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Punjab Univ. J. Zool., Vol. 32 ( 2 ), pp. 209 - 215 , 201 7 ISSN 1016 - 1597 (Print) ISSN2313 - 8556 ( online)

Original Artic le

Effects of on serum proteins profile in male active and passive smokers

Nabila Roohi, Mehjabeen , S amina Ashraf *

Department of Zoology, University of the Punjab, Lahore , Pakistan

Article history Abstract Received: March 28, 201 7 The present study deals with the analysis of serum total proteins and fractions Revised: December 1 0 , 2017 (albumin, total globulins, gamma globulins and non - gamma globulins) of active and Accepted: December 1 1 , 2 017 passive smokers. Blood samples of 180 cigarette smokers were collected from different locations of Lahore and 60 healthy non - smokers from University of the Authors’ Contribution Punjab, Quaid - e - Azam campus Lahore, in terms of comparable age, height, weight NR: Supervise the research; and socioeconomic set up. Serum protein profile was analyzed using commercially M: contributed in analytical available kits by using automated clinical chemistry analyzer. Serum total proteins work, data, interpretation and have shown a significant decrease in smok ers in comparison with controls. A drafting of article , SA: significant depression in serum albumin was observed in smokers due to increased contributed in collection and excretion of albumin in bound state with the chemicals, whereas, total globulins, processing of data gamma globulins and non - gamma globulin serum proteins have shown significant increase in smokers. Our findings provide primary information about fractions and Key words total serum proteins of cigarette smokers. The decrease in albumin justifies the Albumin reduction in total serum proteins and elevation in globulins. Smoking cess ation T otal proteins plays a significant role in recovery of these proteins. Total globulins Gamma globulins Cigarette smokers

To cite this article: ROOHI, N., MEHJABEEN, ASHRAF, S., 2017. Effect of cigarette smoking on serum proteins profile in male active and passive smokers . Punjab Univ. J. Zool., 3 2 ( 2 ): 209 - 215 .

