Indmedica Home | About Indmedica | Medical Jobs | Advertise On Indmedica
Search Indmedica Web
Indmedica - India's premier medical portal

Biomedical Research

Oxidative imbalance in smokers with and without hypertension

Author(s): Meera K.S.

Vol. 22, No. 3 (2011-07 - 2011-09)

Meera K.S.

Department of Biochemistry, M S Ramaiah Medical College, Bangalore – 560054, Karnataka, India.

Abstract

Cigarette smoking is a leading risk factor for coronary and vascular disease. Smokers are exposed to increased load of reactive oxidants which can promote peroxidation of lipids and lipoprotein resulting in increased arterial pressure. Therefore we intended to determine the ROS mediated endothelial dysfunction by assessing the extent of lipid peroxidation and to study the possible role of erythrocyte catalase activity and serum total bilirubin in smokers with and without hypertension. The study group included essential hypertensive smokers (n=22) and nonsmokers (n=22) and normotensive smokers (n=22) as cases and nonsmokers (n=22) as controls. Fasting blood sample were collected from both cases and controls. Eryth-rocyte catalase activity, serum total bilirubin, serum malondialdehyde (MDA) level were measured. ANOVA and Pearson’s correlation were used for statistical analysis. The present study showed significantly decreased erythrocyte catalase activity and serum total bilirubin was on the higher side of the physiological range. There was a significant rise in MDA levels in smokers with and without hypertension as compared to controls. The present study showed progressive increase in oxidative stress in smokers and hypertensives.

Key Words: Smokers, Catalase, MDA, Total bilirubin
Accepted March 07 2011

Introduction

Cigarette smoking is an established leading risk factor for coronary and vascular disease. Smoking adversely affects the prognosis in patients with previous myocardial infarc-tion or angina pectoris by enhancing the effects of hyper-tension, hypercholesterolemia and metabolic perturbation of insulin resistance. The acute effects of smoking include transient increase in heart rate, blood pressure, decrease in serum high density lipoprotein level, impaired glucose tolerance and altered insulin sensitivity. [1]

Cigarette smoke is a complex mixture of toxic agents and included among these are free radicals, redox cycling agents, cytotoxic aldehydes and other carcinogens like polycyclic aromatic hydrocarbons, benzpyrenes and ni-trosamines. Each puff of cigarette smoke contains more than 1014 low molecular weight free radicals which can directly or indirectly initiate and propagate lipid peroxida-tion [2]. Cigarette smoking and hypertension increase the predisposition for the development of atherosclerosis and its clinical complications. A dysfunctional endothelium is due to reduced nitric oxide [NO] availability and in-creased production of reactive oxygen species (ROS) like superoxide ion (O2−) and hydrogen peroxide (H2O2).They are considered an early indicator of atherothrombotic damage and of cardiovascular events [3]. Increased load of reactive oxidants promotes peroxi-dation of lipids and lipoproteins. Several defense mecha-nism exists which can reduce the damages brought about by the ROS. These defense mechanisms are crucial to reduce the detrimental effect of ROS and preserving the cellular function at the optimum. ROS are unstable and have very short life span, therefore by products of lipid peroxidation or depletion of endogenous antioxidants have been used as a marker of free radical generation. Serum malondialdehyde (MDA) a three carbon com-pound reflects both autoxidation and oxygen mediated peroxidation of poly unsaturated fatty acids in particular. It reflects the oxidative status of the biological system. MDA causes damage to low density lipoproteins (LDL) which in turn can be taken up by macrophages via scav-enger receptors and forms foam cells. Due to increased production of ROS and increased oxidative stress, lipid peroxidation products are found to be elevated in smokers [4]. Catalase (E.C.1.11.1.6) is a major antioxidant defense component directly catalyzing the decomposition of H2O2 toH20 and sharing the function with glutathione peroxi-dase. Increased erythrocyte catalase activity is found in smokers and hypertensives. Bilirubin, the downstream product of heme degradation has a very effective antioxidant and anti inflammatory properties. The antioxidant properties of bilirubin are responsible for reduced risk for cardiovascular disease in individuals with slightly in-creased serum bilirubin [5].

Therefore we intended to determine the ROS mediated endothelial dysfunction by assessing the extent of lipid peroxidation and study the possible role of erythrocyte catalase activity and serum total bilirubin in smokers with and without hypertension.

