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Atherogenic changes of low-density lipoprotein susceptibility to oxidation, and antioxidant enzymes in pulmonary tuberculosis
Atherosclerosis 217 (2011) 268–273
Contents lists available at ScienceDirect
Atherosclerosis journal homepage: www.elsevier.com/locate/atherosclerosis
Atherogenic changes of low-density lipoprotein susceptibility to oxidation, and antioxidant enzymes in pulmonary tuberculosis Nariman Nezami a,∗ , Amir Ghorbanihaghjo b , Nadereh Rashtchizadeh c , Hassan Argani d , Arash Tafrishinejad a , Sona Ghorashi e , Babak Hajhosseini f a
Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran Research Center for TB and Pulmonary Diseases, Tabriz University of Medical Sciences, Tabriz, Iran c Biotechnology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran d Department of Nephrology, Shahid Beheshti Medical University, Tehran, Iran e Islamic Azad University, Tabriz Branch, Young Researchers Club, Tabriz, Iran f Center for Special Minimally Invasive and Robotic Surgery, Stanford University Medical Center, Palo Alto, CA, USA b
a r t i c l e
i n f o
Article history: Received 24 December 2010 Received in revised form 2 March 2011 Accepted 18 March 2011 Available online 29 March 2011 Keywords: Tuberculosis Pulmonary Low-density lipoprotein cholesterol Oxidation Glutathione peroxidase Superoxide dismutase Oxidative stress Malondialdehyde
a b s t r a c t Objectives: Tuberculosis remains one of the most common infectious diseases and a leading cause of mortality world wide. There is some evidence for the possible involvement of Mycobacterium tuberculosis in atherosclerosis. We aim to investigate total antioxidant capacity (TAC), red blood cell superoxide dismutase (SOD) activity, whole blood glutathione peroxidase (GPX) activity, low-density lipoprotein (LDL) susceptibility to oxidation, and malondialdehyde (MDA) levels in patients with pulmonary tuberculosis (PTB). Methods: Forty-five males with active PTB (case group) and 45 healthy age-matched males (control group) were enrolled in the study. TAC, SOD and GPX activities were determined by commercial ELISA kits. MDA levels were measured using the thiobarbituric acid method. LDL susceptibility to oxidation was assessed by measuring lag phase duration. Results: TAC, SOD and GPX activities, and lag phase duration in the case group were significantly lower than the control group (p = .002, p = .004, p = .008, and p = .004, respectively; independent), while the MDA levels was higher in case group (p = .024). Conclusions: Our findings suggest a higher susceptibility of LDL to oxidation and higher levels of lipid peroxidation, and therefore, a possible higher risk of atherosclerosis in patients with PTB. © 2011 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Emergence of multidrug-resistant strains of Mycobacterium tuberculosis (Mtb) in association with a growing number of highly susceptible immuno compromised individuals arising from the human immune deficiency virus (HIV) pandemics has resulted in a resurgence of tuberculosis as one of the most prevalent infectious diseases and a leading cause of mortality world wide [1]. During the last decade, some serological [2] and experimental [3] studies have revealed the presumptive role of several infectious agents in the inflammatory mechanism of atherosclerosis. Some studies have suggested the possible association between Mtb and atherosclerosis [4].
∗ Corresponding author at: Clinical Pharmacy Laboratory, Drug Applied Research Center, Tabriz University of Medical Sciences, Pashmineh, Danehsgah Street, Post code: 5165665811, Tabriz, Eastern Azerbaijan, Iran. Tel.: +98 411 3363234; fax: +98 411 3363231. E-mail address: [email protected] (N. Nezami). 0021-9150/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.atherosclerosis.2011.03.025
Tuberculosis causes innate immune cells to produce reactive nitrogen intermediates (RNI) and reactive oxygen species (ROS). The higher levels of free radicals in circulation predispose patients with active tuberculosis including the pulmonary tuberculosis (PTB) to oxidative stress and a wide variety of damages [5]. Oxidative stress resulted from an overproduced free radicals and impaired antioxidant defense mechanisms has been suggested to play role in the pathogenesis of atherosclerotic ischemic heart disease [6,7]. It has been shown that advanced oxidation of protein products through oxidation of amino acid residues acts as a mediator for oxidative stress and monocyte activation. Also, low-density lipoprotein (LDL) oxidative modification plays a pivotal role in initiation and progression of atherosclerosis by stimulating the foam cell formation, eliciting the endothelial dysfunction, and simulating the molecule expression on endothelial cells and monocytes [8–10]. Recent studies have focused on the free radicals and radicalscavenging antioxidants in tuberculosis infection [11]. These studies have reported a decreased total antioxidant capacity (TAC), an increased malondialdehyde (MDA), a decreased paraoxonase
N. Nezami et al. / Atherosclerosis 217 (2011) 268–273
enzyme activity, and an abnormal lipids and lipoproteins in patients with PTB [4,12]. To the best of our knowledge, the present study is the first study to evaluate and report the association between PTB and red blood cell superoxide dismutase (SOD) and whole blood glutathione peroxidase (GPX) activities, and LDL susceptibility to oxidation (as a predicting factor of atherosclerosis). Additionally, we aim to evaluate TAC and MDA levels in patients with active PTB. 2. Methods Forty-five patients with newly diagnosed active PTB, and before starting any anti-tuberculosis therapy, were enrolled as a case group. The control group consisted of 45 healthy individuals. The case and control groups were carefully matched based on their sex, age, smoking status, history of systemic diseases other than TB, and geographical area of residence. All 90 participants were nonsmoker males, and had no history of systemic diseases, except for newly diagnosed TB in the case group. Patients were admitted to the Tabriz Research Center for TB and Pulmonary Diseases during two years period (March 2002–March 2004). In all cases, diagnosis of Mtb was confirmed using chest