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ANALYSIS OF SERUM PROTEIN PATTERN BY ELECTROPHORESIS IN VALVULAR HEART DISEASE.

Byline: Zahid Hussain Siddiqui and Abdul Majeed Cheema

ABSTRACT

In the present study the blood samples of patients diagnosed for valvular heart disease (VHD) were obtained from the Punjab Institute of Cardiology, Lahore. Blood samples of the normal subjects of comparable age group with an absent history of cardiac ailment were also collected for the control comparison. The sera of all categories were separated and used for the study of the protein profile with sodium dodecyle sulfate polyacrylamide gel electrophoresis (SDS-PAGE) in first dimension. Gene Genius Bio-imaging Gel Documentation System was used for the quantification of various protein fractions.

This provides the data of molecular weight and percent raw volume for each of the fraction. The protein fractions that showed significant variations were separated by using the technique of electroblotting and electroelution and run on isoelectric focusing (IEF) in second dimension to determine the isoelectric points.

Significant increase was observed in apolipoprotein B, alpha-2-macroglobulin, ceruloplasmin and immunoglobulin heavy chain in VHD patients compared to normal subjects. These results show that level of apolipoprotein B, alpha-2-macroglobulin, ceruloplasmin and immunoglobulin heavy chain are strong predictor of VHD and can also be used for its diagnosis.

Key words: Valvular heart disease, Protein fractions, Electrophoresis.

INTRODUCTION

Valvular heart disease (VHD) is cardiac dysfunction produced by structural and/or functional abnormalities of single or multiple cardiac valves [1]. The clinical consequences depend on the valve involved, the degree of impairment, the rate of its development and the rate and quality of compensatory mechanism [2]. Valvular abnormalities may be caused by congenital disorders or by a variety of acquired diseases [3] like rheumatic fever and rheumatic heart disease [4]. Other important causes are myxomatous degradation, calcification and dilation of aorta [3].

Valvular heart disease can be diagnosed on the basis of clinical history of streptococcal infection, rheumatic fever/rheumatic heart disease, and high anti M protein antibodies [5], chest X-ray, ECG, echocardiography and cardiac catheterization. During the last few years interest has focused on proteins released into the serum following injury/infection for the diagnosis of cardiovascular disease [6, 7]. Elevated level of C-reactive protein [8] and fibrinogen was demonstrated in the serum of heart valve disease compare with control group [8, 9]. Patients suffering from acquired valvular heart disease have shown that blood serum levels of ceruloplasmin, copper and collagen like protein were statistically significant higher than those found in controls [10]. Accumulation of oxidized low density lipoprotein was demonstrated in the heart valves that illustrate atherosclerosis as an additional mechanism accelerating valvular degeneration [11].

Serum antitropomyosin antibodies in patients with valvular heart disease were significantly higher than those of normal controls [12]. There was an increased frequency of elevated antibody levels in patients with valvular heart disease which play a role in the pathogenesis of the valve lesion [13]. Significantly higher values of immunoglobulins (Ig) such as Ig G, Ig A, C3 and C4 were observed in patients of valvular heart disease who died later [14]. Anti M protein antibody titer was found in children with mitral regurgitation [5]. It has been found that tissue and plasma Pentraxin3 levels were increased in patients with aortic stenosis [15].

Applications of newer analytical procedures proceed with the time and one of the useful methodologies involves the electrophoretic system. It is a powerful technique for the analysis of proteins including serum proteins which can be used to study the variations in the protein profiles in VHD patients. Presently two-dimensional gel electrophoresis (2-DE), with one dimension on isoelectric focusing (IEF) and other on sodium dodecyle sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and vice versa, is mostly employed in such analysis. 2-DE demonstrated that three protein spots were significantly over-expressed and 4 were significantly down-regulated in the bicuspid valve patients compared to the tricuspid valve patient [16}. SDS-PAGE is likely to yield protein distribution profile consistent with the patient's clinical status and may have an important role in diagnostic investigation [17].

Present study was carried out to investigate the protein patterns in the sera of VHD patients and their comparison with those of healthy subjects through SDS-PAGE and 2-DE and characterization of the proteins specifically found in the serum of VHS patient in our local population for prediction and diagnosis of VHD.