Materials and Methods

The study population consisted of 88 males who were grouped as follows:

Group 1: 22 normotensive nonsmokers as controls
Group 2: 22 normotensive smokers
Group 3: 22 nonsmokers with Essential hypertension
Group 4: 22 smokers with Essential hypertension

These are the individuals who visited M S Ramaiah medi-cal college teaching hospital, Bangalore. The study was approved by institutional Ethical board. The clinical his-tory of the study population was taken including details of personal habits like smoking and alcohol intake. The number of cigarette smoked per day varied in smokers but in all cases were above 8 cigarettes per day and the re-ported length of smoking was greater than 12 months. Newly diagnosed essential hypertensive patients recruited for the study had diastolic pressure greater than 90 mmHg and/or systolic pressure greater than 140 mmHg. Secon-dary form of hypertension was excluded by routine diag-nostic procedures. The study groups were not on any drug regimen like anti-hypertensives, lipid lowering drugs, antibiotics, NSAID group of drugs, multivitamins and antioxidant supplementation. Patients with diagnosed dia-betes mellitus, cardio-vascular disease, impaired renal function, gastrointestinal, liver diseases and other chronic diseases were excluded from the study.

Blood samples were collected after overnight fasting in appropriate vacutainers. Hemoglobin was determined immediately after collecting whole blood sample by Drabkins method. The serum and the erythrocyte sedi-ments were separated and various parameters were ana-lysed. Erythrocyte catalase activity was assayed in hemo-lysate by the UV-method described by Aebi[6].The cata-lase activity was expressed as k(rate constant of first order reaction, absolute activity) and k/gmHb(specific activ-ity).Serum MDA,TBA-reactive substance was estimated using 0.67% TBA and 40% TCA. The pink color adduct was measured spectrophotometrically at 530 nm. The MDA content was calculated using the molar extinction coefficient coefficient 1.56×1057. Serum total bilirubin

Statistical Analysis

The results are expressed as Mean±S.D. ANOVA and Post hoc tukey test were used for statistical analysis. Pearson’s correlation coefficient was calculated and for all determinants p<0.05 was considered significant. All statistical analysis was performed using SPSS 15.0 ver-sion software.

Results

The age distribution of the various subjects studied are shown in Table1,with normotensive nonsmokers (Group I ), normotensive smokers(Group II), Essential hyperten-sive nonsmokers(Group III) and Essential hypertensive smokers(Group IV).There was not much difference in the mean age between the various study groups.

The mean systolic and diastolic blood pressures in nor-motensive smokers were higher than normotensive non smokers. Similarly, Essential hypertensive smokers had higher systolic and diastolic blood pressure than Essential hypertensive nonsmokers as shown in Table 2. Erythro-cyte catalase activity was significantly reduced and serum MDA level was significantly raised in smokers and non smokers with hypertension. Normotensive smokers had increased MDA levels and reduced catalase activity as compared to normotensive non smokers. As compared to group I total bilirubin gradually increased and hemoglo-bin gradually decreased in all other groups. (Table 2).Pair wise comparison shows a significant difference in dia-stolic pressure between Group I and Group II. However, there is a significant difference in both systolic and dia-stolic pressure between Group I and Group III, Group I and Group IV, Group II and Group III and between Group II and Group IV. There was significant reduction in eryth-rocyte catalase activity and significant increase in S.MDA and total bilirubin in hypertensive smokers as compared to normotensive smokers and non smokers. There was increase in S.MDA and total bilirubin in hypertensive smokers as compared to nonsmoker hypertensive. Hemo-globin was reduced in cases as compared to controls (Ta-ble 3).

Significant correlation was found between MDA and sys-tolic blood pressure in hypertensive smokers(r=0.454, p<0.05) (Fig 1).There was positive correlation between catalase activity and systolic blood pressure in hyperten-sive smokers (r=0.474, p<0.05)(Fig 2). Similarly there is inverse correlation between total bilirubin and diastolic blood pressure in normotensive smokers (r=-0.473, p<0.05) (Fig 3) and positive correlation between MDA and diastolic blood pressure(r=0.514.p<0.05). There is negative correlation between serum MDA and erythrocyte catalase activity in normotensive smokers (r= -0.543, p<0.05) (Fig 4).