INTRODUCTION approximately 4000 compounds, 200 of which are toxicants and 80 are known . igarette sm oking is an important risk Cigarette smoke includes l arge quantities of factor for coronary, peripheral and oxidants, free radicals that generates oxidative atherosclerotic vascular disease. It alters stress and p olycyclic aromatic hydrocarbons that C are highly lipid soluble (Carel and Eviatar, 1985; the process of cell division in cardiac muscles. Additionally there is dose dependent response Abel et al ., 2005; De Heens et al .,2008). These between the quantity of smoked, chemicals tend to accumulate in the liver cardiovascular morbidity and fatality. Smoking progressively (Osman et al ., 2017). increases absorption and inhalation of numerous Chemicals in cigarette smoke have detrimental metabolites and compounds deleterious effect on liver tissues, therefore, (Qulander et al ., 2012). Though to bacco has altering some of the serum biochemical detrimental effects on the human health, it is still parameters related to liver efficiency (Abdul - highly consumed globally (Benowitz et al ., Razaq and Ahmed, 2013). The risk of in 1988). It has been considered as causative smokers vary on the basis of number of agent of lethal diseases such as , cigarettes smoked per dayand duration of respiratory disorders, variety of infections and smoking ( Janson et al ., 2001 ; Lubin et al ., heart diseases (Z hong et al ., 2008; Mehta et al ., 2007). Proteins are the most important 2008). Many biochemical parameters and body compounds that are present in human serum systems are affected by cigarette smoke. abundantly and play a key role for the Moreover, cigarette smoke contains maintena nce of subtle acid - alkaline balance of the body. In addition, proteins are source of 1 71 - PUJZ - 71028230 /1 7 /0 209 - 0215 Copyright 201 7 , Dept. Zool., P.U., Lahore, Pakistan *Corresponding author: [email protected] 210 N.ROOHI ET AL. energy for the tissues and muscles when number of enzymatic functions in the plasm a in adequate amount of carbohydrate is not addition to natural and acquired immunity ingested. The human serum protein level shows against the invading organisms (Al - Bayatee and a significant decrease in smokers (Alsalhen an d Shanawa, 2014). Abdalsalam, 2014). Serum total protein contains The current study was conducted to albumin and globulin (DeGowin et al ., 2004). assess and evaluate the effect of cigarette The quantitative determination is usually done smoke on human serum protein. by automated analyzer (Gates and Fink, 2008).In human serum, about 50 - 60% of MATERIALS AND METHODS proteins are represented by human serum albumin (Zunszain et al ., 2003). Through numerous studies, remarkable reduction in Subjects serum albumin level has been detected in Protein analyses were carried out on smokers. Globulins are heterogeneous group of serum samples from 240 male subjects (age: protein and second to albumin on the basis of 30 - 50) classified into four groups with 60 available amount in serum with the solubility rate subjects in each group. Subjects were lower than the albumin (Marieb and Hoehn, categorized according to the number of smoked 2010). Globulins acts to compensate the cigarettes per day into three smokers groups reduced level of albumin in the serum by (pas sive, moderate and heavy smokers, 60 increased production of immunoglobulins by the subjects each) and non - smokers (control lymphocytes and failure of these cells to group,60 subjects).A proforma was designed to synthesize globulins leads t o advanced diseases obtain a detailed clinical history for each subject that have been involved in reduced protein of study. Each subject provided the details of his synthesis and accelerates the catabolism of age, blood pressure, body weig ht, body height proteins (Pisters and Brennan, 1990; Inui, 2002). and other physical measurements. Participants Cigarette smoke affects the organs, were selected on the basis of their clinical even those not in direct contact with the smoke history, smoking status and number of and it alters the enzymatic activity of the organs consumed cigarettes per day. The exclusion playing an important role in anabolic metabolism criteria for the study include existing physical of proteins (Abdul - Razaq and Ahmed, 2013). diseases like hepat itis, cancer, autoimmune Cigarette smoke contains numerous oxidants disease, cardiovascular disorders, diabetes etc. and prooxidants that are capable of producing Sixty healthy non - smokers volunteered were free radicals by neutrophils and gener ates selected as control group. Body mass index oxidative stress in the body which in turn (BMI) was measured using the following formula: produces highly reactive oxygen and nitrogen 2 2 species (Cedergren et al ., 2007; Arinola, 2011). BMI (Kg/m ) = Weight (Kg) / (Height (m) ) Once formed, these oxidizing species are eliminated by the body’s antioxidants like Super Experimental design oxide dismutase (SOD), G lutathione (GSH), Whole blood was collected in a test tube Glutathione peroxidase (GSH - Px) and catalase and allowed to clot by leaving it undisturbed at (Valko et al ., 2007; Seven et al ., 2008). When for 15 - 30 minutes. Clot was free radicals are overproduced, the level of removed by centrifuging at 3000 rpm for 15 antioxidants begins to reduce and damage minutes in a refrigerated centrifuge. All the biomolecules such as lipids, proteins and DNA samples were analyzed for albumin and total (Seven et al ., 2008). proteins using Chema Diagnostic kits. Serum Albumin, one of the well - known acute gamma globulin proteins were assessed by phase protein,has antioxidant properties and it precipitation from serum with ammonium can be considered as amarker of inflammation sulphate - sodium chloride reagent (Wolfson et (Pongpaew et al ., 2001). al ., 1948). Chemistry analyzer (Model 5010, Globulins are antibodies that are Robert Riele GmbH & Co KG. D - 13467 Berlin, produced by the lymphocytes. Free radicals in Germany) was used for quantitative cigarettes smoke enhance the production of determinationof proteins. Total serum globulins globulins that help the body to fight against the were measured by deducting the values of oxidative stress. Gamma globulins are the most albumin from total protein whereas, non - gamma important globulins, synthesized by the B globulin proteins were determined by deducting lymphocytes or plasma cells and perform a EFFECT OF SMOKING ON SERUM PROTEINS 211 t he values of gamma globulins from globulins for control and smokers groups. Variations were each sample. assessed by inter - group comparisons. The mean value of systolic blood pressure showed a Statistical analysis significant increase in chain smokers when Statistical analysis was performed by compared with controls and passive smokers. using GraphPad Prism version 5.00.Data were Diastolic Blood pressure showed a significant analyzed statistically using one - way ANOVA and decrease in passive smokers in comparison to presented as Mean±SEM. Tukey’s post hoc controls and a significant increase in chain mul tiple comparison tests were employed for smokers when compared to passive smokers. A comparison amongst the groups of the study. significant increase was observed in body mass Differences were assumed to be significant at p index of chain smokers when compared with < 0.05. moderate smokers. Significant reduction was observed in total proteins level in passive, RESULTS moderate and chain smoker s when compared to controls and smokers groups. Significant Serum protein profile including total decrease in albumin level was observed in proteins, albumin, total globulins, gamma passive and moderate and chain smokers when globulins, non - gamma globulins along with compared to controls and smokers group. anthropometric parameters were analyzed for