MATERIALS AND METHODS

Twenty patients from the Punjab Institute of Cardiology, Lahore, with a diagnosis of VHD (based upon Chest X-ray and echocardiography) were selected and their blood samples were collected. Five ml blood was collected from each patient with the help of syringe. Blood samples of same number of healthy subjects with negative family history of CVD were also collected for use as controls. Serum was separated by centrifugation and stored at -70 degC until used for analysis. For SDS-PAGE, the serum samples were diluted in phosphate buffer (pH 7.2) and proteins were denatured by heating with loading dye (1.54 g dithiothreitol, 2 g sodiumdodocyle sulfate, 8 mL of 1.0 M Tris HCl; pH 6.8, 10 mL of glycerol and 20 mg of bromophenol blue dye) in boiling water bath for two minutes before loading on the gel. Lypholized mixture of proteins SDS-6H for high (205-45 kDa) and SDS VII-L for low (66-14.2 kDa) molecular weight proteins (Sigma Chemicals) were used as molecular weight markers.

It was reconstituted, separately, in 1.5 mL of sample buffer (0.0625M Tris HCl pH 6.75, 2% SDS, 5% mercaptoethanol, 10% glycerol and 0.001% bromophenol blue). Heated in a boiling water bath for 2 minutes and stored in aliquots at -70degC. Polyacrylamide gels, 5 % for high and 12 % for low molecular weight proteins, were prepared [18]. Protein size marker and each of the samples were loaded in separate wells and gels were electrophoresed at 20 mA and 200 volts in a cooling chamber maintained at 4degC. Electrophoresis was stopped, immediately, after dye seemed to diffuse in the buffer in the lower chamber. Following electrophoresis, the 5% gel was stained with coomassie brillient blue for 30 minutes and 12 % gel for two hours. After staining the gels were destained until the clearance of blue background. Protein fractions of different molecular weights were visible in the form of blue bands on a transparent background.

Gels were photographed and their images were saved for protein quantification by Gene Genius Bio-imaging Gel Documentation System provides the data of molecular weights against protein markers and the total area covered by each of the protein fractions. The data was employed in finding the enhancement or reduction and the appearance and disappearance of particular protein fractions for comparison of the healthy individuals and VHD patients.

Table-1: Average raw volumes (%) exhibited by electrophoretically separated serum protein fractions of control and valvular heart disease (VHD) groups and their percentage differences.

Molecular###Average raw###Average raw###Percentage

weight of###volume (%) of###volume (%) of###difference in

proteins###protein fractions###protein fractions###protein fractions

(kids)###in control group###in VHD group###in VHD group

270###2.45 +- 0.21###3.55 +- 0.23###45

190###7.37 +- 0.30###10.49 +- 0.58###42

186###3.50 +- 0.26###4.30 +- 0.23###23

135###4.25 +- 0.23###5.30 +- 0.20###25

115###5.23 +- 0.37###5.97 +- 0.27###14

100###3.25 +- 0.24###5.68 +- 0.38###75

77###10.21 +- 0.23###10.62 +- 0.27###04

66###26.52 +- 0.18###27.21 +- 0.21###03

54###14.55 +- 0.31###15.16 +- 0.34###04

45###10.64 +- 0.41###10.77 +- 0.21###01

36###2.22 +- 0.12###2.11 +- 0.13###05

28###9.48 +- 0.48###8.63 +- 0.62###09

24###12.42 +- 0.29###12.81 +- 0.66###03

23###3.38 +- 0.25###3.41 +- 0.27###01

17###1.34 +- 0.05###1.09 +- 0.09###19

14###0.96 +- 0.11###1.03 +- 0.12###07

Increase; Decrease; P less than 0.05; P less than 0.01

Samples containing significant quantities of desired protein fractions were run on SDS-PAGE and the unstained gel was electroblotted on polyvinylidene diflouride (PVDF) membrane [19]. The required protein band from PVDF membrane was excised and electroeluted [20]. Each of the eluted protein was freeze dried and reconstituted in the buffer when used for isoelectric focusing.10 uL solution D (10 % w/v sodium dodecyle sulfate in 2.3 % w/v dithioerythreitol) was added to 60 uL eluted protein solution, mixed and heated at 95degC for 5 minutes. Brought to room temperature and added 5 uL solution E (8 M urea, 4 % CHAPS (3-[(3cholamidopropyl)dimethylammonio]-1-Propanesulfonate), 40 mM Tris HCl and 65 mM dithioerythretol, traces of bromophenol blue). The eluted protein was subjected, afterwards, to isoelectric focusing [21] in order to determine its isoelectric point/s against isoelectric focusing markers.

RESULTS

No new fractions were detected in VHD group when compared to healthy group. However, most of the protein fractions of high molecular weight expressed significantly greater in their intensities in this group compared to the control group.