Table 1. Age distribution of subjects studied

Age in years Group I Group II Group III Group IV
No % No % No % No %
21-30 0 0.0 2 9.1 0 0.0 0 0.0
31-40 11 50.0 10 54.5 1 4.5 3 13.6
41-50 7 31.8 8 36.4 12 54.5 9 40.9
51-60 4 18.2 2 9.1 8 36.4 8 36.4
61-65 0 0.0 0 0.0 1 4.5 2 9.1
Total 22 100.0 22 100.0 22 100.0 22 100.0
Mean ± SD 42.55±7.68 40.05±7.47 48.68±6.94 50.36±7.89

Table 2. Mean and SD of SBP, DBP , Catalase , MDA , T.Bilirubin and Hb

Variables Group I Group II Group III Group IV Significance
SBP (mm Hg) 122.18±6.47 126.45±5.38 153.14±8.91 157.82±13.32 F=88.694; P<0.001٭٭
DBP (mm Hg) 77.18±6.64 82.09±3.68 93.82±3.59 96.45±6.29 F=68.153; P<0.001٭٭
Catalase (k/gm Hb) 128.67±21.56 119.99±20.5 87.03±21.3 81.12±19.25 F=28.753; P<0.001٭٭
MDA (nmoles/dl) 92.23±20.80 106.56±25.23 238.47±41.56 268.4±45.58 F=146.117; P<0.001٭٭
T.Bilirubin (μmoles/dl) 8.84±1.92 9.14±1.69 10.33±1.8 11.76±2.09 F=10.971; P<0.001٭٭
Hb (gm %) 11.85±1.2 11.22±1.18 10.81±1.34 10.54±1.43 F=4.310; P<0.001٭٭

Table 3: Pairwise comparison of SBP , DBP , Catalase, MDA , T.Bilirubin and Hb between groups

Variables I-II I-III I-IV II-III II-IV III-IV
SBP (mm Hg) 0.404 <0.001٭٭ <0.001٭٭ <0.001٭٭ <0.001٭٭ 0.322
DBP (mm Hg) 0.014٭ <0.001٭٭ <0.001٭٭ <0.001٭٭ <0.001٭٭ 0.348
Catalase (k/gmHb) 0.507 <0.001٭٭ <0.001٭٭ <0.001٭٭ <0.001٭٭ 0.779
MDA (nmoles/dl) 0.527 <0.001٭٭ <0.001٭٭ <0.001٭٭ <0.001٭٭ 0.028٭
T.Bilirubin (μmoles/dl) 0.952 0.050٭ <0.001٭٭ 0.163 <0.001٭٭ 0.063+
Hb (gm%) 0.365 0.043٭ 0.006٭٭ 0.720 0.310 0.901

Numbers are P values obtained Post-hoc Tukey test

Fig 1

Figure 1.: Correlation between systolic blood pressure and S. MDA in Group IV

Fig 2

Figure 2. Correlation between systolic blood pressure and erythrocyte catalase activity in Group III.

Fig 3

Figure 3. Correlation between diastolic blood pressure and S. Total bilirubin in Group II.

Fig 4

Figure. 4. Correlation between erythrocyte catalase ac-tivity and S. MDA in Group II

Discussion

Cigarette smoking is associated with increased production of ROS which in turn can initiate lipid peroxidation and proceed as self perpetuating chain reactions. An increase in ROS generation especially reduces the bioavailability of NO by inactivating it and consequently increasing the vascular tone and blood pressure [8]. The other mecha-nism by which smoking can contribute to the elevation in arterial pressure includes α 1-adrenoreceptor mediated vasoconstricition, vasopressin release and direct toxic effect on endothelial cells by reducing prostacyclin pro-duction and increasing leucocytes adhesion to the endo-thelial cells ; which can predispose to the development of hypertension over a period of time [9,10].

Erythrocyte catalase activity is significantly reduced in both hypertensives and smokers. But the reduction is more marked in hypertensive smokers (Table 2). An in-crease in ROS generation especially O2− by endothelial and vascular smooth muscle cells results in oxidant dam-age of the tissues. Erythrocytes in blood act as a sink for H2O2 and O2− generated in tissue. CAT also protects erythrocytes against H2O2 which is generated by the dis-mutation of O2− and by auto oxidation of hemoglobin. CAT has higher Km for H2O2 and becomes more impor-tant at higher concentration of H2O2 than glutathione per-oxidase during increased oxidative stress [11].The possi-ble mechanism for decrease in CAT activity may be due to inhibition of the enzyme by O2− by generating ferroxy catalase, which does not decompose H2O2 rapidly thereby resulting in further damage to cells. The resulting increase in H2O2 concentration can inactivate superoxide dismu-tase leading to higher O2−.levels. The increase of O2− increases arterial pressure by inactivating NO and produc-ing peroxy nitrite, a stronger and relatively long lived oxidant which is cytotoxic and can initiate lipid peroxida-tion without the requirement of transition metals[8]. The reduced capacity of CAT and superoxide dismutase to neutralize ROS results in increased generation of hydroxyl radical, which initiates the peroxidation of polyun-saturated fatty acids. Cigarette smoke contains peroxyl radical and acetaldehyde. Increased concentration of per-oxyl radical induces lipid peroxidation and acetaldehyde is found to deplete the cells of reduced glutathione mak-ing the cells more vulnerable to peroxidative damage [13].