X-ray and at least one of the following confirmatory tests: positive Ziehl–Neelsen staining of sputum and/or a growth of the organism in Lowenstein–Jensen medium. All healthy controls had negative Tuberculin Skin Test (TST < 5 mm). Further exclusion criteria were defined as coexistent respiratory disorders, cardiovascular diseases, diabetes mellitus, liver disorders, systemic rheumatologic disorders such as rheumatoid arthritis or lupus, malignancy, hyperlipidemia, systolic or diastolic hypertension, renal dysfunction, anemia (defined as hemoglobin >12), antioxidant supplements (e.g., vitamins or statins) intake, and positive antibody against HIV. The study protocol was approved by the Ethic Committee and the Institutional Review Board of Tabriz University of Medical Sciences, assuring its compliance with the Helsinki Declaration. All participants gave informed consent before participating in the study. Venous blood samples were obtained from all participants after 12 h overnight fasting. After low-speed centrifugation, serums were separated immediately. Blood samples were collected in two parts: 3 ml for immediate analysis of lipid profile; and 5 ml for measuring main study outcomes, which has subsequently divided into three microtubes of red blood cell, serum, and plasma and stored at −70 ◦ C. Serum levels of cholesterol (Chol), triglyceride (TG), highdensity lipoprotein cholesterol (HDL-C), and LDL cholesterol (LDL-C) were determined by using commercial reagents in an automated chemical analyzer (Abbott analyzer, Abbott Laboratories, IL, USA) [13]. Serum MDA levels, as indicator of lipid peroxidation, were measured by the thiobarbituric acid (TBA) method [14]. The LDL fraction was isolated from plasma by ultracentrifugation (Beckman Optima TLX, Beckman Coulter Inc., Minnesota, USA) at 15 ◦ C and 100,000 rpm for 4 h with the TLA-100.3 rotor. LDL oxidation (50 g) was determined as the production of conjugated dienes induced by Cu2+ (5 M) every 5 min at 234 nm in one ml phosphate buffer solution at 37 ◦ C under ultraviolet exposure (CECIL 8000, CECIL instrument, Cambridge, UK) and the results were recorded as lag phase. TACs in serum samples were measured using the spectrophotometric method (CECIL 8000, CECIL instrument, Cambridge, UK) as recommended by the RANDOX TAS kit (RANDOX Lab. Ltd., Antrim, UK; lot no. 091668). Whole blood GPX activity was measured with the method described by Paglia and Valentine [15] using the RANDOX Ransel kit (Randox Lab. Ltd., Antrim, UK; lot no. 084547). The enzyme
269
Table 1 Clinical findings in the case group (patients with pulmonary tuberculosis). Clinical findings
No
Percent
Unexplained weight loss Fatigue Fever Night sweats Chills Loss of appetite Coughing >3 weeks Hemoptysis Chest pain/costal pain with breathing or coughing
21 28 37 30 28 35 42 26 31
46% 62.5% 82% 67% 62.5% 78.5% 93.5% 58% 69%
reaction in the tube, which contained NADPH, reduced glutathione (GSH), sodium azide, and glutathione reductase—was initiated by the addition of H2 O2 . The absorbance was read at 340 nm and expressed as U/g Hb. Copper–Zinc (Cu–Zn) SOD activity was determined according to the method described by Sun et al. [16] using the RANDOX Ransod kit (Randox Lab. Ltd., Antrim, UK; lot no. 106974). The principle of this measurement was based on the inhibition of nitroblue tetrazolium (NBT) reduction by the “xanthine–xanthine oxidase system” as a superoxide-generator system. Activity was assessed in the ethanol phase of the red blood cell extract sample after 1.0 ml ethanol/chloroform mixture (5/3, v/v) was added to the same volume of red blood cell extract and was centrifuged. A measurement unit of SOD was expressed as the enzyme amount causing 50% inhibition in NBT reduction rate. Results were expressed as U/g Hb. Statistical analyses were performed by SPSS version 13.0 for windows software package (SPSS Inc., Chicago, USA). Results are presented as mean values with a standard deviation. Statistical difference between groups and correlations were estimated by using independent Sample t-tests and Pearson Correlation respectively. The results were considered statistically significant when the P value was less than 0.05. 3. Results As a result of matching, the mean age of two groups was not significantly different; the mean age of cases and controls was 39.00 ± 10.40 and 37.40 ± 9.10 years, respectively. The average body mass index (BMI) in both groups was within normal ranges; however, it was significantly lower in the case group compared to the control group (22.76 ± 4.21 vs. 24.57 ± 4.88; p = .002). Clinical findings of the case group are shown in Table 1. As demonstrated in Table 2, there were no significant differences between the case and control groups in terms of serum Chol, TG, HDL-C, and LDL-C levels. Serum TAC, and SOD and GPX activities were significantly lower in cases compared to controls. Lag phase was significantly shorter in the case group compared to the control group while the serum MDA levels were significantly higher. Shorter lag phase in the case group indicated a higher susceptibility of LDL to oxidation. The comparison of the two groups with p values is indicated in Table 3. Table 2 Comparison of the case and control groups in terms of lipid profiles. Case group Cholesterol (mmol/l) Triglyceride (mmol/l) HDL-Ca (mmol/l) LDL-Cb (mmol/l) a
4.38 1.45 1.07 2.63
± ± ± ±
0.77 1.01 0.13 0.73
Control group 4.50 1.49 1.09 2.72
± ± ± ±
0.65 0.41 0.15 0.54
p value >0.05 >0.05 >0.05 >0.05
High-density lipoprotein cholesterol (HDL-C) Low-density lipoprotein cholesterol (LDL-C). Independent sample t-test was used for comparing. b
270
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Fig. 1. The correlations between the lag phase and antioxidant enzymes activities, total antioxidant capacity and malondialdehyde level in the case group: (A) a direct linear correlation between lag phase and red blood cells superoxide dismutase activity (P = .011, r = +.376); (B) a direct linear correlation between lag phase and whole blood glutathione peroxidase activity (P < .001, r = +.604); (C) a direct linear correlation between lag phase and total antioxidant capacity (P = .019, r = +.347); (D) a reverse linear correlation between lag phase and malondialdehyde levels (P < .001, r = −.698).