The fractions of 270, 190, 135 and 100 kDa exhibited highly significant increase of 45 %, 42 %, 25 % and 75 % (P less than 0.01) respectively. The percent raw volume expressed by these fractions of 270, 190, 135 and 100 kDa in healthy subjects were 2.45 +- 0.21 %, 7.37 +- 0.30 %, 4.25 +- 0.23 % and 3.25 +- 0.24 % respectively. The same fractions exhibited values in pathological group were 3.55 +- 0.23 %, 10.49 +- 0.23 %, 5.30 +- 0.20 % and 5.68 +- 0.38 % respectively. The fraction of 186 kDa exhibited significant increase of 23 % (P less than 0.05) in patients compared to control group. The average raw volume exhibited by this fraction was 3.50 +- 0.26 % and 4.30 +- 0.23 % in normal and valvular heart disease group respectively. No significant variations were observed in low molecular weight protein fractions in this group when compared with the control group (Table-1; Fig. 1-2).

The protein fractions showing considerable variations were subjected to isoelectric focusing. High molecular weight protein fraction of 270 kDa was resolved into two bands corresponding to isoelectric points 5.9 and 6.1. The fraction of 190 kDa was resolved into five bands whose isoelectric points were determined as 5.4, 5.5, 5.7, 5.9 and 6.1. The fraction of 135 kDa was resolved into two bands whose isoelectric points were determined as 4.8 and 5.2. Lastly the fraction of 100 kDa was resolved into six fractions corresponding to isoelectric points 7.1, 7.2, 7.5, 7.7, 7.8 and 7.9 (Fig. 3).

From the above data regarding the molecular weight and the isoelectric points, each of the proteins was then identified using human plasma protein map [22]. Protein fractions of 270, 190, 135 and 100 kDa were found to be apolipoprotein B, alphs-2-macroglobulin, ceruloplasmin and immunoglobulin heavy chain respectively.

DISCUSSION

The aim of the present study was to find the variations in the serum protein profile in the patients of valvular heart disease in local sampled population because in recent years proteomics is a rapidly growing research area and has increased the understanding of many diseases and protein composition represents the functional status of biological compartment. Due to resolution and sensitivity the technique of two- dimensional gel electrophoresis (2-DE) is powerful tool for the analysis and detection of protein from complex biological sources [21]. Variations in the serum protein profile in VHD were therefore detected by sodium dodecyle sulfate polyacrylamide gel electrophoresis (SDS-PAGE) in first dimension. Isoelectric focusing (IEF) was performed, in second dimension electrophoresis (2-DE), of those proteins which exhibited significant variations and could be diagnostically significant in identifying VHD.

The level of apoplipoprotein B (270 kDa) have been found to be elevated in the present study by 45 % in patients of valvular heart disease. These results are in agreement with observations of many authors. Accumulation of oxidized low density lipoprotein (Apolipoprotein B containing lipoprotein) was demonstrated in the heart valve that illustrates atherosclerosis as an additional mechanism accelerating valvular degradation [11].

The excess circulatory levels of any lipoprotein can be caused by one or two factors, either excess production or decreased catabolism [23]. It is caused by increased apolipoprotein B synthesis by the liver [24, 25] and also due to defect in low density lipoprotein (LDL) receptor, leading to inadequate hepatic uptake of LDL and markedly increasing circulatory LDL or apolipoprotein B, a component of LDL [26]. An important reason of increased LDL level is defective apolipoprotein B in which a substitution of glutamine for arginine at position 3500 results in a form of apolipoprotein B that binds poorly to the receptor and result in reduced LDL clearance and increase LDL or apolipoprotein B concentration in blood [27]. Another mutation of apolipoprotein B in which a substitution of cystine for arginine at position 3531 impairs binding of apolipoprotein B to the LDL receptor [28].

It is speculated that number of LDL receptor is not fixed and modified by genetic defects, dietary intake of saturated fat, cholesterol and calories and certain pharmacological agents. Thus the interaction of genetic and environmental factors control the number of lipoprotein receptors. These interactions may explain different responses within populations to similar dietary constituents [23, 29]. High use of alcohol [30] and hypertension [31] also the cause of increased level of apolipoprotein B. Smoking [32, 33] increase concentration of LDL and testosterone increase LDL-cholesterol in blood [34]. From the above discussion it is suggested that the higher level of apolipoprotein B may be used as diagnostic marker for cardiovascular disease. It is also indicated that the factors responsible for the enhancement of apolipoprotein B are genetic, dietary and environmental. Management of these factors may be helpful in reducing the chances of the disease.

An elevation of 42 % was exhibited by alpha-2-macroglobulin (190 kDa) in patients of valvular heart disease. Previous studies showed higher concentration of alpha-2-macroglobulin and two other protease inhibitors which significantly reduced during valvular heart disease [35]. Increased level of globulin has been reported in three patients of valvular heart disease and other immunologic abnormalities [36]. It was postulated that valvular heart disease is associated with an underlying immunologic disorder or autoimmune process. It has also been demonstrated that alpha-2-macroglobulin has a vital but unclear role in immunological and inflammatory process [37].

