Lipid Peroxidation in Bronchial Asthma Anita Sharma, Satish Bansal and R.K. NagpaP

Departments of Pediatrics and IBiochemistry, Pt. B.D. Sharma Postgraduate Institute of Medical Sciences, Rohtak, Haryana, India.

Abstract. Objective : To study the levels of free oxygen radicals in children with bronchial asthma during an attack and symptom free interval. Methods : Serum Malondiadehyde (MDA) levels were studied in 25 children between the age of 6 years-14 years who presented with an acute attack of bronchial asthma. In each patient, serum MDA levels were measured at the time of admission, 24-48 hours after good response to treatment and after a symptom free interval of 3 weeks. Results were compared with control group. Results : In study group serum MDA levels were highest at the time of admission, which decreased significantly at 24-48 hours with treatment. After a 3 weeks symptom free interval serum MDA levels had decreased further but were still higher than healthy control group. Conclusion : Lipid peroxidation is increased in bronchial asthma dudng an acute attack and symptom free period. [Indian J Pedlatr 2003; 70 (9) : 715-717] E-mail: [email protected] Kay words : Free oxygen radicals; MDA; Bronchial asthma

Free oxygen radicals have been incriminated as the causative agent in a n u m b e r of diseases including bronchial asthma. TM Although all the tissues in the body are vulnerable, by virtue of its location, anatomy and functions the epithelial tissue of the lung is the most vulnerable,5~because lung is exposed to oxidants of ambient air 7 and also to those which are generated by inflammatory cells on the epithelial surface.14~ Bronchial asthma is defined as a chronic inflammatory disorder of airways in which bronchial mucosa is infilterated by a number of inflammatory cells.8 These cells secrete a number of chemicals including free oxygen radicals which are responsible for chronic inflammation and increased hyper-responsiveness of airways.14~ Free oxygen radicals can cause widespread damage of all biological membranes by attacking their proteins, lipids, nucleic acids and glycoconjugates. Lipid peroxidation is the oxidation process of polyunsaturated fatty acids present in the cell membrane which yields peroxide radicals-lipid hydroperoxides and aldehyde products such as M a l o n d i a l d e h y d e (MDA). These products in turn enhance the vascular permeability and leucocyte chemotaxis and alter the prostaglandin synthesis and histamine release thus perpetuating inflammation2-1~ Lipid peroxidation and degradation products formed by free radicals are an important measures of a ongoing oxidative damage. Serum MDA estimation is an important method to determine this ongoing lipid peroxidation and therefore oxidative damage. H In view of the scanty literature on the status of lipid

Reprint requests : Dr. Anita Sharma, 39/9J Medical Campus, Rohtak-124001,Haryana. Indian Joumal of Pediatrics, Volume 70--September, 2003

peroxidation by measuring serum MDA during an acute attack and in symptom free period of bronchial asthma this study was planned.

MATERIALS AND METHODS Twenty five children (Group I) of either sex between the age group of 7-14 years, who presented in acute attack of bronchial asthma as per classification of International Pediatric Asthma Consensus group were taken. For each patient, serum MDA levels were measured thrice : (la) at the time of presentation, (lb) after 24--48 hours, when the acute attack was well controlled by standard treatment and (lc) lastly after a symptom free intervals of 3 weeks and ensuring that patient was off bronchodilators and steroids for at least 7 days prior to estimation of serum MDA levels. Guidelines of International Pediatric Asthma Consensus groups were followed for the diagnosis, treatment and assessing the response of treatment.8 Exclusion C r i t e r i a : Children who had received any bronchodilator therapy six hours before reporting to hospital, smokers, suspected foreign body inhalation and ~tildren with associated pneumonia were exluded from study. Control group (Group ]I) comprised of 25 age matched children, who had been hospitalized for elective surgery and had no symptoms suggestive of bronchial asthma presently or in the past. Only one serum MDA level estimation was done in them. All the children who fulfilled the inclusion criteria were enrolled after a written consent from parents. A detailed history and physical examination were carried out. In the s t u d y group, laboratory test including 715

Anita Sharma et al complete hemogram, total eosinophil count, X-ray chest PA, M a n t o u x test, stool and urine e x a m i n a t i o n was carded out and was approved by the Postgraduate Board of Studies of the Institution. M e t h o d of MDA Estimation : 5 ml venous blood was taken for estimation. Malondialdehyde (MDA) level was estimated by the method of Placer et at22 The test is based on the principle that Thiobarbituric acid (TBA) reacts with MDA to form a stable p i n k c o l o u r w h i c h has an a b s o r p t i o n m a x i m u m at 535 n m and 548 nm, and is estimated by calorimeter. Because of its simplicity and sensitivity the TBA method is the most widely used test for lipid peroxidation.

S U M

,4

M D A

3

.

.