Moreover, in the case group, a significant direct linear correlation between lag phase and SOD and GPX activities and TAC (p = .011, r = +.376; p < .001, r = +.604; p = .019, r = +.347, respectively), and a significant reverse linear correlation between lag phase and MDA (p < .001, r = −.698) were observed (Fig. 1). In the control group, a similar significant direct linear correlations between lag phase and SOD and GPX activities and TAC (p < .001, r = +.679; p < .001, r = +.712; p = .008, r = +.390, respectively), and a significant reverse linear correlation between lag phase and MDA (p < .001, r = −.776) were observed (Fig. 2). Table 3 Comparison of the case and control groups in terms of TAC, SOD, and GPX. Case group a
TAC (mmol/ml) SODb (U/g Hb) GPXc (U/g Hb) Lag phase (min) MDAd (nmol/ml) a b c d
1.33 1143.57 34.38 61.23 3.09
± ± ± ± ±
Control group 0.19 403.03 13.03 10.20 1.11
1.46 1441.23 43.70 69.29 2.63
± ± ± ± ±
0.18 609.28 16.36 14.80 0.75
Total antioxidant capacity Red blood cells superoxide dismutase activity Whole blood glutathione peroxidase activity Malondialdehyde. Independent sample t-test was used for comparing.
p value 0.002 0.008 0.004 0.004 0.024
There was also a significant direct linear correlation between SOD and GPX activities in both case and control groups (p = .001, r = +.483 and p < .001, r = +.938), (Fig. 3A and B). 4. Discussion Mtb is an intracellular pathogen that grows and replicates in host’s macrophages. Following phagocytosis, phagocytes undergo a respiratory burst [17] producing huge amounts of ROS and RNI to essentially destroy the ingested microorganisms [12,17]. The increased levels of several circulating markers of free radical activity were shown in PTB indicating an enhanced oxidative stress in affected individuals [18]. The present study demonstrates a lower TAC as an indicator of an enhanced oxidative stress in PTB patients. TAC reflects interaction between free radicals and antioxidants (e.g., Zn, Vitamin E and C, and antioxidant enzymes such as SOD and GPX) [19]. The lower TAC in PTB could be partially due to depleted levels of nonenzymatic antioxidants such as Zn and Vitamin C and E in these patients [11,12]. Vitamin E and C are the first antioxidants to be depleted upon exposure to both environmental and inflammatory oxidants [12]. Reduced serum levels of Zn in TB patients could be a result of redistribution of Zn outside plasma into other tis-
N. Nezami et al. / Atherosclerosis 217 (2011) 268–273
271
Fig. 2. The correlations between lag phase and antioxidant enzymes activities and malondialdehyde levels in the control group: (A) a direct linear correlation between lag phase and red blood cells superoxide dismutase activity (P < .001, r = +.679); (B) a direct linear correlation between lag phase and whole blood glutathione peroxidase activity (P < .001, r = +.712); (C) a direct linear correlation between lag phase and total antioxidant capacity (P = .008, r = +.390); (D) a reverse linear correlation between lag phase and malondialdehyde levels (P < .001, r = −.776).
Fig. 3. The relationship between two major antioxidant enzymes, red blood cells superoxide dismutase and whole blood glutathione peroxidase activities in the case (A) and control (B) groups. There is a direct linear correlation between red blood cells superoxide dismutase and whole blood glutathione peroxidase activities in the both case and control groups (P = .001, r = +.483; P < .001, r = +.938).
272
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Fig. 4. The three phases of LDL oxidation: lag phase, propagation phase, and decomposition phase.
