DOI: 10.5958/j.2319-5886.2.2.

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International Journal of Medical Research & Health Sciences

www.ijmrhs.com
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Volume 2 Issue 2 April - June

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Coden: IJMRHS

Copyright @2013

ISSN: 2319-5886
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Received: 12 Feb 2013 Research article

Revised: 10 Mar 2013

Accepted: 13 Mar 2013

ROLE OF -1-ANTITRYPSIN AND OXIDATIVE STRESS IN EMPHYSEMA *Anita M Raut1, Suryakar AN2, Smita D.Mhaisekar3, Dilip Mhaisekar4 Dept of Biochemistry Dr. Vikhe Patil Institute of Medical Sciences, Ahmednagar, Maharashtra. Prof. & Registrar , MUHS, Nashik. 3Mhaisekar Hospital , Nanded. 4 Prof & Head, Resp. Med. Dr. Shankarrao Chavan Govt. Medical College , Nanded.
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*Correspondence author e- mail: anitaraut009@gmail.com

ABSTRACT

In the lungs, deficiency of alpha-1antitrypsin can lead to the development of emphysema. Emphysema involves destruction of the lungs air sacs (alveoli), where oxygen from the air is exchanged for carbon dioxide in the blood. Alpha 1-antitrypsin is a protein produced by the liver and that circulates in the blood. Among other functions, the protein protects the lungs so they can function properly. In people with the disorder, the liver produces too little or no alpha 1 -antitrypsin. Without enough alpha-1 antitrypsin, neutrophil elastase is free to destroy air sac tissue. 60 emphysema patients were included in the study. 100 healthy non-smokers were served as controls. Their baseline clinical examination, -1antitrypsin (AAT), malondialdehyde (MDA) and vitamin E were measured. The mean levels of - 1 antitrypsin and vitamin E in the patients at baseline were lower (P< 0.001) than the controls. The malondialdehyde were high (P<0.001) in the emphysema patients compared to controls. We found decreased levels of 1- antitrypsin and vitamin E and increased levels of malondialdehyde (MDA). Thus the present study confirmed the existence of oxidative stress and altered levels of alpha 1antitrypsin in emphysema patients. Keywords: Emphysema, -1-antitrypsin, Vitamin E, Oxidative stress, ROS, Malondialdehyde
INTRODUCTION

In the lungs, alpha-1 antitrypsin deficiency produces emphysema, a chronic progressive lung disease. The disease often becomes noticeable between the ages groups of 30 to 40 years in smokers and 10 to 15 years later in non-smokers. Alpha 1-antitrypsin deficiency damages the tiny air sacs (alveoli) in the lungs. When the alveoli are damaged, the lungs aren't able to expand and contract well enough for the person to breathe

normally. Patients may feel short of breath, and they may cough or wheeze. As the lungs deteriorate, many patients develop lung diseases, such as emphysema, or chronic bronchitis. The severe, early onset of emphysema that occurs in patients with circulating deficiency of alpha 1antitrypsin. - 1 antitrypsin attests to the importance of the protease inhibitor in maintaining lung parenchymal integrity. It has
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led to the powerful concept of protease:antiprotease balance being crucial to alveolar homeostasis.1 Body tissues and blood normally contain powerful enzymes known as proteases that can attack foreign substances within the body, which may be harmful, such as tobacco smoke. However, these protease enzymes must be carefully regulated because they could attack and damage normal tissues rather than the intended target resulting in local tissue damage. Blood and tissues have a protease inhibitor that binds the enzyme to prevent unrestricted and potentially harmful protease activity. The commonest protease inhibitor in the blood is alpha-1 antitrypsin and its role is to protect the tissues from protease attack. These proteins play an important role in controlling inflammation, coagulation and repair mechanisms in the body. 2 Oxidative stress increases oxidant generation, which cannot be neutralized with antioxidant defence mechanisms. Lipids, proteins and deoxyribonucleic acid are components of the cell that are more sensitive to oxidative damage. Oxygen radicals can modify amino acid side chains, form protein aggregates, cleave peptide bonds, and make proteins more susceptible to proteolytic degradation. It has been shown that neutrophils have a principal effect or role in pulmonary tissue damage. Neutrophil elastase can damage air spaces by degrading elastin, and a variety of extracellular membrane proteins, proteoglycans, and glycoproteins. Neutrophil elastase can also stimulate inflammation by increasing interleukin-8 synthesis. Additionally, neutrophil elastase can activate or inactivate inhibitors of neutrophil collagenase, and secretory leukoprotease proteinase inhibitor. Apart from neutrophils, oxidative stress causes activation of other phagocytes and severe inflammatory response ensues.3 The reactive oxygen species (ROS) are generated during normal cellular activity and may exist in excess in some pathophysiological conditions, such as inflammation or reperfusion injury. These molecules oxidize a variety of cellular