. ~ J '

,

I 4

1

0 0

I 2

I 6

i 8

I 10

I 12

14

Duration of disease (in years) Fig. 1. Correlation between serum MDA levels a n d d u r a t i o n of disease

RESULTS

Serum MDA levels of control (Group II) and patients (study group I) are given in Table 1. Table 1. Serum MDA Levels (lamolFl) in Sudy Group and Control

Group

Group Study group (I) (n=25) la (Before treatment) lb (After treatment) lc (After 3 weeks follow up) Control group (II) (n=25)

Serum MDA (mean _+SD)

p value

3.6 _+1.5 3.05 -+ 1.5 2.5 _+1.3 2.07 + 1.02

<0.001" <0.001" >0.05*

*Compared to control; Statistical analysis I a v s Ib p<0.001; Ia vs Ic p<0.001; Ib vs I p<0.001

At the time of presentation serum MDA levels of study group (la) were significantly higher than the controls (Group II). Study group showed a statistically significant decrease in serum MDA levels after 24-48 hours with the control of acute attack (lb) but the levels were still higher than the control group. After a symptom free interval of 3 weeks in study group (lc), serum MDA levels had decreased further but were still higher than the control group. H o w e v e r , the difference was not statistically significant. Four patients had clinical cyanosis at the time of p r e s e n t a t i o n . Their m e a n s e r u m MDA levels w e r e significantly higher than those who had no cyanosis (4.9 + 0.9 Bmol/l; 3.5_+2.5 Bmol/1; p<0.01 respectively). Serum MDA levels were significantly higher in patients where duration of illness was more than four years as compared to those where duratio was less than four years (3.7 _+1.09 pmol/1; 1.6 -+ 0.7 ~tmol/1; p<0.001). There was a high positive correlation (r=+0.912)-between the duration of bronchial asthma and serum MDA levels (Fig 1). DISCUSSION Free o x y g e n radicals ( s u p e r o x i d e anion, h y d r o g e n p e r o x i d e and h y d r o x y l radicals) are p r o d u c e d by 716

sequential incomplete reduction of oxygen molecules and are promptly scavenged by antioxidant enzymes present in vivo. Since they are short lived in circulation it is difficult to directly detect them 14 and they have been assessed indirectly b y measuring free oxygen radicals mediated lipid peroxidation. 9 Lipid peroxidation is the most important mechanism of cellular injury. Serum MDA level is an i m p o r t a n t m e t h o d to estimate lipid peroxidation? ,~ Although some workers have studied the role of free oxygen radicals in bronchial asthma and have reported higher level of superoxide anions 3,1~16and lower levels of antioxidant defens~ mechanism ~7in bronchial asthma, lipid peroxodation by estimation of MDA has not been comprehensively studied in bronchial asthma. In the p r e s e n t s t u d y , s e r u m MDA levels w e r e measured serially at the time of presentation, after 24-48 hours of good response to standard treatment and after a symptom free period of three weeks. Serum MDA levels were highest at the time of acute attack and significantly decreased with treatment. Serum MDA levels further decreased after a symptom free interval of three weeks. But the serum MDA levels continued to remain higher than the controls indicating thereby the relation of level of s e r u m MDA levels w i t h that of the u n d e r l y i n g inflammatory process in bronchial asthma. Other workers ]s,19have studied the generation of active form of o x y g e n (AFO), (i) l e u k o c y t e p e r o x i d e lipid oxidation (PLO) by measuring plasma MDA levels and (iI~ antiperoxide plasma activity (APA) by its resistance to the injection of free o x y g e n radicals o x i d a t i o n of hydrogen peroxide in acute attack of asthma. Both these studies have reported increased levels of plasma MDA and decreased levels of plasma APA in acute attack of bronchial asthma as c o m p a r e d to controls. Like the present study, they also noted that during remission the levels of plasma MDA were approximating the normal, but never equalled the control. Furthermore, both studies have reported the role of antioxidants when given along w i t h the b r o n c h o d i l a t o r t h e r a p y . T h e r e was m o r e Indian Journal of Pediatrics, Volume 70--September, 2003

Lipid Peroxidation in Bronchial Asthma p r o n o u n c e d lowering of plasma M D A levels in the g r o u p given antioxidants along with bronchodilators c o m p a r e d to t h o s e b r o n c h i a l a s t h m a p a t i e n t s w h o r e c e i v e d o n l y s t a n d a r d t h e r a p y b u t w i t h o u t antioxidants. Like o t h e r s t u d i e s 16,18w e also o b s e r v e d a p o s i t i v e correlation b e t w e e n the d u r a t i o n of illness a n d s e r u m M D A levels. This c o u l d be p r o b a b l y d u e to c o n s t a n t production of free oxygen radicals by underlying inflammation. In the present study, serum MDA levels were significantly higher in 4 patients w h o had clinical cyanosis at t h e t i m e o f p r e s e n t a t i o n . T h e e x t e n t o f l i p i d p e r o x i d a t i o n (cell m e m b r a n e d a m a g e ) d e n o t e s t h e a m o u n t of free o x y g e n radicals generated w h i c h have not been p r o b a b l y scavengered b y defense mechanism. Lipid p e r o x i d a t i o n m a y n o t be related to the p r i m a r y tissue injury, b u t m a y amplify the original injury. CONCLUSION Children of bronchial asthma had increased lipid peroxidation as indicated b y s e r u m M D A levels. S e r u m M D A levels are correlated with the severity a n d duration of illness. Their diagnostic and prognostic role needs to be evaluated further. REFERENCES

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