Smoking increases endothelial angiotensin II production. Angiotensin II activates NADH/NADPH oxidase and pro-tein kinase C activity in vascular cells thereby increasing O2− production and decreasing NO availability which also may attribute to endothelial dysfunction[8]. MDA, a marker of oxidative stress due to increased per-oxidation of lipids is significantly increased in both smokers and hypertensives in the present study. However, it is increased three-fold in smokers with hypertensives as compared to normotensives. The increased production of MDA may be due to increased formation of reactive oxi-dants by smoking. Peroxidation of lipids brings about changes in the molecular structure of the lipids and these changes becomes more marked when the damaged lipids are the constituents of the biological membrane disrupting the cohesive lipid layer arrangement and structural or-ganization. The lipid peroxides in general, enhances pros-taglandin synthesis which is another source of free radical and associated decrease in NO production are well known risk factor for atherosclerotic complication [15]. Increased serum MDA in hypertensives suggests an association be-tween increased oxidative stress. The slight increase of MDA level in normotensive smokers as compared to non-smokers indicates the non specific nature of MDA as a marker of any disease.

The significant positive correlation between both diastolic and systolic blood pressure with MDA in hypertensive smokers suggests the role of lipid peroxidation in causing endothelial dysfunction and increasing arterial pressure. The inverse correlation between MDA and erythrocyte catalase activity can be explained by the fact that as cata-lase activity is reduced, lipid peroxidation is favored and consequently serum MDA level raises. The increased MDA level further inactivates antioxidant enzymes like catalase and superoxide dismutase under oxidative stress.

Bilirubin by virtue of its radical scavenging property re-duces the risk of cardiovascular disease. Its antioxidant activity and cardio protective potential are attributable to both unconjugated and conjugated bilirubin[16]. Bilirubin acts as co-antioxidant with α-tocopherol and inhibits oxi-dation of LDL. Smoking reduces the antioxidant potential of bilirubin by oxidant damage and hence erasing some of the beneficial effect of bilirubin [17]. However, in the present study high bilirubin levels (in the upper limit of the reference range) as found in smokers and hyperten-sives can be reasoned out as due to increased induction of heme oxygenase (HO-1). HO-1 can be induced by heme cytokines, oxidative stress and others [16,18]. Heme primes the endothelial for oxidant damage by the release of catalytically active iron into the aqueous environment of the tissue. Iron, a transition metal can generate O2− and other ROS. Increased expression of HO-1 in endothe-lial and smooth muscle cells generate bilirubin which renders protection against oxidants. The induction of HO-1 is even more beneficial when catalase and superoxide dismutase activity is compromised or glutathione levels are reduced. The increased level of bilirubin within physiological limits can reduce arterial pressure by scav-enging O2− in the vasculature, inhibiting NADPH oxi-dase and protein kinase C activity [19]. The other possible mechanism by which HO-1 induction can reduce arterial pressure is by decreasing vasculature resistance by the HO-driven carbon monoxide. Ju chin et.al has reported a negative correlation between bilirubin and the incidence of hypertension [19]. In the present study a negative cor-relation was found between bilirubin and diastolic pres-sure in smokers. In the other groups significant correla-tion was not found between bilirubin and arterial pressure which may be reasoned out, as some of the other antioxi-dants may have been used up which may have sparing action on lipid soluble antioxidants.

Hemoglobin is found to be reduced in both smokers and hypertensives as compared to normotensives (Ta-ble2).This can be explained as either due to poor diet or smoking or both inspite of high bilirubin level, the anti-oxidant role of bilirubin in the present study appears to be ineffective as shown by elevated serum MDA level. The elevation of MDA levels may be due to increased ROS in smokers and hypertensives. The decrease in erythrocyte catalase activity leads to ineffective breakdown of H2O2 which can further increase lipid peroxidation and impair endothelial dysfunction thereby initiating the process of atherogenesis.