Table I: Average values (mean±SEM) of anthropo metric parameters, serum total proteins and its fractions (g/dl) in study groups.

Parameters Control Passive Moderate Chain Systolic BP 116.2 ± 0.826 114.8 ± 1.334 118.5 ± 1.160 122.0 ± 1.160 Diastolic BP 79.50 ± 0.837 75.67 ± 1.019 78.33 ± 0.757 80.67 ± 1.059 Body mass index 21.81 ± 0.218 23.01 ± 0.531 21.46 ± 0.403 23.25 ± 0.540 Total proteins 11.01 ± 0.168 10.46 ± 0.136 9.457 ± 0.084 9.063 ± 0.080 Albumin 6.542 ± 0.121 6.020 ± 0.141 4.640 ± 0.081 3.943 ± 0.081 Total globulins 4.445 ± 0.080 4.272 ± 0.084 5.207 ± 0.098 5.145 ± 0.094 Gamma globulins 2.982 ± 0.056 3.352 ± 0.048 3.570 ± 0.075 3.552 ± 0.044 Non - gamma 1.563 ± 0.037 1.077 ± 0.040 1.330 ± 0.031 1.925 ± 0.068 globulins Passive; passive smokers, Moderate; moderate smokers, Chain; chain smokers

Table II: Percentage differences and ‘p’ value summary of anthropometric parameters, serum total proteins and its fractions in compared groups.

Control Control Control Passive Passive Moderate Parameters vs vs vs vs vs vs Passive Moderate Chain Moderate Chain Chain Systolic BP 2% ↓ 2% ↑ 5% ↑** 3% ↑ 6% ↑*** 3% ↑ Diastolic BP 5% ↓* 2% ↑ 1% ↑ 3% ↑ 7% ↑** 3% ↑ Body mass 5% ↑ 2% ↓ 7% ↑ 7% ↓ 1% ↑ 8% ↑* index Total proteins 5% ↓* 14% ↓*** 18% ↓*** 10% ↓*** 13% ↓*** 4% ↓ Albumin 8% ↓** 29% ↓*** 40% ↓*** 23% ↓*** 34% ↓*** 15% ↓*** Total globulins 4% ↓ 17% ↑*** 16% ↑*** 22% ↑*** 20% ↑*** 2% ↓ Gamma 12% ↑*** 20% ↑*** 19% ↑*** 6% ↑* 6% ↑ 1% ↓ globulins Non - gamma 31% ↓*** 15% ↑** 23% ↑*** 23% ↑** 78% ↑*** 45% ↑*** globulins ↑ Increase, ↓ Decrease, * p< 0.05, ** p< 0.01,*** p<0.001 212 N.ROOHI ET AL.

