Background: Pleural effusion is a common complication of various diseases. Free radicals are known to produce damage in many biological tissues. Free radicals exert their cytotoxic effect by causing lipid peroxidation which is believed to be responsible for the exudation of fluid into the pleural space.Aim: Evaluation of the association of benign and malignant pleural effusion with the free radical induced pleurisy by measurement of lipid peroxidation products and nitric oxide activity.Methods: Case control study was conducted on 50 cases of benign pleural effusion, 50 cases of malignant pleural effusion and 15 cases of healthy controls. Malondialdehyde (MDA) levels were measured by spectrophotometric method with TBA. Nitric Oxide activity was measured by spectrophotometric method using Griess reaction.Results: Our results showed significant increase of MDAs level in both groups of patients: benign and malignant in comparison with control group. Significant increase in the concentrations of nitrate/nitrite depicting total nitric oxide was observed in benign as well as malignant group in comparison to control healthy group.Conclusion: These results suggest that determination of biomarkers of oxidative stress products may be useful in the diagnosis and treatment of patients

Hari D. Khanna

J.K. Mishra

Manish Shankar

Tandon R.

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Journal of Stress Physiology & Biochemistry, Vol. 8 No. 2 2012, pp. 138-144 ISSN 1997-0838Original Text Copyright © 2012 by Tandon, Mishra, Shankar and KhannaORIGINAL ARTICLELipid Peroxidation Products and Nitric Oxide in the Evaluation of Benign and Malignant Pleural EffusionTandon1 R., J.K. Mishra2, Manish Shankar2 and Hari D. Khanna3*1 Department of Medicine, Institute of Medical Sciences, Banaras Hindu University, Varanasi-221005 (India)2 Department of T.B and Respiratory Diseases, Institute of Medical Sciences, Banaras Hindu University, Varanasi-221005 (India)3 Department of Biophysics, Institute of Medical Sciences, Banaras Hindu University, Varanasi-221005 (India)*E-mail:hdkhanna@yahoo.co.inReceived March 20 2012Background: Pleural effusion is a common complication of various diseases. Free radicals are known to produce damage in many biological tissues. Free radicals exert their cytotoxic effect by causing lipid peroxidation which is believed to be responsible for the exudation of fluid into the pleural space.Aim: Evaluation of the association of benign and malignant pleural effusion with the free radical induced pleurisy by measurement of lipid peroxidation products and nitric oxide activity.Methods: Case control study was conducted on 50 cases of benign pleural effusion, 50 cases of malignant pleural effusion and 15 cases of healthy controls. Malondialdehyde (MDA) levels were measured   by   spectrophotometric   method   with   TBA.   Nitric   Oxide   activity  was   measured   by spectrophotometric method using Griess reaction.Results: Our results showed significant increase of MDAs level in both groups of patients: benign and malignant in comparison with control group. Significant increase in the concentrations of nitrate /nitrite depicting total nitric oxide was observed in benign as well as malignant group in comparison to control healthy group.Conclusion: These results suggest that determination of biomarkers of oxidative stress products may be useful in the diagnosis and treatment of patients. Key words: Pleural effusion, benign, malignant, free radical induced pleurisyJOURNAL OF STRESS PHYSIOLOGY & BIOCHEMISTRY Vol. 8 No. 2 2012Lipid Peroxidation Products and Nitric Oxide...ORIGINAL ARTICLELipid Peroxidation Products and Nitric Oxide in the Evaluation of Benign and Malignant Pleural EffusionTandon1 R., J.K. Mishra2, Manish Shankar2 and Hari D. Khanna3*1 Department of Medicine, Institute of Medical Sciences, Banaras Hindu University, Varanasi-221005 (India)2 Department of T.B and Respiratory Diseases, Institute of Medical Sciences, Banaras Hindu University, Varanasi-221005 (India)3 Department of Biophysics, Institute of