OUTCOME OF TOBACCO SMOKING HABIT ON LIPID PEROXIDATION AND ANTIOXIDANT PARAMETERS IN ANEMIC INDIVIDUALS

Nirjala Laxmi Madhikarmi1*, Madhav Gautam2 Received : 02 March, 2021 Accepted : 22 May, 2021 Published : 04 November 2021 ISSN: 2542-2758 (Print) 2542-2804 (Online) 1481 Birat Journal of Health Sciences Vol.6/No.2/Issue 15/May-Aug., 2021 Original Research Ar cle


INTRODUCTION
Globally 2 billion people are es mated to be suffering from anemia and smoking is a public health problem throughout 1 the globe and is known to affect an oxidants level. Globally, one in three adults, or 1.2 billion people smoke and about 2,3 82% reside in low-and middle-income countries. Free radicals are con nuously produced by the body's normal use of oxygen. Cells use oxygen to generate energy and free radicals are produced by the mitochondria. A normal cell balances between forma on and removal of free radicals. However, balance is shi ed towards more forma on of free radicals as cigare e smoke produces number of 4,5 hazardous chemicals compounds including free radicals. Approximately, cigare es produce 7000 chemicals, each puff 15 contains 10 low molecular weight free radicals which 3,6 ini ate and propagate lipid. Studies has been carried out in smoking, anemia, an oxidants, free radicals in separate form but lack of combined research on anemic smokers against an oxidants-free radicals made us chose this topic. In smoking and anemia context we designed to inves gate impact of stress induced by cigare e on lipid peroxida on, enzyma c and non-enzyma c  20.45 ± 4.59 kg/m . Blood was collected in EDTA vials and centrifuged at 3000 rpm for 20 minutes for the separa on of plasma and plain vials for serum extrac on. Hemoglobin concentra on less than 13.5g/dl for men, or <12g/dl for women is the normal criteria for segrega ng anemia but smokers have elevated hemoglobin level. So various hematological parameters were assessed included; red blood cell count; 4.21 3 million/mm , mean corpuscular volume (MCV); 87.2 fL mean corpuscular hemoglobin concentra on; 34.26 g/dl, white 3 blood cell; 6672 cells/mm were the selec on criteria for anemia. Vitamin A was es mated by the method of Bessy et al, it was extracted into n-heptane layer and the absorbance was read 9 at 327nm. Vitamin C was determined using dinitrophenyl hydrazine (DNPH) by Natelson method, the orange-red color 10 complex formed was read at 520nm. Vitamin E was measured by Baker and Frank Method, red colored complex 11 was measured at 520nm. The total an oxidant ac vity (TAA) was determined by Benzie and Strain method, blue 12 colored complex was read at 593nm. Lipid peroxida on product, thiobarbituric acid reac ve substances (TBARS) was assessed by Satoh method, the pink colored complex 13 was measured at 535nm. The lipid hydroperoxide (LPHO) 14 was determined using Jiang method at 560nm. Nitrite and nitrate concentra on were determined by Cortas and Wakid 15 using ac vated cadmium. Superoxide dismutase (SOD) was determined by Kakkar method, the nico namide adenine dinucleo de-phenazine methosulfate-nitro blue tetrazolium 16 formazan complex was measured at 560nm. Catalase (CAT) 17 ac vity was es mated by Sinha method, glutathione 18 peroxidase (GPx) was analyzed by Rotruck method. 19 Glutathione was es mated by Beutler and Kelley method. All the data were processed and analyzed using SPSS 20.0 (Sta s cal Package for the Social Sciences). All the results are expressed as mean ± standard devia on, the Independent sample t-test for was used for comparing this means values with sta s cal significance at p<0.05. The controls selected were of healthy rather than nonsmoking anemic because when we did analysis, the difference was only in low hemoglobin level in anemic individuals. The anemic non-smoking subjects had lower an oxidants status as well as higher oxidants values which did not show any significance between my case and controls.