Total globulins level showed a body (Yu et al ., 1997 and Pessione et al ., 2001). significant increase in moderate and chain Albumin is the most abundant protein in the smokers when compared to controls and serum, it comprises more than half of the blood passive smokers. Gamma globulins showed a serum proteins (Sokolowska et al ., 2009). Non - significant increase in passive, moderate and smokers have normal level o f albumin in the chain smokers when compared to controls and blood that plays a role in the transport of an increase was observed in moderate smokers biomolecules. Chemicals in cigarette smoke when compared to passive smokers. Significant have both direct and indirect effect on serum reduced level of non - gamma globulins was protein profile. These directly alters the binding observed in passive smokers when compared to properties of albumin and results in its controls and significant increase was observed degradation in the liver and loss of albumin by in non - gamma globulins level of moderate and the kidney (hypo - albuminuria) (Chase et al ., chain smokers when compared to controls and 1991; Ramamurthy et al ., 2012).Most of the passive smokers (Table I - II). proteins are present in the serum in free and bound state. Albumin binds water, cations, fatty DISCUSSION acids, bilirubin, thyroxine, hormones a nd various that are metabolized in the liver. Reduced albumin in serum is a sign of poor health status Smoking has been considered as one of (Farrugia, 2010). Albumin synthesis takes place the 10 greatest contributors to global health in the liver and a marked reduction in serum problems and it is considered as harmful agent albumin is observed in moderate and chain causing disease with multiple organ dysfunction smokers as c ompared to controls. It has a well - and deaths. If present trends were allowed to marked clearance rate of chemicals from the continue unchecked and the government blood. Albumin is degraded into amino acids in continued to be a silent spectator, the number of the serum and hence is available for energy deaths annually could touch the 10 million marks (Thakore et al ., 1994). (Leone, 2005). Albumin has antioxidant properties that Proteins are major extracellular are modified by carb onylation induced by component of the and acts as cigarette smoke chemicals (Clerici et al ., 2014). a dynamic system with various biological High level of free radicals results into increased functions (Gorinstein et al ., 2000). Modern proteolytic activity (Tetley, 2006). Albumin proteomics and improvement in assay of protein excretion rate is about 2.8 times higher in markers are useful for the study of proteins in smokers than non - smokers with development controls and smokers samples (Mathelin et al. , and progression of diabetic renal damage 2006). Marked variation has been observed in (Chase et al ., 1991). The human serum protein smokers on the basis of serum proteins level, it includes globulin in addition to albumin. is not the am ount of total proteins but the Globulins are antibodies that are produced in changes at individual protein level that are response to stimulation of the immune system. important (DeGowin et al ., 2004). Serum total Total globulins level is elevated in m oderate and protein level is constant; the decrease in one chain smokers in comparison to controls and type of protein (albumin) through catabolismis passive smokers. It consists of gamma and non - compensated by the increase in the secon d gamma fraction of globulins. Globulins are (globulin) by increase synthesis (Al - Joudi, 2005). produced by the lymphocytes that are activated It provides information regarding a patient’s by immune system when a body becomes prone general status. It was observed that serum to infection by the e xposure of volatile and protein concentration showed a marked polycyclic aromatic hydrocarbons released from reduction in smokers with an increase in the the cigarette smoke (De - Gowin et al ., 2004; Al - degree of dose and consumption rate (Salive et Joudi, 2005; Marieb and Hoehn, 2010). al ., 1992). Globulins are present in free or bound state in Most of the proteins are synthesized in plasma, it is metabolized and excreted in free - the liver and by mature lymphocytes. Cigarette