Medical Sciences, Banaras Hindu University, Varanasi-221005 (India)*E-mail:hdkhanna@yahoo.co.inReceived March 20 2012Background: Pleural effusion is a common complication of various diseases. Free radicals are known to produce damage in many biological tissues. Free radicals exert their cytotoxic effect by causing lipid peroxidation which is believed to be responsible for the exudation of fluid into the pleural space.Aim: Evaluation of the association of benign and malignant pleural effusion with the free radical induced pleurisy by measurement of lipid peroxidation products and nitric oxide activity.Methods: Case control study was conducted on 50 cases of benign pleural effusion, 50 cases of malignant pleural effusion and 15 cases of healthy controls. Malondialdehyde (MDA) levels were measured   by   spectrophotometric   method   with   TBA.   Nitric   Oxide   activity  was   measured   by spectrophotometric method using Griess reaction.Results: Our results showed significant increase of MDAs level in both groups of patients: benign and malignant in comparison with control group. Significant increase in the concentrations of nitrate /nitrite depicting total nitric oxide was observed in benign as well as malignant group in comparison to control healthy group.Conclusion: These results suggest that determination of biomarkers of oxidative stress products may be useful in the diagnosis and treatment of patients. Key words: Pleural effusion, benign, malignant, free radical induced pleurisyPleural effusion is a common occurrence in a number   of   pulmonary   and   extra-pulmonary diseases, being often the only manifestation of the disease (Light et al.1973). Pleural effusion occurs in a variety of diseases pertaining to respiratory system,   cardiovascular   system,   renal   and   other systems. Determining the cause of pleural effusion is not always easy. The first and most important step in the evaluation of pleural effusion is to differentiate   them   as   transudates   and   exudates (Storey et al. 1976; Guleria et al 2003). Recently, usefulness of MDA has been studied to differentiate transudates   from   exudates.   MDA   is   a   lipid peroxidation   product,   produced   by   oxidative JOURNAL OF STRESS PHYSIOLOGY & BIOCHEMISTRY Vol. 8 No. 2 2012139Tandon et aldeterioration   of   unsaturated   fatty   acids.   Two sources have been suggested for MDA pleural fluid (Gupta 2002). The first possible source is plasma proteins.   When   pleura   are   inflamed,   there   is increased leakage of plasma proteins into pleural space. Plasma MDA is mostly bound to plasma proteins.   Due   to   increased   leakage   of   plasma proteins into pleural space, MDA enters into pleural space, leading to increased concentration in pleural fluid.   The  second   possible   source   is   local production by inflammatory cells which are present in increased number in conditions like tuberculosis, malignancy etc (Gupta 2002).Nitric oxide (NO), determined as its immediate metabolite nitrite, were measured by Agrenius et al, 1994 in pleural fluid, in order to investigate if they were involved in pleural inflammation and pleural fluid exudation.An attempt  has  been made  to evaluate  the pleural fluid in benign and malignant effusion to have an insight on the correlation of MDA and nitric oxide in the disease process. MATERIALS AND METHODSCase control study comprising of 50 cases of benign pleural effusion and 50 cases of malignant pleural effusion was carried out in the Department of   Medicine   and   Department   of   T.B   and Respiratory Diseases, Institute of Medical Sciences, Banaras Hindu University. All the selected patients presented with pleural effusion of inflammatory origin, and of neoplastic origin diagnosed clinically, radiologically and cytologically were taken for the study. Exclusion criteria comprised of HIV positive patients, Diabetes Mellitus, Rheumatoid Arthritis and other immune mediated diseases, and patients who were consuming antioxidants for prolonged duration of more than four weeks. 