sues, reduction of the hepatic production of the Zn-carrier protein (␣2-macroglobulin), and/or rising of the metallothionein production [20]. Poor nutritional intake is another important factor that reduces the level of antioxidant micronutrients such as Zn and Vitamin E and C in patients with PTB [21]. There are other factors that are associated with, or can have a similar presentation as poor nutritional intake such as poor socio-economic status, anorexia [11,22], impaired absorption of nutrients, or increased liver metabolism [23]. The poor nutritional status of PTB patients was demonstrated by lower BMI in the present study. Our study shows a reduced TAC in patients with PTB associated with lower activities of SOD. SOD is the primary enzyme involved in the enzymatic antioxidant defense system [24]. Decreased level of SOD activity has been demonstrated in the disseminated stage of miliary and infiltrative tuberculosis [25]. Furthermore, Mtb encodes a type of SOD, which detoxifies exogenous ROS and contributes to the survival of bacteria [26]. Mtb’s self-produced SOD may further interfere with production of host’s SOD by depleting the host’s Zn sources. Another enzyme which displays an antioxidative capacity and protects cells from toxic effects of ROS and RNI is GPX (a GSHdependant enzyme) [27]. GSH may be depleted by acting against ROS, RNI and Mtb, and therefore, GPX activity reduces in PTB. The results of the present study reveal an enhanced LDL susceptibility to oxidation (reduced lag phase) and increased MDA (as one of the end-products of lipid peroxidation), and their association with decreased SOD and GPX activities and TAC in PTB patients. A typical LDL particle has a core, which is surrounded by polar lipids. Fatty acids in LDL particle are protected by antioxidants against free radical attacks. There are three phases in LDL oxidation: lag phase, propagation phase, and decomposition phase (Fig. 4). Those LDL particles that are rich in terms of antioxidants, are more resistant to oxidation and have longer lag phase during the oxidation process, and vice versa [28]. During the lag, little oxidation occurs due to the functional antioxidant defense mechanisms and presence of antioxidant molecules such as a-tocopherol and b-carotene. At the end of the lag phase, antioxidant property of LDL diminishes and polyunsaturated fatty acids (PUFA) in LDL particle are rapidly oxidized into lipid hydroperoxides; MDA is an indicator of this phase, which is known as the propagation phase. During the final phase (decomposition phase), unstable lipid hydroperoxides start to decompose and lipid peroxide concentration decreases [28,29]. It has been previously shown that a decrease in activity of antioxidative enzymes (e.g., SOD and GPX) may result in intensification of free radical lipoperoxidation in the blood and other tissues during atherogenesis [6]. Also, it has been proven that an oxidative stress may damage intracellular and circulating lipids [6]. Studies in the field of atherogenesis have mainly focused on the oxidation of LDL [30]. This modification is considered to play
a pivotal role in the initiation and progression of atherosclerosis, because the oxidized LDL facilitates foam cell formation [8], elicits endothelial dysfunction [9], and stimulates an expression of the adhesion molecules on endothelial cells and monocytes [10]. Based on these previously proved facts and considering the new findings of our study, we believe that a more susceptible LDL in PTB patients could predisposes them to the higher risk of atherosclerosis. The main limitations of this study include: relatively small sample size, using male participants only, and undetermined nutritional status and serum biomarkers including albumin, leptin, ferritin, transferritin, uric acid, and more importantly, ox-LDL. Also, an assessment of ROI and RNI levels might have improved the quality of the present work. In conclusion, low TAC and red blood cell SOD and whole blood GPX activities in PTB patients are indicators of an oxidantantioxidant imbalance in these patients, which is associated with an enhanced susceptibility of LDL to oxidation and higher levels of lipid peroxidation; this condition predisposes PTB patients to a higher risk of atherosclerosis. Therefore, we suggest that antioxidant therapy could be considered in PTB patients; however, further randomized prospective studies with larger population of patients are warranted to assess the exact safety and efficacy of antioxidant therapy in PTB patients.
References [1] Iademarco MF, Castro KG. Epidemiology of tuberculosis. Semin Respir Infect 2003;18:225–40. [2] Kiechl S, Egger G, Mayr M, et al. Chronic infections and the risk of carotid atherosclerosis: prospective results from a large population study. Circulation 2001;103:1064–70. [3] George J, Shoenfeld Y, Gilburd B, Afek A, Shaish A, Harats D. Requisite role for interleukin-4 in the acceleration of fatty streaks induced by heat shock protein 65 or Mycobacterium tuberculosis. Circ Res 2000;86:1203–10. [4] Selek S, Cosar N, Kocyigit A, et al. PON1 activity and total oxidant status in patients with active pulmonary tuberculosis. Clin Biochem 2008;41:140–4. [5] Halliwell B, Aruoma OI. DNA damage by oxygen-derived species Its mechanism and measurement in mammalian systems. FEBS Lett 1991;281:9–19. [6] Lankin V. Free radical lipoperoxidation during atherosclerosis. Free Radic Biol Med 1994;16:8. [7] Halliwell B, Gutteridge JM. Role of free radicals and catalytic metal ions in human disease: an overview. Methods Enzymol 1990;186:1–85. [8] Huh HY, Pearce SF, Yesner LM, Schindler JL, Silverstein RL. Regulated expression of CD36 during monocyte-to-macrophage differentiation: potential role of CD36 in foam cell formation. Blood 1996;87:2020–8. [9] Selwyn AP, Kinlay S, Libby P, Ganz P. Atherogenic lipids, vascular dysfunction, and clinical signs of ischemic heart disease. Circulation 1997;95:5–7. [10] Takei A, Huang Y, Lopes-Virella MF. Expression of adhesion molecules by human endothelial cells exposed to oxidized low density lipoprotein. Influences of degree of oxidation and location of oxidized LDL. Atherosclerosis 2001;154:79–86. [11] Wiid I, Seaman T, Hoal EG, Benade AJ, Van Helden PD. Total antioxidant levels are low during active TB and rise with anti-tuberculosis therapy. IUBMB Life 2004;56:101–6. [12] Lamsal M, Gautam N, Bhatta N, Toora BD, Bhattacharya SK, Baral N. Evaluation of lipid peroxidation product, nitrite and antioxidant levels in newly diagnosed and two months follow-up patients with pulmonary tuberculosis. Southeast Asian J Trop Med Public Health 2007;38:695–703. [13] Ghorbanihaghjo A, Rashtchizadeh N, Rohbaninoubar M, Vatankhah A, Rafi A. Oxidative stress in patients with pulmonary tuberculosis. Saudi Med J 2006;27:1075–7. [14] Yagi K. Assay for blood plasma or serum. Methods Enzymol 1984;105:328–31. [15] Paglia DE, Valentine WN. Studies on the quantitative and qualitative characterization of erythrocyte glutathione peroxidase. J Lab Clin Med 1967;70:158–69. [16] Sun Y, Oberley LW, Li Y. A simple method for clinical assay of superoxide dismutase. Clin Chem 1988;34:497–500. [17] Nathan C, Shiloh MU. Reactive oxygen and nitrogen intermediates in the relationship between mammalian hosts and microbial pathogens. Proc Natl Acad Sci USA 2000;97:8841–8. [18] Jack CI, Jackson MJ, Hind CR. Circulating markers of free radical activity in patients with pulmonary tuberculosis. Tuberc Lung Dis 1994;75:132–7. [19] Evans P, Halliwell B. Micronutrients: oxidant/antioxidant status. Br J Nutr 2001;85(Suppl. 2):S67–74. [20] Filteau SM, Tomkins AM. Micronutrients and tropical infections. Trans R Soc Trop Med Hyg 1994;88(1–3):26. [21] Karyadi E, Schultink W, Nelwan RH, et al. Poor micronutrient status of active pulmonary tuberculosis patients in Indonesia. J Nutr 2000;130:2953–8.