state (Al - Joudi, 2005). Globulins bind with smoke causes adverse effects on the organs hemoglobin, transport metals, such as iron, in that are not in direct contact with the smoke, the blood. They are present in free or bound such as the liver, as it is involved in the state in the plasma. Non - gamma globulins proces sing of toxins to be eliminated from the include IgA, IgM, IgE and IgG that show a EFFECT OF SMOKING ON SERUM PROTEINS 213 marked increase in moderate and chain smokers male in El - beida City, Libya. smoke rs as compared to controls (DeGowin et Int. Curr. Pharm. J ., 3 (7): 291 - 295. al ., 2004). ARINOLA, O.G., AKINOSUN, O.M. AND OLANIYI, J.A., 2011.Passive and active Conclusion cigarette smoking: Effects on In this study a marked decrease in plasmalevels of antioxidant vitamins, serum protein level was observed in cigarette immunoglobulin classes and acute smokers when compared to control subjects. phas e reactants. Afr ican J. Biotechol., Heavy smoking was found to be associated with 10 (32): 6130 - 6132. low albumin concentration, low total protein BENOWITZ, N.L., PORCHET, H., SHEINER, L. levels and higher globulins levels. Cigarette AND JACOB, P., 1988. smoke can affect hepatic functions due to absorption and cardiovascular effects deleterious effect of free radicals and toxicants with smokeless use: on liver that alter the synthesis of proteins and comparison with cigarettes and nicotine reduces the protein content of the serum. gum. Clin. Pharmacol. Ther ., 44 (1): 23 - Increased globulins levels in smokers 28. compensate for the reduced albumin level in CAREL, R.S. ANDEVIATAR, J., 1985. Factors serum and hence total protein level remains affecting leukocyte count in healthy constant. Albumin and globulins level vary as adults. Prev. Med., 14 (5): 607 - 619. the duration and dose of smoking changes. CEDERGREN, J., FORSLUND, T., Thus the study concludes that smoke rs have SUNDQVIST, T. AND SKOGH, T., higher risk of liver dysfunction than that of non - 2007. Intracellular oxidative activation in smokers. synovial fluid neu trophils from patients with rheumatoid arthritis but not from Acknowledgements other arthritis patients. J. Rheumatol ., Authors gratefully acknowledge the 34 (11): 2162 - 2170. volunteers of the study for their cooperation in CHASE, H.P., GARG, S.K., MARSHALL, G., blood sampling . BERG, C.L., HARRIS, S ., JACKSON, W.E. AND HAMMAN, R.E., 1991. REFERENCES Cigarette s moking i ncreases the r isk of a lbuminuria a mong s ubjects w ith t ype I Diabetes. JAMA , 265 (5): 614 - 617. ABDUL - RAZAQ, S.N. AND AHMED, B.M., 2013. CLERICI, M., COLOMBO, G., SECUNDO, F., Effect of cigarette smoking on liver GAGLIANO, N., COLOMBO, R., function test and some other related PORTINARO, N., GIUSTARINI . , D., parameters. Zanco. J. Med. Sci., MILZANI, A., ROSSI, R. AND DALLE - 17 (3):556 - 562. DONNE, I., 2014. Cigarette smoke ABEL, G.A., HAYS, J.T., DECKER, P .A., induces alte rations in the - binding CROGHAN, G.A., KUTER, D.J. properties of human serum albumin. AND ANDRIGOTTI, N.A., 2005. Effects Blood Cells Mol. Dis ., 52 (4): 166 - 74. of biochemically confirmed smoking DE HEENS, G.L., KIKKERT, R., AARDEN, L.A., cessation on white blood cell count. VAN DER VELDEN, U. ANDLOOS, Mayo. Clin. Proc., 80 (8):1022 - 1028. B.G., 2008. Effects of smoking on the ex AL - BAYATTE, L. A .A. AND SHNAWA, I.M.S., vivo cytokine production in periodontitis. 2014.The effect of Senescence and J. Periodontal. Res ., 44 (1): 28 - 34. smoke on human immunoglobulin DEGOWIN, R.L., LEBLOND, R.F. AND concentrations. J. Asian. Sci. Res. , 4 (5): BROWN, D.D., 2004. DeGowin’s 222 - 229. diagnostic examination 8 th ed. McGraw AL - JOUDI, F.S., 2005. Prognostic value of an Hill Professional Publishers, 948 - 959. index for serum globulin compensation FARRUGIA, A., 2010. Albumin Usage in Clinical in colons and breast . Singapore Medicine: Tradition or Therapeutic? Med. J. , 46 (12): 710 - 713. Transfus. Med. Rev. , 24 (1): 53 - 63. ALSALHEN, K.S. AND ABDALSALAM, R.D., GATES, R.A. AND FINK, R.M., 2008. Oncology 2014. Effect of cigarette smoking on nursing secrets 3 rd ed. Westline liver functions: a comparative study Industrial Drive St.Louis, Missouri conducted among smokers and n on - Publishers, p. 523. 214 N.ROOHI ET AL.