constituents, but sulfur-containing amino acid residues are especially susceptible. While reversible cysteine oxidation and reduction is part of well-established signalling systems, the oxidation and the enzymatically catalysed reduction of methionine is just emerging as a novel molecular mechanism for cellular regulation. The oxidation of methionine to methionine sulfoxide in signalling proteins such as ion channels affects the function of these target proteins. Methionine sulfoxide reductase, which reduces methionine sulfoxide to methionine in a thioredoxin-dependent manner, is therefore not only an enzyme important for the repair of age- or degenerative disease-related protein modifications. It is also a potential missing link in the post-translational modification cycle involved in the specific oxidation and reduction of methionine residues in cell signalling proteins, which may give rise to activity-dependent plasticity changes in cellular excitability and causes deficiency of alpha1antitrypsin.4 Study of the role of alpha 1-antitrypsin in protection against oxidative stress and inflammation in the lung cells of emphysema patients not only limits the damage but also inhibits inflammatory responses. Aims & objectives In view of the above concept the present work was planned to study alpha 1-antitrysin and non enzymatic antioxidants as well as oxidative balance during inflammation in emphysema patients. MATERIALS & METHODS Study design In the present study total 160 patients were included after taking the consent form, between the age 25-60 years, either sex, who attend the OPD of Dr. Vikhe Patil Memorial Hospital during the period from Jan-Dec 2012. Patient randomly divided into 2 groups. Group 1 Control healthy subjects (N=100), Group 2 emphysema patients (N=60). The control
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subjects were completely healthy nonsmokers and showed no abnormality on clinical examinations and were completely symptom less. 5ml blood sample was collected from each patient. Serum was separated from blood by centrifugation at 3000 RPM for 10 minutes at room temperature. All the samples were analysed on the same day of collection. The serum analysed for following biochemical analysis: 1. The level of serum total lipid peroxide in terms of Malondiadehyde (MDA) was determined by the Kei Satoh method.5
RESULTS

2. The activity of 1- antitrypsin (-1-AT) was assayed by immuno diffusion method of Macini using Kits of Bioscientifica Co. Argentina.6 3. Serum Vitamin E ( Tocopherol) was estimated by the method of Baker and Frank.7 Stastics: The statistical analysis was performed by using student t test and P values < 0.001 were interpreted as very statistically significant. The values were expressed as mean SD.

Table.1: Illustrate the levels of MDA , AAT & Vitamin C in the healthy controls and emphysema Sr No. Parameters Healthy controls n=100 Emphysema patients n=60 1. Sr.MDA (mol/L) 1.660.28 4.20.57* 2. -1antitrypsin (mg/dl) 14623.12 37.410.1* 3. Sr. Vitamin E (mol/L) 1.030.23 0.360.05* n= number of cases, All values are expressed in mean SD, *Significant
DISCUSSION

Table No. 1 display malondialdehyde (MDA) levels in healthy controls and emphysema patient. Oxygen radicals are toxic and are thought to be involved in the pathogenesis of a variety of diseases. In recent years, an increasing amount of research has focused on the proposal that an oxidant/antioxidant imbalance occurs in smokers and in patients with lung diseases as part of the pathogenesis of the condition. Nonetheless, obtaining definitive proof that oxidants contribute to lung disease or that antioxidant therapy is beneficial in lung disease remains problematic. Unfortunately, because of the great variability that exists in the individuals who smoke and difficulties in measuring oxidative status, many challenges remain, in understanding, treating and preventing lung disease. Reactive oxygen species (ROS), such as the superoxide anion liberated by phagocytes recruited to sites of inflammation, are proposed