N. Nezami et al. / Atherosclerosis 217 (2011) 268–273 [22] Jetan CA, Jamaiah I, Rohela M, Nissapatorn V.Tuberculosis: an eight-year (2000–2007) retrospective study at the University of Malaya Medical Centre (UMMC), Kuala Lumpur, Malaysia. Southeast Asian J Trop Med Public Health 2010;41:378–85. [23] Koyanagi A, Kuffo D, Gresely L, Shenkin A, Cuevas LE. Relationships between serum concentrations of C-reactive protein and micronutrients, in patients with tuberculosis. Ann Trop Med Parasitol 2004;98:391–9. [24] Fridovich I. Superoxide radical and superoxide dismutases. Annu Rev Biochem 1995;64:97–112. [25] Safarian MD, Karapetian ET. Dynamics of the activity of antioxidant enzymes in the blood of patients with pulmonary tuberculosis. Probl Tuberk 1990:60–1. [26] Spagnolo L, Toro I, D’Orazio M, et al. Unique features of the sodC-encoded superoxide dismutase from Mycobacterium tuberculosis, a fully functional
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copper-containing enzyme lacking zinc in the active site. J Biol Chem 2004;279:33447–55. Seres T, Knickelbein RG, Warshaw JB, Johnston Jr RB. The phagocytosisassociated respiratory burst in human monocytes is associated with increased uptake of glutathione. J Immunol 2000;165:3333–40. Esterbauer H, Wag G, Puhl H. Lipid peroxidation and its role in atherosclerosis. Br Med Bull 1993;49:566–76. Esterbauer H, Striegl G, Puhl H, Rotheneder M. Continuous monitoring of in vitro oxidation of human low density lipoprotein. Free Radic Res Commun 1989;6:67–75. Chisolm GM, Steinberg D. The oxidative modification hypothesis of atherogenesis: an overview. Free Radic Biol Med 2000;28: 1815–26.
Contents lists available at ScienceDirect
Atherosclerosis journal homepage: www.elsevier.com/locate/atherosclerosis
Atherogenic changes of low-density lipoprotein susceptibility to oxidation, and antioxidant enzymes in pulmonary tuberculosis Nariman Nezami a,∗ , Amir Ghorbanihaghjo b , Nadereh Rashtchizadeh c , Hassan Argani d , Arash Tafrishinejad a , Sona Ghorashi e , Babak Hajhosseini f a
Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran Research Center for TB and Pulmonary Diseases, Tabriz University of Medical Sciences, Tabriz, Iran c Biotechnology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran d Department of Nephrology, Shahid Beheshti Medical University, Tehran, Iran e Islamic Azad University, Tabriz Branch, Young Researchers Club, Tabriz, Iran f Center for Special Minimally Invasive and Robotic Surgery, Stanford University Medical Center, Palo Alto, CA, USA b
a r t i c l e
i n f o
Article history: Received 24 December 2010 Received in revised form 2 March 2011 Accepted 18 March 2011 Available online 29 March 2011 Keywords: Tuberculosis Pulmonary Low-density lipoprotein cholesterol Oxidation Glutathione peroxidase Superoxide dismutase Oxidative stress Malondialdehyde
a b s t r a c t Objectives: Tuberculosis remains one of the most common infectious diseases and a leading cause of mortality world wide. There is some evidence for the possible involvement of Mycobacterium tuberculosis in atherosclerosis. We aim to investigate total antioxidant capacity (TAC), red blood cell superoxide dismutase (SOD) activity, whole blood glutathione peroxidase (GPX) activity, low-density lipoprotein (LDL) susceptibility to oxidation, and malondialdehyde (MDA) levels in patients with pulmonary tuberculosis (PTB). Methods: Forty-five males with active PTB (case group) and 45 healthy age-matched males (control group) were enrolled in the study. TAC, SOD and GPX activities were determined by commercial ELISA kits. MDA levels were measured using the thiobarbituric acid method. LDL susceptibility to oxidation was assessed by measuring lag phase duration. Results: TAC, SOD and GPX activities, and lag phase duration in the case group were significantly lower than the control group (p = .002, p = .004, p = .008, and p = .004, respectively; independent), while the MDA levels was higher in case group (p = .024). Conclusions: Our findings suggest a higher susceptibility of LDL to oxidation and higher levels of lipid peroxidation, and therefore, a possible higher risk of atherosclerosis in patients with PTB. © 2011 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Emergence of multidrug-resistant strains of Mycobacterium tuberculosis (Mtb) in association with a growing number of highly susceptible immuno compromised individuals arising from the human immune deficiency virus (HIV) pandemics has resulted in a resurgence of tuberculosis as one of the most prevalent infectious diseases and a leading cause of mortality world wide [1]. During the last decade, some serological [2] and experimental [3] studies have revealed the presumptive role of several infectious agents in the inflammatory mechanism of atherosclerosis. Some studies have suggested the possible association between Mtb and atherosclerosis [4].