RESULTS
A total of 300 subjects were selected for the study, among them 150 were anemic individuals with smoking habit and the rest 150 were the healthy age-gender matched healthy controls. The matched age of cases and controls was 45.35 ± 12.55 years, and an equal number of male and female ra o could not be found because of the lack of anemic female smokers as compared to male. The number of s cks intake of males were more as compared to females in our study group. Hemoglobin level was found to be higher in anemic smokers. The associa on of raised hemoglobin in anemic smoking was found to be sta s cally significant as compared with their healthy counter parts. Likewise, the an oxidant defence system too, was found to diminished in the anemic smokers. Significant eleva ons were found in free radical analysing parameters such as thiobarbituric acid reac ve substances, lipid hydroperoxide and nitric oxide and nitrite levels in anemic smokers which is shown (Table 1).

DISCUSSION
The prevalence of anemia ranges from 15-78% and the prevalence of smoking and tobacco use is 56.5% in men and 19.5% in women and is considered higher in comparison to [3][4][5][6][7] other countries. Each year seven million people are dying of tobacco smoking worldwide with several associated complica ons, among them anemia is one. Smoking has more effect on the health because it is associated with [1][2][3] higher number of cigare es smoked. The present study indicates marked increase in reac ve oxygen species produc on as reflected by elevated oxidant and decreased [5][6][7][8] an oxidants parameters in anemic smokers. 20-50yrs me is the cri cal me for the health and future development [20][21][22][23][24] and cigare e smoking can influence lifelong health risk.
Smoking is the powerful risk factor for anemia because it contains toxic vigorous molecules like aldehydes, heavy metals, hydrogen cyanide, low molecular weight phenols, nitrosamines, polycyclic aroma c hydrocarbons, metallic [23][24][25][26] ions, hydroxyl radicals. The large volume of oxidants from the smoke phase as well as the tar phase of cigare e smoke produces copious amounts of ROS, which are responsible for increased lipid peroxida on and its harmful consequences. Oxida ve stress occurs when there is imbalance between oxidant agents, insufficient an oxidant defence mechanism. Reac ve oxygen species generated by cigare e smoke plays an important role in eleva on of oxida ve stress, and malondialdehyde, nitric oxide, lipid hydroperoxide end [25][26][27][28][29][30] products is one of the indicators of oxida ve stress. Our study corroborates with that of Ugbebor et al, who found significantly higher hemoglobin concentra on in 25 smokers. The high Hb concentra on in smokers may be since smoking causes excessive produc on of carbon monoxide leading to forma on of carboxy Hb. As Hb has 200 mes more affinity to carbon monoxide than oxygen, there is a stark unavailability of Hb for Oxygen carriage, shi ing the 25,26 Hb-O dissocia on curve to the le . As a compensatory 2 mechanism, the body tries to produce more Hb by 1,3,4 increasing the rate of erythropoiesis. This can a ribute to the higher membrane peroxida on and altered RBC volume coupled with the cellular oxida ve stress, makes RBC more 28,[31][32][33][34] fragile in smokers with anemia. Red blood cells are at increased risk from oxida ve processes for a diverse reason. It is con nually exposed to high oxygen [1][2][3][4][22][23][24] tensions, and Hb is suscep ble to auto oxida on.
RBCs are unable to repair damaged components by resynthesis. Therefore, RBC is completely dependent on the an oxidant enzymes catalase, superoxide dismutase, and glutathione peroxidase through out 120 days of its life span. These enzyme systems build up an efficient defence system and are par cularly involved in the detoxifica on of the cell from [31][32][33] oxygen-free radicals. Erythrocyte glutathione peroxidase (GPx) ac vity plays amore sensi ve and be er indicator of smoking induced oxida ve stress in anemic individuals. GPx detoxifies the H O produced by superoxide dismutase 2 2 (SOD) ac on and converts lipid hydroper oxides to nontoxic alcohols. Hence, GPx is considered to act as a chain-breaking an oxidant. Our findings also supported the results who have studies the influence of gender, age and cigare e smoking on an oxidant enzymes and have reported significantly reduced GPx ac vity in smokers. Significant lower ac vity of GPx and SOD in the erythrocytes of smokers, while catalase (CAT) ac vity remains unchanged was