In conclusion, the present study indicates marked increase in ROS production as reflected by elevated MDA levels with concomitant decrease in erythrocyte catalase activity in smokers and hypertensives. Increase in total bilirubin level within physiological limits is not able provide de-fense to cellular damage by ROS in smokers and hyper-tensives. Smoking further compounds the endothelial dys-function and oxidative stress associated with hyperten-sion. However, additional studies using larger sample size with the inclusion of various other antioxidants which contribute to the radical trapping antioxidant parameter (TRAP) and other clinically relevant endothelium dys-function markers are needed to substantiate these studies. Oxidative imbalance in smokers with and without Hypertension

References

  1. Frati AC, Iniestra F, Arizo CR. Acute effects of ciga-rette smoking on glucose tolerance and other cardio-vascular risk factors.Diabetes care 1996; 19: 112-118.
  2. Chruch DF, Pryor WA. Free radical chemistry of ciga-rette smoke and its toxicological implications. En-vioron Health Perspect 1985; 64:111-126.
  3. de la Sierra A, Larrouse M. Endothelial dysfunction is associated with increased level of biomarkers in essen-tial hypertension. Journal of Human Hypertension 2010; 24: 373-379.
  4. Volkonen M, Kuusi T. Passive smoking induces atherogenic changes in low density lipoprotein. Circu-lation 1998; 97: 2012-2016.
  5. Vitek L, Schwertner HA. The heme catabolic pathway and its protective effect on oxidative stress mediated disease. Adv Clin Chem 2007; 43:1-57.
  6. Aebi HE. Catalase. Bergmeyers Methods of enzymatic analysis. Weinhein. Verlang Chemic.1983. 273-278.
  7. Sadasivudu B. Serum malondialdehyde, insulin, glu-cose and lipid profile in hypertension. Med Sci Res1997; 25: 631-633.
  8. Reckethoff JF, Zhang H, Srivastava K, Roberts II J, Morrow JD, Romero CJ. A subpressor dose of angio-tensin II increases plasma F (2)-isoprostanes in rats. Hypertension 2000; 35: 476-479.
  9. Campisi R, Czernin J, Schoder H, Sayre JW, Ma-rengo FD, Phelps ME.Effect of long term smoking on myocardial blood flow, coronary vasomotion and vasodilator capacity. Circulation 1998; 98:119-125.
  10. Narkiewicz K, van de Borne JHP, Hausberg M, Cooley RL, Winneford MD, Davison DE. Cigarette smoking increases sympathetic outflow in humans. Circulation 1998; 98: 528-534.
  11. Mulholland CW, Elwood PC, Davis A, Thurnham DI, Kennedy O, Coulter J. Antioxidant enzymes, inflam-matory indices and lifestyle factors in older man-a co-hort analysis. QJM 1999; 92(10): 579-585.
  12. Sozmen B. Effect of N-dicyclopropylmethyl amino-2-oxazoline(S-3341) an antioxidant status and nitric ox-ide in hypertensive patients. Curr Med Res Opinion 1998; 14: 89-96.
  13. Nadiger HA, Mathew CA, Sadasivudu B. Serum malondialdehyde (TBA reactive substance) level in cigarette smokers. Atherosclerosis1987; 64: 71-73.
  14. William B. Angiotensin II and the pathophysiology of CV remodeling. Am J Cardiology 2001; 87(Supp):10C-17C.
  15. Armas –Padilla MC, Armas-Hernandez MJ, Sara-Canache B, Cammarata R, Pancheo B, Guerreo J, et al. Nitric oxide and malondialdehyde in human hyperten-sion. Am J Ther 2007; 14(2):172-176.
  16. Mayer M. Association of serum bilirubin concentration with risk of coronary artery disease. Clin Chem 2000; 46: 1723-1727.
  17. Neuzil J, Stocker R. Free and albumin bound Bilirubin are efficient co-antioxidant for alpha-tocopherol inhib-iting plasma and low density lipoprotein lipid peroxida-tion. J Biol Chemi 1994; 269(24): 16712-16719.
  18. Zeng B, Lin G, Ren X, Zhang Y, Chen H. Over expres-sion of HO-1 in mesenchymal stem cells promotes an-giogenesis and improves myocardial function in in-fected myocardium. Biomed Sci 2010; 17: 80-85.
  19. Jun Chin H, Song YR. The bilirubin level is negatively correlated with the incidence of hypertension in Nor-motensive Korean population. J Korean Med Science 2009; 24 (Suppl 1): 850-856.
Access free medical resources from Wiley-Blackwell now!

About Indmedica - Conditions of Usage - Advertise On Indmedica - Contact Us

Copyright © 2005 Indmedica