∗ Corresponding author at: Clinical Pharmacy Laboratory, Drug Applied Research Center, Tabriz University of Medical Sciences, Pashmineh, Danehsgah Street, Post code: 5165665811, Tabriz, Eastern Azerbaijan, Iran. Tel.: +98 411 3363234; fax: +98 411 3363231. E-mail address: [email protected] (N. Nezami). 0021-9150/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.atherosclerosis.2011.03.025
Tuberculosis causes innate immune cells to produce reactive nitrogen intermediates (RNI) and reactive oxygen species (ROS). The higher levels of free radicals in circulation predispose patients with active tuberculosis including the pulmonary tuberculosis (PTB) to oxidative stress and a wide variety of damages [5]. Oxidative stress resulted from an overproduced free radicals and impaired antioxidant defense mechanisms has been suggested to play role in the pathogenesis of atherosclerotic ischemic heart disease [6,7]. It has been shown that advanced oxidation of protein products through oxidation of amino acid residues acts as a mediator for oxidative stress and monocyte activation. Also, low-density lipoprotein (LDL) oxidative modification plays a pivotal role in initiation and progression of atherosclerosis by stimulating the foam cell formation, eliciting the endothelial dysfunction, and simulating the molecule expression on endothelial cells and monocytes [8–10]. Recent studies have focused on the free radicals and radicalscavenging antioxidants in tuberculosis infection [11]. These studies have reported a decreased total antioxidant capacity (TAC), an increased malondialdehyde (MDA), a decreased paraoxonase
N. Nezami et al. / Atherosclerosis 217 (2011) 268–273
enzyme activity, and an abnormal lipids and lipoproteins in patients with PTB [4,12]. To the best of our knowledge, the present study is the first study to evaluate and report the association between PTB and red blood cell superoxide dismutase (SOD) and whole blood glutathione peroxidase (GPX) activities, and LDL susceptibility to oxidation (as a predicting factor of atherosclerosis). Additionally, we aim to evaluate TAC and MDA levels in patients with active PTB. 2. Methods Forty-five patients with newly diagnosed active PTB, and before starting any anti-tuberculosis therapy, were enrolled as a case group. The control group consisted of 45 healthy individuals. The case and control groups were carefully matched based on their sex, age, smoking status, history of systemic diseases other than TB, and geographical area of residence. All 90 participants were nonsmoker males, and had no history of systemic diseases, except for newly diagnosed TB in the case group. Patients were admitted to the Tabriz Research Center for TB and Pulmonary Diseases during two years period (March 2002–March 2004). In all cases, diagnosis of Mtb was confirmed using chest X-ray and at least one of the following confirmatory tests: positive Ziehl–Neelsen staining of sputum and/or a growth of the organism in Lowenstein–Jensen medium. All healthy controls had negative Tuberculin Skin Test (TST < 5 mm). Further exclusion criteria were defined as coexistent respiratory disorders, cardiovascular diseases, diabetes mellitus, liver disorders, systemic rheumatologic disorders such as rheumatoid arthritis or lupus, malignancy, hyperlipidemia, systolic or diastolic hypertension, renal dysfunction, anemia (defined as hemoglobin >12), antioxidant supplements (e.g., vitamins or statins) intake, and positive antibody against HIV. The study protocol was approved by the Ethic Committee and the Institutional Review Board of Tabriz University of Medical Sciences, assuring its compliance with the Helsinki Declaration. All participants gave informed consent before participating in the study. Venous blood samples were obtained from all participants after 12 h overnight fasting. After low-speed centrifugation, serums were separated immediately. Blood samples were collected in two parts: 3 ml for immediate analysis of lipid profile; and 5 ml for measuring main study outcomes, which has subsequently divided into three microtubes of red blood cell, serum, and plasma and stored at −70 ◦ C. Serum levels of cholesterol (Chol), triglyceride (TG), highdensity lipoprotein cholesterol (HDL-C), and LDL cholesterol (LDL-C) were determined by using commercial reagents in an automated chemical analyzer (Abbott analyzer, Abbott Laboratories, IL, USA) [13]. Serum MDA levels, as indicator of lipid peroxidation, were measured by the thiobarbituric acid (TBA) method [14]. The LDL fraction was isolated from plasma by ultracentrifugation (Beckman Optima TLX, Beckman Coulter Inc., Minnesota, USA) at 15 ◦ C and 100,000 rpm for 4 h with the TLA-100.3 rotor. LDL oxidation (50 g) was determined as the production of conjugated dienes induced by Cu2+ (5 M) every 5 min at 234 nm in one ml phosphate buffer solution at 37 ◦ C under ultraviolet exposure (CECIL 8000, CECIL instrument, Cambridge, UK) and the results were recorded as lag phase. TACs in serum samples were measured using the spectrophotometric method (CECIL 8000, CECIL instrument, Cambridge, UK) as recommended by the RANDOX TAS kit (RANDOX Lab. Ltd., Antrim, UK; lot no. 091668). Whole blood GPX activity was measured with the method described by Paglia and Valentine [15] using the RANDOX Ransel kit (Randox Lab. Ltd., Antrim, UK; lot no. 084547). The enzyme
