat was disrupted by intravenous infusion of the hypertonic saline and changes in enzymes--namely, superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and glutathione reductase (GR)--were evaluated in the brain microvessels at 30 min after the intravenous administration of hypertonic saline, being the time of peak effect. There was a significant increase in the activities of CAT (40%), GPx (26%), and SOD (16%) over the control values. In addition, within 90 min BBB was found to be reestablished and the levels of enzymes reverted to normal. Malondialdehyde (MDA) levels and activity of lactate dehydrogenase (LDH) remained unaltered during and following disruption, suggesting that there was no change in the membrane lipid environment. Similarly, there was no cell lysis. The results suggest that the disruption of BBB following hypertonic saline administration might be due to an increase in the generation of free radicals in the brain microvessels.

Interaction of nitric oxide synthase inhibitors and their D-enantiomers with rat neutrophil luminol dependent chemiluminescence response.

1. Formyl-methionyl-leucyl-phenylalanine (FMLP) or arachidonic acid (AA) induced luminol dependent chemiluminescence (LCL) response of rat polymorphonuclear leukocytes (PMNLs) was found to be inhibited by nitric oxide synthease inhibitors and their D-enantiomers. 2. Rat PMNLs LCL response was inhibited by NG-nitro-L-arginine methyl ester (L-NAME), D-NAME, NG-monomethyl-L-arginine (L-NMMA) or D-NMMA, in a concentration- and time-dependent manner. 3. It was observed that both L- and D-enantiomers of the arginine analogues (1000 microM) did not inhibit AA induced lucigenin-dependent chemiluminescence (LUCDCL) response and cytochrome c reduction, used for estimating the NADPH-oxidase activity in the cells and in the cell free system, respectively. 4. None of the L- and D-enantiomers had any effect on either rat basal PMNLs or AA-induced oxygen consumption. 5. In addition, neither the L nor D-enantiomers of NAME altered either AA-induced release or the activity of myeloperoxidase from rat PMNLs azurophilic granules. 6. The results obtained indicate that the attenuation of the LCL response by L- and D-enantiomers of arginine analogues, is a non-specific effect as there was no inhibition of NADPH-oxidase and MPO activity, MPO release or oxygen consumption. Therefore, the data obtained indicate that these agents should be used with caution to analyse the role of nitric oxide in rat PMNLs LCL response.

Nitrite content and antioxidant enzyme levels in the blood of schizophrenia patients.

RATIONALE: Recent studies have suggested augmentation in the inflammatory response as well as involvement of nitric oxide (NO) in mood disorders. Polymorphonuclear leukocytes (PMN), NO and free radicals have been associated with inflammatory response; however, the status of NO in the PMN has not been investigated so far in schizophrenia patients. OBJECTIVES: The present study was undertaken to investigate levels of nitrite (a metabolite of NO), malonaldehyde (MDA, lipid peroxidation product) and antioxidant enzymes such as superoxide dismutase (SOD), catalase and glutathione peroxidase (Gpx) in the PMN of schizophrenia patients. METHODS: Patients with schizophrenia (n=62) were diagnosed according to DSM-IV and were free of anti-psychotic medications/ECT for at least 3 months. Mean age of the patients was 29.06+/-1.17 years, with a male to female ratio of 4:1, and mean duration of illness was 3.7+/-0.6 years. The control group consisted of 82 healthy subjects with a mean age of 37.0+/-1.26 and a male to female ratio of 5:1. PMN were isolated from the blood. Nitrite, MDA and antioxidant enzymes were estimated by standard biochemical techniques in the PMN of normal healthy controls and schizophrenia patients. Platelet and plasma nitrite levels were also estimated in controls and schizophrenia patients. RESULTS: Nitrite content in the PMN was reduced to 68%, while plasma and platelet nitrite content in schizophrenia patients was not significantly changed in comparison to controls. Malonaldehyde (MDA) content in PMN was significantly augmented in schizophrenia patients but activity of SOD, catalase and Gpx remain unaltered. CONCLUSION: Results obtained indicate a significant decrease in NO synthesis and an increase in MDA in the PMN of schizophrenia patients, while antioxidant enzyme activities were not altered in the PMN of schizophrenia patients. This suggests that the decrease in PMN NO synthesis by PMN might lead to oxidative stress in schizophrenia patients.