reported by study of 26 Orhan et al. Our study as well as few researchers have clearly exhibited that erythrocyte GPx and SOD ac vi es serves as a potent marker of an oxidant defence in smoking 28,29,34,35 and anemic subjects. During oxida ve stress, the glutathione (sul ydryl) groups protects other cellular structures against free radical induced oxida on and disulphide band forma on. Free radicals present in cigare e increases peroxida on products in smokers' blood and vascular ssues are chiefly responsible for these deleterious effects. Increased exposure to cigare e smoke was shown to decrease plasma an oxidants in vitro and to lower vitamin C levels in the plasma and leucocytes of smokers. Many studies have confirmed higher RBC lipid peroxida on in smokers as related to non-smokers. In addi on, it has been confirmed that glutathione levels decrease in biological ssues due to exposure to cigare e 35 smoke. Anemia and smoking have emerged as major public health problem worldwide. Remarkably, there appears a link between smoking and levels of haemoglobin. Smoking increases erythropoiesis by producing more carboxy haemoglobin and making Hb unavailable for oxygen transport, shi s the Hb dissocia on curve in the le side, resul ng in a reduc on in ability of Hb to deliver oxygen to the ssue. For compensa on of decreased oxygen delivering capacity, higher haemoglobin level is maintained by smokers. Smoking decreases the levels of vitamin C which, in turn, predisposes the individual to iron deficiency anemia due to decrease in the absorp on of iron. Smoking is also known to cause macrocytosis mainly by altering the levels of vitamin B and folic acid. Oxida ve stress, 12 inflamma on, bone marrow depression, and gastri s caused by smoking also result in anemia, induces change in hematological parameters thus acts as a predisposing factor [24][25][26][27][31][32][33][34][35] for mul system diseases. Tobacco smoking is the most important risk factor accompanying with chronic bronchi s, emphysema, oxida ve stress, inflamma on, bone marrow depression, gastri s, exacerbates respiratory disease in children (parental smoking), causes pediatric deaths from low birth weight, short gesta on, respiratory distress syndrome and sudden infant death syndrome (maternal smoking). Numerous health problems; hematological and physiological changes occurs and a achment of peroxidants to RBC 24,27,36 membranes increases RBChemolysis. Our results undoubtedly show an appreciable increase in the malondialdehyde (MDA) levels in the erythrocytes ofanemic smokers undertaken in this study in comparison to non-anemicsmokers. The high ranges of MDA, lipid hydroperoxides and nitric oxide levels in erythrocytes of anemic smokers reveals the consequence of uncontrolled lipid peroxida on and suppressed enzyma c and non- 3 6 -3 9 enzyma c an oxidant defence.
The process of uncontrolled lipid peroxida on in biological system due to toxic chemicals and oxidants presented by cigare e smoke may be accompanied with loss of essen al polyunsaturated fa y acids, forma on of toxic hydroperoxides and other free radicals damaging macro-molecules like lipids, proteins, DNA, etc. Based on the findings of the present study, it may be concluded that smoking is associated with the genera on of free radicals, abnormali es and peroxida on of vital body molecules which implies increased risk not only the anemic smokers but also their passive smokers who might be their family members, colleagues, etc. However, addi onal studies with further ssue damage parameters are needed to evaluate the oxida ve stress in anemic smokers. Further, the study can be carried out increasing the sample size and mul -centric study can be done to highlight the effects of an oxidants and lipid peroxida on on anemic smokers is recommended.

CONCLUSIONS
Our study on smoking anemic individuals showed increased hemoglobin status as compared with their controls. Regardless of high concentra on of hemoglobin, cases showed low status of non-enzyma c an oxidants and high levels of oxidants as compared to controls. The invasion of the RBC membrane by oxidants, cigare e smoking alters the cellular metabolic func on and can ul mately lead to early erythrocyte hemolysis.

LIMITATIONS OF STUDY
Limita ons of the present study included: smoker anemic pa ents was difficult to find, and so were less in number, study group and healthy par cipants were confined to our ins tu on only as per NHRC rule. Anemic female with smoking cases were very few, so male to female ra o could not be maintained.