269
Table 1 Clinical findings in the case group (patients with pulmonary tuberculosis). Clinical findings
No
Percent
Unexplained weight loss Fatigue Fever Night sweats Chills Loss of appetite Coughing >3 weeks Hemoptysis Chest pain/costal pain with breathing or coughing
21 28 37 30 28 35 42 26 31
46% 62.5% 82% 67% 62.5% 78.5% 93.5% 58% 69%
reaction in the tube, which contained NADPH, reduced glutathione (GSH), sodium azide, and glutathione reductase—was initiated by the addition of H2 O2 . The absorbance was read at 340 nm and expressed as U/g Hb. Copper–Zinc (Cu–Zn) SOD activity was determined according to the method described by Sun et al. [16] using the RANDOX Ransod kit (Randox Lab. Ltd., Antrim, UK; lot no. 106974). The principle of this measurement was based on the inhibition of nitroblue tetrazolium (NBT) reduction by the “xanthine–xanthine oxidase system” as a superoxide-generator system. Activity was assessed in the ethanol phase of the red blood cell extract sample after 1.0 ml ethanol/chloroform mixture (5/3, v/v) was added to the same volume of red blood cell extract and was centrifuged. A measurement unit of SOD was expressed as the enzyme amount causing 50% inhibition in NBT reduction rate. Results were expressed as U/g Hb. Statistical analyses were performed by SPSS version 13.0 for windows software package (SPSS Inc., Chicago, USA). Results are presented as mean values with a standard deviation. Statistical difference between groups and correlations were estimated by using independent Sample t-tests and Pearson Correlation respectively. The results were considered statistically significant when the P value was less than 0.05. 3. Results As a result of matching, the mean age of two groups was not significantly different; the mean age of cases and controls was 39.00 ± 10.40 and 37.40 ± 9.10 years, respectively. The average body mass index (BMI) in both groups was within normal ranges; however, it was significantly lower in the case group compared to the control group (22.76 ± 4.21 vs. 24.57 ± 4.88; p = .002). Clinical findings of the case group are shown in Table 1. As demonstrated in Table 2, there were no significant differences between the case and control groups in terms of serum Chol, TG, HDL-C, and LDL-C levels. Serum TAC, and SOD and GPX activities were significantly lower in cases compared to controls. Lag phase was significantly shorter in the case group compared to the control group while the serum MDA levels were significantly higher. Shorter lag phase in the case group indicated a higher susceptibility of LDL to oxidation. The comparison of the two groups with p values is indicated in Table 3. Table 2 Comparison of the case and control groups in terms of lipid profiles. Case group Cholesterol (mmol/l) Triglyceride (mmol/l) HDL-Ca (mmol/l) LDL-Cb (mmol/l) a
4.38 1.45 1.07 2.63
± ± ± ±
0.77 1.01 0.13 0.73
Control group 4.50 1.49 1.09 2.72
± ± ± ±
0.65 0.41 0.15 0.54
p value >0.05 >0.05 >0.05 >0.05
High-density lipoprotein cholesterol (HDL-C) Low-density lipoprotein cholesterol (LDL-C). Independent sample t-test was used for comparing. b
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Fig. 1. The correlations between the lag phase and antioxidant enzymes activities, total antioxidant capacity and malondialdehyde level in the case group: (A) a direct linear correlation between lag phase and red blood cells superoxide dismutase activity (P = .011, r = +.376); (B) a direct linear correlation between lag phase and whole blood glutathione peroxidase activity (P < .001, r = +.604); (C) a direct linear correlation between lag phase and total antioxidant capacity (P = .019, r = +.347); (D) a reverse linear correlation between lag phase and malondialdehyde levels (P < .001, r = −.698).
Moreover, in the case group, a significant direct linear correlation between lag phase and SOD and GPX activities and TAC (p = .011, r = +.376; p < .001, r = +.604; p = .019, r = +.347, respectively), and a significant reverse linear correlation between lag phase and MDA (p < .001, r = −.698) were observed (Fig. 1). In the control group, a similar significant direct linear correlations between lag phase and SOD and GPX activities and TAC (p < .001, r = +.679; p < .001, r = +.712; p = .008, r = +.390, respectively), and a significant reverse linear correlation between lag phase and MDA (p < .001, r = −.776) were observed (Fig. 2). Table 3 Comparison of the case and control groups in terms of TAC, SOD, and GPX. Case group a
TAC (mmol/ml) SODb (U/g Hb) GPXc (U/g Hb) Lag phase (min) MDAd (nmol/ml) a b c d
1.33 1143.57 34.38 61.23 3.09
± ± ± ± ±
Control group 0.19 403.03 13.03 10.20 1.11
1.46 1441.23 43.70 69.29 2.63
± ± ± ± ±
0.18 609.28 16.36 14.80 0.75
Total antioxidant capacity Red blood cells superoxide dismutase activity Whole blood glutathione peroxidase activity Malondialdehyde. Independent sample t-test was used for comparing.
