Treatment Of Hdl With Mse Biology Essay

Introduction

Burning mosquito coils indoors generates smoke that can control mosquitoes efficiently. This practice is currently used in Asia, Africa, and South America. However, the smoke may contain pollutants of health concern. Allethrin and prallethrin are the chief constituents of various mosquito repellent- insecticides in India. Other common ingredients are wood flour, coconut shall powder and starch/binders. When a mosquito coil is burned, the insecticides evaporate with the smoke. Submicrometer particles, Carbonyl compounds (acetaldehyde, formaldehyde) and polycyclic aromatic hydrocarbons (PAHs) are also released during combustion.

Accumulating evidence suggests that exposure to pollutants; ultrafine particles present in ambient air could elevate the risk of cardiovascular disease and even cardiovascular mortality. Plasma profile of chronic exposure to mosquito repellent showed increase in concentration of VLDL cholesterol, triglyceride and also elevated lipid peroxidation. Thus it can be indicative to coronary and cardiovascular risk.

Many studies are evidence for strong inverse relationship between the serum concentration of HDL and incidence of coronary heart diseases. HDL particle have antiatherothrombotic action including anti-inflammatory, antioxidant, and profibrinolytic activities. This protective ability of HDL comes from the presence anti-inflammatory enzyme Paraoxonase1 associated with it. Human paraoxonase 1 (arylesterases, EC 3.1.8.1, hPON1) is a calcium dependent enzyme with a molecular weight of 43kD. The enzyme is synthesized in the liver and then secreted into the blood, and exhibits broad-substrate specificity.

Paraoxonase 1 plays a part in detoxification of organophosphate compound by hydrolyzing it. PON1 can also minimize the Atherosclerosis by preventing the oxidation of low density lipoproteins (LDLs) as well as high density lipoproteins (HDLs) (10). It has ability to protect lipoprotein particles from free radical oxidation because it can hydrolyse oxidized cholesterol esters, phosphatidyl-choline core aldehydes, and degrade hydrogen peroxide (12, 13, and 14).

Therefore, in this study we investigated protective role of PON1 against modification of HDL caused by mosquito coil smoke.

Materials and Methods

Chemicals:

Experimental apparatus:

The experimental apparatus is shown in Figure 1. The smoke was collected in 20ml of Tris Buffer (100mM, pH 8) from the outlet tube of the apparatus setup shown below. Tested mosquito coils brand purchased from Mysore India. Based on the information on the product had active ingredients such as d trans allethrin, wood flour, coconut shall powder and starch/binders.

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Preparation of mosquito coil smoke extracts (MSE):

Smoke from a mosquito coil was passed through 20ml of 0.1M Tris Buffer pH8, containg 2mM CaCl2 by aspiration for about 2 hour.

Preparation of HDL

HDL was isolated from serum by density gradient centrifugation according to the method of Redgrave et al. (9). 6ml of serum was mixed with solid KBr such that the density was about 0.5 g/ml. 18ml of saline was layered on top of the serum sample. The tubes were centrifuged using thermo scientific ultracentrifuge in a fixed angle rotor for 3hours at 45000 rpm (200 000 g) at 4°C. After centrifugation, the tubes were placed in the vertical position. The fractions were aspirated from the top. HDL appears as an orange-layer at the bottom of the tube. HDL containing fractions were dialyzed in the dark for 6 to 8hours against 100mM Phosphate Buffer Saline.

Treatment of HDL with MSE

Two hundred microlitre of HDL was added to 2ml Tris Buffer (pH8, 0.1M) containing CaCl2 (2mM) with or without MSE. The reaction mixture with or without MSE were incubated at 37 oC for various time period .

Paraoxonase Activity

PON1 activity was measured by adding Enzyme to Tris buffer (100 mmol/L, pH 8.0) containing 2mmol/L CaCl2 and 2mmol/L paraoxon (O,O-diethyl-O-p-nitrophenylphosphate, Sigma Chemical Co). The rate of generation of p-nitrophenol was determined at 412 nm, 25°C, with the use of a continuously recording spectrophotometer (Shimadzu). The amount of generated 4-nitrophenol was calculated from the molar extinction coefficients at pH 8.0, which was 17,100M-1 cm-1. One unit of paraoxonase activity is defined as 1 µmol of 4-nitrophenol formed per min under the above assay conditions.

Lipid peroxidation assay

The extent of lipid peroxidation was measured by the formation of malondialdehyde (MDA) by the method of Buege and Aust (1978). One milliliter of Mosquito coil smoke extract treated HDL (1:6 v/v) was taken in a test tube to which 1.5ml of 20% acetic acid (pH adjusted to 3.5 with NaOH), 1.5ml of 0.8% TBA and 0.05ml 20% TCA are added. Kept in boiling water bath for 15 min and the contents were allowed to cool and then centrifuged at 1000g for 10 min. The supernatant was transferred into a separate test tube and the absorbance of the sample was read at 535 nm by a UV/Visible spectrophotometer against the reagent blank assuming the molar extinction coefficient to be 1.56 X 105.

Result:

Fig.1 HDL associated PON1 activity in the presence or absence of MSE at various time period. 200 μl of HDL is incubated with 1.8 ml MSE for 5 hr.

HDL associated PON1 activity at various concentrations of Allethrin after 1 hr incubation. HDL was incubated at 37oC with various concentration of Allethrin.

Figure 3. The effect of MSE on HDL oxidation. Kinetic analysis of MDA and conjugated diene formation in the HDL incubated with Mosquito Smoke Extract. 1ml of HDL was incubated with 5ml of mosquito smoke extract for 5 hr.

Discussion

Pesticides are an undeniable part of modern life, used to protect everything from home to agricultural field from specific insects and pest. Mosquito coil is the common pesticide using in most part of the world without considering its harms on health. It has been reported that one mosquito coil exhaust smoke can be equal to the smoke of 127 cigarettes.

Narendra et al studied the biochemical profile of those who chronically exposed to mosquito repellant. They found elevated lipid peroxides, VLDL, Triglyceride, SGOT and SGPT level in their profile. Elevated Lipid peroxidation is also observed in rats exposed to allethrin-based mosquito coil smoke for 15–180 days. Kale et al. (1999) undertook to determine pyrethroid-induced lipid peroxidation (LPO) and found the increase of antioxidant enzyme such as superoxide dismutase (SOD) and catalase (CAT) in response to the increased oxidative stress. The particular reason for the lipid peroxidation is not clear.

HDL and LDL particles would not go far away from this oxidation. Oxidation of HDL and LDL considered being risk factor for cardiovascular diseases. Although HDL having anti-inflammatory, antioxidant and pro-fibrinolytic activities, it can themselves undergo oxidative modification their by lose its protective role. But, there are enzymes associated with HDL which can revert back the changes and helps in gaining back HDL to its normal function. In this regard, contribution of Paraoxonase1 is more comparing to other HDL associated enzymes. Paraoxonase1 that may cleave oxidized lipids and thereby inhibit HDL from oxidation.

Our data showed increased MDA level and decreased PON1 activity was observed at initial hours (1-2hr) of MSE incubation. PON1 activity was found to be recovered at later hours (3-5hr) along with the declining MDA level. Neutral effect of Allethrin on PON1 activity substantiates the involvement of exhaust particles on HDL oxidation.

In conclusion, HDL associated PON1 is involved in protecting the HDL from Mosquito smoke induced HDL oxidation.