to be a major cause of the cell and tissue damage, including apoptosis, associated with many chronic inflammatory diseases.8,9 Lung cells, in particular alveolar epithelial type II cells, are susceptible to the injurious effects of oxidants. Lungs are continuously exposed to oxidants, either generated endogenously by metabolic reactions or exogenously, such as air pollutants or cigarette smoke Inhaled environmental oxidants exacerbate the underlying inflammation in inflammatory lung diseases. Ozone is a potent oxidant, which causes cellular damage by lipid peroxidation as well as loss of functional groups on biomolecules. Inhalation of ozone may lead to an increase in neutrophil numbers, increased airway responsiveness and reduced pulmonary function in normal subjects.10 This has been linked to neutrophil infiltration into the airway epithelium.11 Cigarette smoking, another environmental hazard, also delivers oxidants and free radicals to
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the lungs. Cigarette smoke contains many oxidants and free radicals, both in the gas and the tar phase and causes sequestration of neutrophils into the pulmonary microcirculation and accumulation of macrophages in respiratory bronchioles.12 Once recruited, these cells become activated and generate ROS in response to a sufficient level of stimulus (threshold

concentration). ROS, which may also be released by lung epithelial cells.13,14 may also stimulate inflammatory cells directly, thereby amplifying lung inflammatory and oxidant events. The elevated MDA levels observed in the present study suggest increased ROS production and thereby lipid peroxidation in emphysema patients .

Fig.1: Mechanism of Lipid peroxidation in Emphysema Table No.1 shows the levels of -1 antitrypsin in healthy controls, and emphysema patients. There was a significant decrease in -1 antitrypsin levels of emphysema patients (P<0.001) as compared to healthy controls. -1 antitrypsin (AAT) is important protein, involved in the bodys defense mechanism that protects against tissue damage from the proteolytic enzymes of inflammatory cells. It is well established that a proteinase / antiproteinase imbalance occurs in the lungs of emphysema patients as part of the pathogenesis . The elastase burden may be increased as a result of increased recruitment of leukocytes to the lungs, and there may be a functional deficiency of 1- antitrypsin due to inactivation in the lungs by oxidation, which is considered to contribute to the pathogenesis of this condition. This functional 1- antitrypsin deficiency is thought to be due to inactivation of the alpha1AT by oxidation of the methionine residue at its active15 site by oxidants and from phygocytes, as part of the pathogenesis of emphysema. (Fig. 2) Table No. 1 describe vitamin E in healthy controls and Emphysema patients. As compared to healthy controls vitamin E was significantly decreased (p<0.001). Vitamin E is the most important lipophilic antioxidant in humans. In cell membrane and lipoproteins the essential antioxidant function of vitamin E is to trap peroxyl radicals and to break the chain reaction of lipid peroxidation. Vitamin E will not prevent the initial formation of secondary radicals in a lipid rich environment, but does minimize the formation of secondary radicals. Alphatocopherol is the most potent antioxidant of the tocopherols and is also the most abundant in humans.
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Fig.2: Destruction of alveolar tissue by elastase released from neutrophil It quickly reacts with the excess charge associated with the extra electron being dispersed across the chromnol ring. This resonanceresonance stabilized radical might subsequently react in one of several ways. The reduced vitamin E level in emphysema patients could be due to partly its over consumption as an antioxidant subsequent to increased production of free radicals by cigarette smoke and inflammatory reaction. Vitamin E radical formed by free radical attack interact with vitamin C.16
CONCLUSION ACKNOWLEDGEMENT

Authors are also also grateful to our CEO Dr. Sujay Vikhe Patil and Deputy Director Dr. Abhijit Diwate for their support and encouragement for carrying out this study.
REFERENCES

Ongoing inflammation and oxidative stress results in the deficiency of alpha 1-antitrpsin antitrpsin and damage to airspace structure and disturbance of the normal maintenance of alveolar structure. This strongly supports a role for protease, antiprotease imbalance in the development of emphysema. Furthermore, cigarette smoke activates airway epithelium to trigger airway remodeling. Both of these processes result in airflow obstruction. Macrophages are activated by cigarette smoke and recruit neutrophils and CD81 lymphocytes to cause elastolysis and emphysema.

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