15 age and sex matched   healthy   individuals   were   the   subjects whose   blood   samples   were   taken   as   control subjects. Blood samples and pleural fluids were taken from the study subjects with their informed consent.Estimation of Malondialdehyde - marker of lipid peroxidationMalondialdehyde (MDA) levels in the study cases and controls were assayed by thiobarbituric acid reactive substances  (TBARS) technique of Philpot, 1963; Burge and Aust, 1978. MDA, which is a stable end product of fatty acid peroxidation, reacts with TBA at acidic conditions to form a complex that has maximum absorbance at 532 nm.Measurement of nitrite-nitrate concentration in pleural fluidThe total nitrite concentration in pleural fluid of study subjects, an indicator of NO synthesis, was measured as described by Cuzzocrea et al (1998). The nitrate in the samples was first reduced to nitrite by incubation with nitrate reductase and NADPH at room temperature for 3 h. The total nitrite   concentration   in   the   samples   was   then measured   using  the  Griess  reaction,  by  adding Griess reagent to the sample. Optical density was measured   at   550 nm.   Nitrite   concentration   was calculated   by   comparing   OD550  with   standard solutions of sodium nitrite prepared in distilled water.Statistical Analysis:Statistical analysis was done using statistical software SPSS version 10.0. Data was found to be normally distributed. Paired t’ test, ANOVA test and SNK test were applied and the results were expressed   as   mean   ±   SD   and   p   value   was considered significant at 5% level of significance.RESULTSThe present study was conducted on the pleural fluid from 50 benign and 50 malignant cases treated in the Department of Medicine and Department of T.B and Respiratory Diseases, Institute of Medical JOURNAL OF STRESS PHYSIOLOGY & BIOCHEMISTRY Vol. 8 No. 2 2012140Lipid Peroxidation Products and Nitric Oxide...Sciences, Banaras Hindu University. Study also included 15 healthy individuals who acted as age and sex matched controls. Blood samples were collected from the antecubital vein of the healthy volunteers and the serum was separated from each sample for biochemical analysis. The consent of each individual was taken purely for research work.The average  age of the study subjects was 48.7+13.9, 43.4+18.6 and 52.4+11.6 years in the control, benign and malignant group respectively. The characteristics of the study subjects are given in table 1.Most of the cases had tuberculosis. Disease wise distribution of the studied subjects in benign pleural effusion   is   depicted   in   table   2.   Disease   wise distribution of study subjects in malignant Pleural Effusion patients is depicted in table 3.Status   of   malondialdehyde,   nitrite/nitrate  in patients  of  benign and  malignant pleural effusion shows significantly higher values in comparison to serum concentrations in healthy control volunteers (Table 4). Raised level of malondialdehyde  and nitrite/nitrate in patients is marker for higher levels of free radical damage and  involvement of free radicals in pleurisy. Table 1 Characteristics of the Study SubjectsControl Benign MalignantNumber 15 50 50GenderMale 09 31 28Female 06 19 22Age (Years) 48.7+13.9 43.4+18.6 52.4+11.6SmokersMale 08 16 19Female 00 08 11Table 2 Disease wise distribution of study subjects in benign Pleural Effusion DiseaseNo. of  PatientsSexMale FemaleTuberculosis 27 16 11Parapneumonic effusion 09 05 04Empyema 07 04 03Pancreatitis 02 02 -Hemothorax 01 01 -Liver Abscess 04 03 01Total 50 31 19JOURNAL OF STRESS PHYSIOLOGY & BIOCHEMISTRY Vol. 8 No. 2 2012141Tandon et alTable 3 Disease wise distribution of study subjects in malignant Pleural Effusion DiseaseNo. of  PatientsSexMale FemaleLUNG TUMOURHistological subtypes31 21 10· Small cell carcinoma 05 04 01· Squamous cell carcinoma 12 10 02· Adenocarcinoma 08 03 05· Poorly differentiated or Large cell carcinoma 06 04 02Other  TUMOUR TYPES 19 07 12· Mammary carcinoma 06 - 06· Ovarian carcinoma 02 - 02· Uterine carcinoma 02 - 02· Stomach carcinoma 01 01 -· Malignant