Nitric oxide modulates blood-brain barrier permeability during infections with an inactivated bacterium.

The objective of the present investigation was to study the involvement of NO in regulating the permeability of the blood-brain barrier (BBB) during infections, since NOS is known to be induced following infections. The administration of inactivated Escherichia coli (a source of lipopolysaccharide) or poly (I:C), an interferon inducer, to rats increased the permeability of BBB significantly. This increase was found to be potentiated in the presence of L-arginine, a substrate for NOS, while D-arginine had no such effect. N-nitro L-arginine methyl ester, an inhibitor of NOS, and dexamethasone, an inhibitor of NOS induction, blocked the E. coli-induced effects. These results suggest that during infections, NOS inductions causes the release of large quantities of NO, resulting in increased BBB permeability.

L-citrulline mediated relaxation in the control and lipopolysaccharide-treated rat aortic rings.

The present study was undertaken to investigate relaxant effect of L-citrulline in phenylephrine precontracted endothelium intact thoracic aortic rings obtained from control or lipopolysaccharide (1 mg/kg)-treated rats. L-citrulline produced 40+/-3% (n=36) and 60+/-5% (n=24) relaxations in control and lipopolysaccharide-treated rings, respectively. Nitric oxide (NO) release and cyclic guanosine-3',5'-monophosphate levels from the rings were also increased following treatment with L-citrulline. Inhibition of guanylate cyclase, L-citrulline recycling to L-arginine or denudation of the endothelium, significantly reduced L-citrulline-induced relaxations both in control and lipopolysaccharide-treated rings. Treatment of rings with protein synthesis inhibitors prevented relaxations to L-citrulline. Inhibitor of Ca2+-activated K+ channels, tetrabutylammonium or precontraction of the rings with KCl (80 mM), significantly attenuated L-citrulline mediated relaxations in control and lipopolysaccharide-treated rings. Thus, L-citrulline seems to exert significant relaxation by supplementing the release of NO due to its recycling to L-arginine, which gets further augmented after lipopolysaccharide treatment.

Modulation of polymorphonuclear leukocytes function by nitric oxide.

Recognition of the endothelium-derived relaxation factor as nitric oxide (NO) gave rise to an impression that NO was synthesised only by the endothelial lining of the vessel wall. Later it was found that NO is synthesized constitutively by the enzyme nitric oxide synthase (NOS) in various cells. However, inflammatory cytokines can induce NOS (known as inducible NOS [iNOS]) activity in all the somatic cells. Blood cells, such as eosinophils, platelets, neutrophils, monocytes, and macrophages, also synthesize NO. Among them, polymorphonuclear leukocytes (PMNs) constitute an important proportion and are also the major participants in a number of pathological conditions with suggestive involvement of NO. PMNs can synthesize NO at rates similar to endothelial cells, thus suggesting the importance of PMN-derived NO in various physiological and pathological conditions. Most of the studies so far focus on the peripheral PMNs, while studies on PMNs after emigration are limited, thus warranting systematic studies on PMNs from both sources. The role of the endothelial NOS (eNOS) and functions of NO derived from the endothelial cells has been studied extensively. However, understanding of the PMNs NOS and its regulatory role in their function is unraveling. The present review summarizes the modulatory role of NO on PMNs functions and points out the discrepancies relating to presence of NOS in PMNs. This information will be helpful in understanding the importance of NO in physiological and pathological conditions associated with PMNs.

Alterations in the free radical generation and nitric oxide release from rat peripheral polymorphonuclear leukocytes following thrombosis.