Alpha-2-macroglobulin is protease inhibitor and synthesized in liver. It has a vital but unclear role in immunological and inflammatory processes. Some evidence suggests that hepatic synthesis of alpha-2-macroglobulin increase in order to compensate partially for the decrease in albumin normally active in maintaining oncotic pressure. Increase level of alpha-2-macroglobulin is also associated with estrogen stimulation as in pregnancy or the use of oral contraceptives [37].

Ceruloplasmin (135 kDa) was found to be elevated by 25 % in patients of valvular heart disease. Previous reports also showed higher concentration of ceruloplasmin in patients of cardiovascular disease [38]. Serum level of copper in 45 patients of valvular heart disease was statistically significant higher compared to the 20 controls [10]. This elevated level of copper may suggest the increase in the concentration of ceruloplasmin which is a copper carrying protein. Ceruloplasmin level also rises in disorders producing inflammation or tissue injury. Estrogen reacts with steroid receptors on Hepatocyte to stimulate ceruloplasmin synthesis and plasma level rise substantially in pregnancy or where women take oral contraceptive agents containing estrogen [39].

Ceruloplasmin is copper carrying protein which functions as a ferroxidase and superoxidase scavenger. It is an acute phase protein. The term "acute phase response" encompasses a complex range of physiological changes that occur following infection, inflammation and related conditions. Increase occurs in plasma concentration of ceruloplasmin as a result of increased synthesis, mediated primarily by interleukin-6 and other cytokines. Cytokines secreted by cells involved in inflammation and immunity [40]. It has been reported that ceruloplasmin may prevent lipid peroxidation and free radical production in inflammatory state [37]. This is perhaps its role in an acute phase reaction.

Immunoglobulins heavy chain (100 kDa) exhibited higher raw volumes by 75 % in patients of valvular heart disease. In patients of valvular heart disease the values of immunoglobulin G and immunoglobulin A were reported to be significantly higher than healthy subjects which might be due to immunological reactions [14]. A prominent deposition of immunoglobulins in the valves from patients with antiphospholipid syndrome was observed[41]. History of valvular heart disease was associated with significant higher rates of positive immunoglobulin G and anticardiolipin and with higher immunoreactivity [42]. Increase in serum immunoglobulins are the normal response to infection and chronic bacterial infection cause an increase in serum levels of all immunoglobulins [37]. In a recent study C. pneumoniae specific immunoglobulin G in plasma was identified as risk marker for valvular aortic stenosis.

The data indicated C. pneumaniae infection might influence and aggravate heart valve sclerosis via the formation of circulating immune complexes [43].

Immunoglobulins are a group of plasma proteins that function as antibodies, recognizing and binding foreign antigens by elements of the cellular immune system. Since every immunoglobulin molecule is specific for one antigen, so there are vast number of different immunoglobulins. All show a similar basic structure. On electrophoresis immunoglobulin behave mainly as gamma globulins (heavy chain) but immunoglobulin A and immunoglobulin M may migrate with beta or alpha-2-globulins, because the normal plasma concentration of immunoglobulin G is much higher than that of other immunoglobulins. The gamma globulin band seen on electrophoresis of normal serum is largely due to immunoglobulin G. Increases and decreases of plasma immunoglobulin concentrations can be either physiological or pathological in origin. Increased concentrations of immunoglobulin are seen in both acute and chronic infection.

Increases in plasma immunoglobulin concentrations are common in autoimmune disease, for example rheumatoid disease and in chronic liver disease, some of which have an autoimmune basis. Many different immunoglobulins are produced in these conditions and they give rise to a diffuse increase in the gamma globulin (heavy chain) band on electrophoresis [40].

The results of present study clearly indicated that in VHD patients, alteration in protein fractions occur as a result of displaced molecular homeostasis. This pattern of variations in VHD patients is similar to most of the populations of the world. The technique of electrophoresis, due to its resolution and sensitivity, is being used for the diagnosis of VHD in these different populations of the world. Therefore the technique of electrophoresis, particularly two-dimensional gel electrophoresis, is also very useful for the diagnosis of VHD in our Pakistani population where the work on the diagnosis of cardiovascular disease is merely conventional and highly underdeveloped.

ACKNOWLEDGEMENT

This project was funded by University of the Punjab, Lahore, Pakistan.

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Author:Siddiqui, Zahid Hussain; Cheema, Abdul Majeed
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Date:Mar 31, 2012
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