GORINSTEIN, S., GOSHEV, I., MONCHEVA, HEGAZY, A.M., 2017. Polycyclic S., ZEMSER, M., WEISZ, M., CASPI, aromatic hydrocarbons (PAHS) A., LIBMAN, I., LERNER, H.T., accumulation and histopathological TRAKHTENBERG, S. AND MARTIN - in gills and mantle of BELLOSO, O., 2000. Intrinsic Lanistescarinatus (Molluscs, tryptophan fluorescence of human Ampullariidae) to asse ss crude oil serum proteins and related toxicity. Punjab Univ. J. Zool ., 32 (1): conformational changes . J. Prot. Chem., 039 - 050. 19 (8): 637 - 642. PESSIONE, F., RAMOND, M.J., NJAPOUM, C., INUI, A., 2002. Cancer anorexia - cachexia DUCHATELLE, V., DEGOTT, C., syndrome: current issues in research ERLINGER, S., RUEFF, B., VALLA, and management. CA Cancer J. Clin., D.C. AND DEGOS, F., 2001. Cigarette 52 : 72 - 91. smoking and hepatic lesions in patients JANSON, C., CHINN, S., JARVIS, D., ZOCK, with chronic titis C. Hepatology., J.P., TOREN, K. AND BURNEY, P., 34 (1): 121 - 125. 2001. Effect of passive smoking on PISTERS, P.W. AND BRENNAN, M.F., 1990. respiratory symptoms, bronchial Amino acid metabolism in human responsiveness, lung function, and total cancer cachexia. Annu. Rev. Nutr ., 10 : serum IgE in the European Community 107 - 132. Respiratory Health : a cross - PONGPAEW, P., TUNGTRONGCHITR, R., sect ional study. Lancet., 358 (9299): PHONRAT, B., VUDHIVAI, N., 2103 - 2109. VIROONUDOMPHOL, D. AND LEONE, A ., 2005 Biochemical markers of SCHELP, F.P.,200 1. cardiovascular damage from tobacco in relation to the phenotype of alpha - 1 - smoke. Curr. Pharm. Des ., 11 (17): antitrypsin and serum vitamin C 2190 - 2208. concentration. J. Nutr. Environ. Med. , LUBIN, J.H., ALAVANJA, M.C., CAPORASO, 11 (3): 167 - 173. N., BROWN, L.M., BROWNSON, R.C., QULANDER, S.F., NAYYER, A.Q., ALI, S. AND FIELD, R.W., GARCIA - C LOSAS, M., SHAKIR, H.A., 2012. Lipid profile in HARTGE, P., HAUPTMANN, M., smoking employees of University of HAYES, R.B., KLEINERMAN, R., Azad Jammu and Kashmir KOGEVINAS, M., KREWSKI, D., Muzaffarabad. Punjab Univ. J. Zool ., LANGHOLZ, B., LETOURNEAU, E.G., 27 (2): 79 - 82. LYNCH, C.F., MALATS, N., SANDLER, RAMAMURTHY, V., RAVEENDRAN, S., D.P., SCHAFFRATH - ROSARIO, A., THIRUMENI, S. AND KRISHNAVENI, SCHOENBERG, J.B., SILVERMAN, S., 2012. Biochemical changes of D.T., WANQ, Z., WICHMANN, H.E., cigarette smokers and non - cigarette WILC OX, H.B. AND ZIELINSKI, smokers. Int. J. Adv. Lif. Sci ., 1 : 12 - 22 . J.M.,2007. Cigarette smoking and SA LIVE, M.E., HUNTLEY, J.C. , PHILIPS, C.L., cancer risk: modeling total exposure and GURALNIK, J.M., COHEN, H.J., intensity. Am. J. Epidemiol ., 166 (4): OSTFELD, A.M. AND WALLACE, R.B., 479 - 489. 1992.Serum albumin in older persons: MARIEB, E.N. AND HOEHN, K., 2010. Human Relationships with age and health anatomy and physiology 8 th ed. status. J. Clin. Epidemioli., 45 (3): 213 - PearsonBenjamin Cummings, San 221. Francisco, CA, pp. 636. SEVEN, A., GUZEL, S., ASLAN, M. AND MATHELIN, C., TOMASETTO, C., CROMER, A. HAMURYADUN, V., 2008.Lipid, protein, AND RIO, M.C., 2006.Proteomics and DNA oxidation and antioxidant status in . Gynecol. Obstet. Fertil ., rheumatoid arthritis. Clin. Biochem ., 41 : 34 : 1161 - 1169. 538 - 543. MEHTA,H., NAZZAL, K. AND SADIKOT, R.T., SOKOLOWSKA, M., WSZELAKA , RYLIK, M., 2008. Cigarette smoking and innate POZNANSKI, J. AND BAL, W., 2009. immunity. Inflamm. Res., 57 (11): 497 - Spectroscopic and thermodynamic 503. determination of three distinct binding OSMAN, G., GALAL, M., ABUL - EZZ, A., site for Co (II) in human serum albumin. MOHAMMED, A., ABUL - ELA, M. AND J. Inorg. Biochem., 103 (7): 1005 - 1013. EFFECT OF SMOKING ON SERUM PROTEINS 215

