**1. Introduction**

Hypertension is an emerging public health problem of this millennium and it is a major challenge to disclose the mechanism involved in the coexistence of hypertension and cardiovascular disease to improve the health of the Hypertensive patients. Patients with symptoms of a cardiovascular disease frequently present without striking evidence of cardiac specific enzymes in blood laboratory assessments or specific electrocardiogram findings. Recently, researchers have reported higher mortality risk associated with higher RDW in patient populations with cardiovascular disease (CVD) [1]. Nowadays, also there is a growing interest in characterizing RBC membrane defects in several diseases, as changes in membrane structure also contribute to the pathophysiology of the disease process.

Human erythrocyte contains about 95% of the glutathione which is responsible for scavenging reactive oxygen species [2]**.** Sialic acid is an important factor for

maintenance of the surface electrical charge and stability of biological cellular system [3]. Erythrocytes and Erythrocyte membrane are more vulnerable to peroxidation due to constant exposure to high oxygen tension and richness in polyunsaturated fatty acid, respectively [4]. The interaction of Reactive oxygen species (ROS) with particularly with fatty acids available in membrane can result in undesirable irreversible changes in cellular membrane. The membranes are therefore naturally protected by available anti-oxidative enzymes like superoxide dismutase, catalase, and glutathione peroxidase and vitamins E and A from oxidative damages [5]. By-products of peroxidation have been shown to cause profound alterations in the structural organization and functions of the cell membrane including decreased membrane fluidity, increased membrane permeability, inactivation of membrane-bound enzymes and loss of essential fatty acids [6]. Reactive oxygen species are the responsible moiety that are involved in the generation and progression of atherosclerosis and that contribute to the development of plaque instability in acute MI [7].

Blood viscosity is strongly affected by the surface charge of RBCs and is responsible for the spacing between them. A higher surface charge causes repulsive forces to increases distant RBCs, preventing their close aggregation, lowers the viscosity, and results into very low peripheral resistance to flow. Therefore, it can be hypothesized that stress like condition develops in hypertension generate ROS which can induce membrane deformity and as a result affect the membrane surface charge. The occlusive arterial disease resulting from RBC aggregation may develop due to comparable membrane deformity [8]. Besides vascular and cardiac tissue integrity, blood and especially the RBC, is critical to performing this critical assignment of CV risk assessment considering it makes up over 90% of formed elements within blood. In cardiovascular diseases, impaired blood rheology has been observed. A direct relationship has been found between increased RBC deformability and increased risk for arterial hypertension [9]. Accurate risk stratification of patients with chronic heart failure is critically important to efficiently target the use of evidence-based therapies and identify high-risk patients who may benefit from advanced treatments [10].

We tested the hypothesis that variations in zeta potential and deformation of erythrocytes were associated with risk of adverse cardiovascular outcomes in a population with hypertension that were free of symptomatic heart failure [11]. In the present work we envisaged to study and evaluate morphological changes taking place in RBCs, erythrocyte fragility, lipid per oxidation and zeta potential which can act as invaluable aid in the diagnosis of a hypertension and risk of Cardiovascular Disorder in hypertension patients. Hence, the aim of this study was to test the hypothesis of association of variation in RBC morphology, erythrocyte fragility and zeta potential in hypertension and its relation with the risk of cardiovascular disorder in hypertension patients.

### **2. Morphological characterization**

#### **2.1 Preparation of blood Smear**

Blood smear was prepared with the aid of wedge method [12]. In this method a drop of blood was placed on base closed to one cease of the slide at least 1 cm away from the edged of the slide. Another slide with the smooth end was used as spreader and smear was prepared by moving spreader inclined at 30–45° angle to the blood.

**97**

mobility; *D* = dielectric constant.

various types of biological membranes.

*Zeta Potential as a Diagnostic Tool to Determine the Angina Risk*

beneath a trinocular research microscope (RXT4, Radical).

This Smear was air dried and fixed with Leishman stain and located and observed

To observe the morphological variations in the erythrocyte membrane structure in Erythrocytes of patients suffering with hypertension and MI, erythrocytes were analyzed by scanning electron microscopy. With this motive blood sample was taken in Eppendorf tube containing 10 μl of Heparin (5000 UI/ml) in 900 μl of pH 7.4 phosphate buffer saline. The blood suspension was then centrifuged (1000 rpm for 10 min) and washed with buffer three times. The supernatant was removed and replaced by same volume of buffer. One drop of these separated erythrocytes were then exposed to 500 μl of 2.5% Glutaraldehyde in distilled water overnight at 4°C to fix. Again samples were washed thrice with distilled water and centrifuged. About 40 μl of each sample was placed on glass covered studs and air dried at room temperature. The Scanning electron microscopy (SEM) analysis of prepared samples was accomplished using Jeol,

About 5 g of anhydrous Dextrose (Merck) was solubilized in 100 ml of distilled

About 0.1 ml of blood sample was transferred into 50 ml of freshly prepared 5%

Zeta meter System 4.0 instrument was used to measure the zeta potential of the Erythrocytes [12]. Zeta potential of the system is measured by applying the electric field to the samples using electrodes and determining the mobility/velocity of the particle under the applied field. Zeta potential was calculated according to the simplified Helmholtz-Smoluchowski equation as follows: Mean velocity of the 10

where ν = viscosity of sample in poise at temperature "t"; *EM* = electrophoretic

The electrophoretic cell consists of capillary which is embedded inside a chamber having electrodes at both ends having cavity for sample connection with electrodes. From the cavity of any one end of the electrophoresis cell the Sample is introduced into the capillary to fill it completely. Electrodes are connected to the cell with the applied electric field at specific voltage of 200 V. Due to the applied electric field charged particles move towards oppositely charged electrode. Their velocity under the applied electric field is measured and expressed in terms of electro kinetic potential/zeta potential. Nowadays, this system is preferably used for determining the zeta potential of

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*DOI: http://dx.doi.org/10.5772/intechopen.92373*

**2.2 Scanning electron microscopy**

Japan (Model—JSM 5610LV).

**2.3 Preparation of iso-osmotic dextrose solution**

water to prepare a 5% w/v iso-osmotic Dextrose solution.

**2.5 Zeta potential measurement by zeta meter system 4.0**

readings was used to calculate the zeta potential Eq. (1).

**2.4 Preparation of blood sample for zeta potential measurement**

w/w dextrose solution which is isotonic with the red blood cells [12].

This Smear was air dried and fixed with Leishman stain and located and observed beneath a trinocular research microscope (RXT4, Radical).