melanoma 01 01 -· Non-Hodgkin’s carcinoma 02 02 -· Unknown primary 05 03 02Total 50 28 22Table 4 Status of Malondialdehyde, nitrite and nitrate in benign and malignant pleural effusions in comparison to controlsGroup(Number)MDA(mMol/L)NO2(µMol/L)NO3(µMol/L)Control (15) 0.817+0.013 34.04+7.89 59.47 + 13.79Benign  (50) 1.101 + 0.178* 79.28 +32.056* 125.96 +41.07*Malignant (50) 1.127+ 0.161* 88.96+ 37.16* 148.38 + 5141*Values expressed as Benign vs. Control;   Malignant vs. Control ,   *p < 0.001DISCUSSIONNumerous   physiological   and   pathological processes such as ageing, excessive caloric intake, infections, inflammatory disorders, environmental toxins,  pharmacological   treatments,   emotional   or psychological   stress,   ionizing   radiation,   cigarette smoke and alcohol increase the bodily concentration of oxidizing substances, known as reactive oxygen species (ROS) or, more commonly, free radicals. These are chemical species which are highly reactive owing to the presence of free unpaired electrons. An increase in free radicals compromises the delicate homeostatic   mechanisms   which   involve neurotransmitters, hormones, oxidizing substances and   numerous   other   mediators.   Owing   to   their structure,   which   is   rich   in   double   bonds, polyunsaturated fatty acids (PUFAs) render cellular membranes vulnerable to damage from free radicals, causing peroxidation. The damage induced by lipid peroxidation renders the cell unstable, and therefore compromises   fluidity,   permeability,   signal transduction   and   causes   receptor,   mitochondrial DNA   and   nuclear   alterations   (Halliwell   and Gutterridge, 2007). Nitric oxide, another free radical, is a poly-functional signaling molecule controlling processes of   vasodilation,   platelet   aggregation, immunocytotoxicity and carcinogenesis could either mediate tumourocidal activity or promote tumour growth  (Agrenius  et al 1994). Nitric oxide  is of importance   in   induced   pleural   inflammation. Inflammation and oxidative stress are pathogenic JOURNAL OF STRESS PHYSIOLOGY & BIOCHEMISTRY Vol. 8 No. 2 2012142Lipid Peroxidation Products and Nitric Oxide...mediators of many diseases. The intracellular redox state is a key determinant of cell fate. Oxidative stress plays a pivotal role in a variety of cellular responses.In the present study, higher concentration of both MDA and total nitric oxide was observed in the pleural fluid of malignant patients than those in benign   disease   group.   Comparison   of   mean concentration of MDA and total nitrate in cancer patients and control group; and also between benign and control group reveals a statistically significant difference. This points towards the existence and involvement of free radicals in pleurisy. To assess whether cancer-induced pleurisy is associated with an alteration of nitric oxide (NO)-synthase activity, Alexander et al (2002) measured the levels of nitrate/nitrite (NOx) in blood serum (BS) and pleural effusion (PE) of cancer patients (secondary pleural metastases and mesotheliomas), patients with benign lung diseases, and in BS of healthy donors.. Their results point up the diverse role of NOx in cancer patients and suggest that NOx acts as a signaling mediator during the formation of pleural metastases and might be considered as a non-specific marker in the corresponding PE. Inflammatory and neoplastic diseases frequently involve the pleural space and walls (Kroegel and. Antony,   1997).  