TETLEY, T . D., 2006. Proteinase inhibitors/ globulin in 1 ml of serum. Am. J. Clin. secretory leukopr otease inhibitor and Path., 18 (9):723 - 730. elafin. In:. Encyclopedia of respiratory YU, M.W., HSU, F.C., SHEEN, I.S., CHU, C.M., medicine (Eds. G.J. Laurent,, S.D. LIN, D.Y., CHEN, C.J. AND LIAW, Y.F., Shapiro) . London: Academic Press. 1997.Prospective study of 517 - 522. hepatocellular carcinoma and liver THAKORE, K.N., GAN, J.C. AND ANSARI, cirrhosis in asymptomatic chronic G.A.S., 1994. Rapid plasma clearance hepatitis B virus carriers. Am. J. ofalbumin - adduct in rats. Epidemiol., 145 (11):1039 - 1047. Toxicol . Lett., 71 (1): 27 - 37. ZHONG, C.Y., ZHOU, Y.M. ANDPINKERTON, VALKO, M., LEIBFRITZ, D., MONCOL, J., K.E., 2008. NF - kappaB inhibition is CRONIN, M.T.., MAZUR, M. AND involved in tobacco smoke - induced TELSER, J., 2007. Free radicals and apoptosis in the lungs of rats. Toxicol. antioxidants in normal physiological Appl. Pharmacol ., 230 (2): 150 - 158. functions and human disease. Int. J. ZUNSZAIN, P.A., GHUMAN, J., KOMATSU, T., Biochem. Cell Biol ., 39 (1):44 - 84. TSUCHIDA, E. AND CURRY, S., WOLFSON, W.Q., COHN, C., CALVARY, E. 2003.Crystal structural an alysis of AND ICHIBA, F., 1948. A rapid human serum albumin complexed with procedure for the estimation of total hemin and fatty acid. BMC Struct. Biol ., protein, true albumin, total globulin, 3 : 1186 - 1472. alpha globulin, beta globulin and gamma