p value 0.002 0.008 0.004 0.004 0.024
There was also a significant direct linear correlation between SOD and GPX activities in both case and control groups (p = .001, r = +.483 and p < .001, r = +.938), (Fig. 3A and B). 4. Discussion Mtb is an intracellular pathogen that grows and replicates in host’s macrophages. Following phagocytosis, phagocytes undergo a respiratory burst [17] producing huge amounts of ROS and RNI to essentially destroy the ingested microorganisms [12,17]. The increased levels of several circulating markers of free radical activity were shown in PTB indicating an enhanced oxidative stress in affected individuals [18]. The present study demonstrates a lower TAC as an indicator of an enhanced oxidative stress in PTB patients. TAC reflects interaction between free radicals and antioxidants (e.g., Zn, Vitamin E and C, and antioxidant enzymes such as SOD and GPX) [19]. The lower TAC in PTB could be partially due to depleted levels of nonenzymatic antioxidants such as Zn and Vitamin C and E in these patients [11,12]. Vitamin E and C are the first antioxidants to be depleted upon exposure to both environmental and inflammatory oxidants [12]. Reduced serum levels of Zn in TB patients could be a result of redistribution of Zn outside plasma into other tis-
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Fig. 2. The correlations between lag phase and antioxidant enzymes activities and malondialdehyde levels in the control group: (A) a direct linear correlation between lag phase and red blood cells superoxide dismutase activity (P < .001, r = +.679); (B) a direct linear correlation between lag phase and whole blood glutathione peroxidase activity (P < .001, r = +.712); (C) a direct linear correlation between lag phase and total antioxidant capacity (P = .008, r = +.390); (D) a reverse linear correlation between lag phase and malondialdehyde levels (P < .001, r = −.776).
Fig. 3. The relationship between two major antioxidant enzymes, red blood cells superoxide dismutase and whole blood glutathione peroxidase activities in the case (A) and control (B) groups. There is a direct linear correlation between red blood cells superoxide dismutase and whole blood glutathione peroxidase activities in the both case and control groups (P = .001, r = +.483; P < .001, r = +.938).
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Fig. 4. The three phases of LDL oxidation: lag phase, propagation phase, and decomposition phase.
sues, reduction of the hepatic production of the Zn-carrier protein (␣2-macroglobulin), and/or rising of the metallothionein production [20]. Poor nutritional intake is another important factor that reduces the level of antioxidant micronutrients such as Zn and Vitamin E and C in patients with PTB [21]. There are other factors that are associated with, or can have a similar presentation as poor nutritional intake such as poor socio-economic status, anorexia [11,22], impaired absorption of nutrients, or increased liver metabolism [23]. The poor nutritional status of PTB patients was demonstrated by lower BMI in the present study. Our study shows a reduced TAC in patients with PTB associated with lower activities of SOD. SOD is the primary enzyme involved in the enzymatic antioxidant defense system [24]. Decreased level of SOD activity has been demonstrated in the disseminated stage of miliary and infiltrative tuberculosis [25]. Furthermore, Mtb encodes a type of SOD, which detoxifies exogenous ROS and contributes to the survival of bacteria [26]. Mtb’s self-produced SOD may further interfere with production of host’s SOD by depleting the host’s Zn sources. Another enzyme which displays an antioxidative capacity and protects cells from toxic effects of ROS and RNI is GPX (a GSHdependant enzyme) [27]. GSH may be depleted by acting against ROS, RNI and Mtb, and therefore, GPX activity reduces in PTB. The results of the present study reveal an enhanced LDL susceptibility to oxidation (reduced lag phase) and increased MDA (as one of the end-products of lipid peroxidation), and their association with decreased SOD and GPX activities and TAC in PTB patients. A typical LDL particle has a core, which is surrounded by polar lipids. Fatty acids in LDL particle are protected by antioxidants against free radical attacks. There are three phases in LDL oxidation: lag phase, propagation phase, and decomposition phase (Fig. 4). Those LDL particles that are rich in terms of antioxidants, are more resistant to oxidation and have longer lag phase during the oxidation process, and vice versa [28]. During the lag, little oxidation occurs due to the functional antioxidant defense mechanisms and presence of antioxidant molecules such as a-tocopherol and b-carotene. At the end of the lag phase, antioxidant property of LDL diminishes and polyunsaturated fatty acids (PUFA) in LDL particle are rapidly oxidized into lipid hydroperoxides; MDA is an indicator of this phase, which is known as the propagation phase. During the final phase (decomposition phase), unstable lipid hydroperoxides start to decompose and lipid peroxide concentration decreases [28,29]. It has been previously shown that a decrease in activity of antioxidative enzymes (e.g., SOD and GPX) may result in intensification of free radical lipoperoxidation in the blood and other tissues during atherogenesis [6]. Also, it has been proven that an oxidative stress may damage intracellular and circulating lipids [6]. Studies in the field of atherogenesis have mainly focused on the oxidation of LDL [30]. This modification is considered to play
a pivotal role in the initiation and progression of atherosclerosis, because the oxidized LDL facilitates foam cell formation [8], elicits endothelial dysfunction [9], and stimulates an expression of the adhesion molecules on endothelial cells and monocytes [10]. Based on these previously proved facts and considering the new findings of our study, we believe that a more susceptible LDL in PTB patients could predisposes them to the higher risk of atherosclerosis. The main limitations of this study include: relatively small sample size, using male participants only, and undetermined nutritional status and serum biomarkers including albumin, leptin, ferritin, transferritin, uric acid, and more importantly, ox-LDL. Also, an assessment of ROI and RNI levels might have improved the quality of the present work. In conclusion, low TAC and red blood cell SOD and whole blood GPX activities in PTB patients are indicators of an oxidantantioxidant imbalance in these patients, which is associated with an enhanced susceptibility of LDL to oxidation and higher levels of lipid peroxidation; this condition predisposes PTB patients to a higher risk of atherosclerosis. Therefore, we suggest that antioxidant therapy could be considered in PTB patients; however, further randomized prospective studies with larger population of patients are warranted to assess the exact safety and efficacy of antioxidant therapy in PTB patients.
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