The present investigation was undertaken to study the alterations in free radical generation and release of nitric oxide (NO) from polymorphonuclear leukocytes (PMNLs) following thrombosis. Thrombosis was induced in rats by intravenous injection of collagen and adrenaline. PMNLs were separated from rat blood by using dextran sedimentation and Ficoll-Hypaque. Arachidonic acid (AA), formyl methionine leucine phenylalanine (FMLP) and opsonized zymosan (OZ) induced free radical generation was estimated as luminol (LCL) and Lucigenin (LUCDCL) dependent chemiluminescence. PMNLs nitric oxide synthase (NOS) activity and NO release were measured by using [14C] L-Arginine (L-Arg) and oxy-hemoglobin respectively. LCL and LUCDCL responses in rat PMNLs were significantly attenuated following thrombosis. There was no change in the release of myeloperoxidase enzyme (MPO) from PMNLs obtained following thrombosis. PMNLs NOS activity and NO release were also found to be increased after thrombosis. Pretreatment of rat PMNLs with 10 mM L-NAME (NO precursor) or 100 microM sodium nitroprusside (NO donor), resulted in significant reduction of AA induced LCL response. Results obtained indicate that NO release form PMNLs was augmented while free radical generation response was attenuated after the induction of thrombosis.

Free radical scavenging mechanisms during pulmonary thromboembolism in rats.

In the present investigation alterations in the free radical generating and scavenging enzymes in platelets, neutrophils (PMNLs), heart and lung homogenates following rat pulmonary thromboembolism have been studied. Thrombosis was induced by intravenous infusion of collagen and adrenaline. Levels of malonaldehyde (MDA) were elevated in the PMNLs after thrombosis. Activities of superoxide dismutase (SOD) and catalase (CAT) were found to increase in platelets and PMNLs respectively. However, there was no significant alteration in the lactate dehydrogenase (LDH), lysozyme (LYS), ratio of xanthine oxidase to dehydrogenase (XO/XH) and PMNLs O2- generation before and after thrombosis. Migration of PMNLs following thrombosis was indicated by increased activity of myeloperoxidase (MPO) in the heart. In addition, pretreatment with allopurinol, a xanthine oxidase inhibitor and indomethacin, a cyclooxygenase inhibitor offered protection against thromboembolism induced death/paralysis. Results suggest the involvement of free radicals in thrombosis.

Role of free radicals in pulmonary thromboembolism in mice.

Involvement of free radicals and their scavenging enzymes in mice pulmonary thromboembolism, induced by intravenous infusion of collagen and adrenaline, has been studied. Malonaldehyde (MDA) and activities of xanthine oxidase (XO), catalase (CAT) and superoxide dismutase (SOD) were estimated in platelets, heart and lung homogenates. MDA increased in all the tissues sharply, while animals showed 70-80% thrombocytopenia. Xanthine oxidase activity in these animals increase significantly in heart. However, increased SOD activity and decreased catalase activity was observed in platelets. Intravenous administration of superoxide dismutase (5 mg/kg), catalase (5 mg/kg) and mannitol (200 mg/kg) protected the mice against pulmonary thromboembolism. The importance of free radicals in mice pulmonary thromboembolism has been demonstrated.

Effect of molsidomine and free radical scavengers on the pulmonary thromboembolism in mice.

Molsidomine is a prodrug of nitric oxide (NO) and is used in the treatment of coronary heart disease. Recent studies with NO have shown that it can exert beneficial as well as toxic effects. It was, therefore, considered desirable to evaluate the effect of this drug in an animal model of thrombosis. It was administered to mice, one hour prior to the thrombotic challenge, in a dose range of 1 to 100 mg/kg ip. In lower doses (1-30 mg/kg), it offered protection by 24-38%, and at 50 and 100 mg/kg it protected 54% and 58% animals respectively. Mice pretreated with molsidomine (3, 10 and 30 mg/kg) and free radical scavengers (superoxide dismutase, catalase, mannitol and deferoxamine, suboptimal doses) together offered significantly more protection against thrombosis than either one of them alone. The results suggest that a combination of molsidomine and free radical scavengers should be more effective than molsidomine alone in the treatment of thrombosis and other ischemic disorders.

Status of antioxidants in brain microvessels of monkey and rat.

Levels of certain antioxidants namely reduced glutathione (GSH), ascorbic acid (Vit C), alpha-tocopherol (Vit E) and antioxidant enzymes, superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione reductase (GR) were compared in monkey and rat brain microvessels which constitute the blood-brain barrier (BBB). The BBB of both the species contains appreciable amounts of the antioxidants to protect against oxidative damage. The level of protection in rat seems to be more efficient than monkey since rat microvessels contain higher concentrations of some of the bio-antioxidants. The comparative status of enzymatic and non-enzymatic protective system against oxidation in the brain microvessels has been discussed.