Pleural   involvement   in   certain diseases   is   associated   with  the   infiltration   of   a number of different types of immune cells, such as neutrophils, eosinophils or lymphocytes, in various proportions depending on both the course and the aetiology of the underlying disease. In addition to infiltrating   cells,   mesothelial   cells   have   been demonstrated   to   actively   participate   in   pleural inflammation via release of various mediators and proteins, including platelet-derived growth factor (PDGF),   interleukin-8,   monocyte   chemotactic peptide   (MCP-1),   nitric   oxide   (NO),   collagen, antioxidant enzymes and the plasminogen activation inhibitor (PAI). Furthermore, several inflammatory mediators   have   been   detected   at   increased concentrations within pleural effusions, including lipid mediators, cytokines and proteins (adenosine deaminase,   lysosyme,   eosinophil-derived   cationic proteins, and products of the coagulation cascade). The   presence   of   these   mediators   underline   the concept   of   pleural   inflammation   and   certain cytokines seem to characterize a specific aetiology of pleurisy. The understanding of these processes and   the   sequence   of   events   leading   to   pleural loculation, pleural adhesion or repair are likely to provide the basis for early therapeutic intervention and reduce pleural-associated morbidity.CONCLUSION The results of the study point the diverse role of lipid peroxidation products and nitric oxide in both benign   and   malignant   pleural   effusions. Malondialdehyde assay is the most widely used test in the appreciation of the role of oxidative stress in disease.   Malondialdehyde   is   one   of   the   several products   formed   during   the   radical   induced decomposition of polyunsaturated fatty acids. The results obtained suggest that the combined analysis of   increased   levels   of   nitrate/   nitrite   in   pleural effusions and of histochemical properties of cancer and inflammatory cells may be useful in exploring the   interrelationship   of   functionally   important cellular   characteristics.   Thus   reduction   of endogenous and exogenous cause of oxidative stress is at present the best preventive option. In near future, new insights and interventions in the action of tumour suppressor genes, tumour cell apoptosis and   the   DNA  repair   mechanisms   may   lead   to additional tools against carcinogenesis from oxygen free radicals and lipid peroxidation.REFERENCESLight RW, Erosan YC, Ball WC Jr. (1973) Cells in pleural   fluid:   their   value   on   differential JOURNAL OF STRESS PHYSIOLOGY & BIOCHEMISTRY Vol. 8 No. 2 2012143Tandon et aldiagnosis. Arch Inter Med 132: 854-860Storey DD, Dines DE, Coles DT (1976) Pleural effusions: a diagnostic dilemma.  JAMA  236: 2183-2186Guleria R, Agarwal SR, Sinha S, Pande JN and Mishra   A   (2003)   Role   of   pleural   fluid cholesterol in differentiating transudative from exudative   pleural   effusion.  The   National Medical Journal of India 16(2):6 4-69Gupta KB (2002) Evaluation of pleural and serum levels   in   differentiating   transudative   from exudative pleural effusions. Indian Journal of Tuberculosis 49: 97-100Agrenius, V., Gustafsson, L E., Widström, O (1994) Tumour   necrosis   factor-α   and   nitric   oxide, determined   as   nitrite,   in   malignant   pleural effusion.  Respiratory Medicine  88(10):  743-748.Philpot, J.St.L. (1963) Estimation and identification of organic peroxides. Radiation Res. Supp., 3: 55-70. Burge   JA,   Aust   SD.   (1978)   Microsomal   lipid peroxidation. Methods Enzymol, 52: 302-310.Cuzzocrea, S., Costantino, G., Zingarelli, B and Caputi A.P (1998):  Beneficial effects of Mn (III)   tetrakis   (4-benzoic   acid)   porphyrin (MnTbap), a superoxide dismutase mimetic, in carrageenan-induced   pleurisy.  Free   Radical Biol Med, 26,25–33Halliwell   B   and   Gutteridge   JMC   (2007)  Free Radicals in Biology and Medicine. 4th edition. Oxford: Oxford University Press, UK.Timoshenko AV, Maslakova OV, Werle B, Bezmen VA, Rebeko   VYa   and   Kayser  K (2002) Presentation of NO-metabolites (nitrate/nitrite) in blood serum  and pleural  effusions  from cancer patients with pleurisy.  Cancer Letters 182 (1): 93-99 Kroegel C and Antony VB (1997) Immunobiology of pleural inflammation: potential implications for pathogenesis, diagnosis and therapy.  Eur. Respiratory J 10: 2411–2418.JOURNAL OF STRESS PHYSIOLOGY & BIOCHEMISTRY Vol. 8 No. 2 2012144

Lipid Peroxidation Products and Nitric Oxide in the Evaluation of Benign and Malignant Pleural Effusion

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