Zeta Potential In Hypertension And Cardiovascular Disease Management
The hypertension and the Cardiovascular disease are the factors which affects several organ-systems of our body, and this get initiated with the rheological properties of blood and its formed elements. Red blood cells (RBCs) are moving in circulation are continuously exposed to the Reactive oxygen species (ROS) that are co-transported and circulating within the vascular system. Also RBC membranes are specifically vulnerable to this oxidative stress. In the current study, we investigate morphological and electrochemical characteristics of RBCs obtained from the patients using scanning electron microscopy (SEM), microscopy and Zeta potential. Statistical analyses of data showed that Hypertensive and cardiac patients RBCs possessed significantly reduced zeta potential relative to that of RBCs from healthy individuals (P-value < 0. 0001). SEM imaging of Hypertensive RBCs revealed increased anisocytes and poikilocytes. These parameters were found exacerbated in patients suffering from cardiac blockage. This increased membrane deformability impairs the RBC's capacity to adequately adapt its shape to navigate blood vessels, especially microvasculature, and this decrease is also reflected in the cell's morphology. Changes to morphology and deformability may also indicate the oxidative degradation of polyunsaturated fatty acids in the membrane resulting in the significantly lowered ZP values of hypertensive patients than the healthy volunteers. These findings suggest that hypertension has impact on the RBC and its membrane integrity with potential for exacerbating pathological cellular function, hemorheology. Hence, it can be concluded that ZP can be used as an effective diagnostic tool for the detection of hypertension and cardio-vascular disorder risk.
Introduction
Hypertension is an emerging public health problem of this millennium and it is a major challenge to unfold the mechanism involved in the coexistence of hypertension and cardiovascular disease to improve the long-term health status 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 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. Erythrocytes and Erythrocyte membrane are more vulnerable to peroxidation due to constant exposure to high oxygen tension and richness in polyunsaturated fatty acid, respectively. Reactive oxygen species (ROS) are highly reactive substances that interact with proteins, DNA and lipids, particularly with membrane fatty acids, and provoke irreversible changes. Cellular membranes are protected from oxidative damages by antioxidative enzymes (superoxide dismutase, catalase, and glutathione peroxidase) and vitamins (E and A).
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. Reactive oxygen species are the responsible moeity that are involved in the generation and progression of atherosclerosis and that contribute to the development of plaque instability in acute MI.
Blood viscosity is strongly influenced by the RBC surface charge that governs the spacing between erythrocytes. A higher repulsive surface charge increases spacing between erythrocytes, reduces clumping, lowers viscosity, and lowers peripheral resistance to flow. Therefore, we can consider that hypertension induced stress like conditions can deform RBC membrane and reduce RBC surface charge. This can be correlated with occlusive arterial disease resulting from higher incidence of RBC aggregation. 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, RBC indices have been implicated in impaired blood rheology. A direct relationship has been found between decreased RBC deformability and increased risk for arterial hypertension.
In the present work we envisaged to study and evaluate RBC morphology, osmotic fragility 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, osmotic fragility and zeta potential in hypertension and its relation with the Cardiovascular Disorder in hypertension patients.
Materials and Methods
Materials: Dextrose (Merck), Distilled water, Rectified spirit, Zeta meter system 4. 0 (Zeta Meter inc. ), RXT4 Trinocular Research microscope (Radical), UV-1800 (Shimadzu).
Sample details
The present study comprised of 64 patients with hypertension, 52 patients with acute MI and 50 healthy controls with no history of hypertension and MI. MI Patients who underwent CABG (coronary artery bypass graft) after MI, who underwent angioplasty andPatients advised CABG and angioplasty were included. Both genders and over the age of 20-78yrs were chosen for this study. Patients with cardiomyopathy, thrombolytic disorder, congenital heart disease, renal or hepatic disorder, myocarditis due to bacterial or viral infections were excluded from the study. Control individuals were ethnically matched, free from MI and without history of hypertension and diabetes as determined by the clinical examination and medical history.
Preparation of blood smear for morphological characterization
Blood smear was prepared taking a drop of blood on slide which was fixed and stained using giemsa stain and observed under a trinocular research microscope.
Preparation of red blood cells and erythrocyte membranes for osmotic fragility test
The blood of healthy volunteers, hypertensive patients and hypertensive patients with MI was drawn, and collected in vial containing EDTA solution (anticoagulant). Further, RBCs were separated from plasma by centrifugation at 3000 rpm for 10 minutes, followed by washing them thrice with 310 imOsM tris buffer pH -7. 6, maintaining the temperature at 4 °C throughout the procedure. The washed cells were then suspended in the same buffer and used for testing.
Measurement of osmotic fragility of RBCs
In this test, different strengths of NaCl solution, from 0. 2 % to 0. 9 % concentration were prepared. To 5ml of each solution of varying concentration, a fixed small volume of packed RBCs were added. Similarly, sample was prepared in double distilled water, which was considered as Standard. The amount of lysis in each mixture was compared with standard mixture in double distilled water (100% lysis), at 415 nm using UV-Vis spectrophotometer (JASCO). The percent haemolysis in terms of osmotic fragility was determined.
Preparation of isotonic dextrose solution
A 5 % w/v Dextrose solution was prepared by dissolving 5 g of anhydrous Dextrose (Merck) in 100 ml of distilled water.
Preparation of blood suspension for Zeta potential measurement
About 0. 04 ml of blood sample was transferred into 50 ml of freshly preparation 5% w/w dextrose to maintain osmotic pressure inside and outside the cell.
Estimation of Zeta potential of prepared blood sample by Zeta meter System 4.0
The zeta potential of the RBCs was measured using Zeta meter System 4. 0. Zeta potential is purely an electro kinetic property of the electrical double layer surrounding the sys-tem but the surface of the system itself. The value of zeta potential gives an indication about the stability of the system under study. This quantity is measured by determining the mobility/velocity of the particle under an applied electric field. Mean velocity of the 10 readings was used to calculate the zeta potential according to the simplified Helmholtz-Smoluchowski Eq.
A special capillary cell called electrophoretic cell is used for the measurement of zeta potential. The capillary is embedded inside a chamber having electrodes at either of the two ends. Sample is placed from any one end of the electrophoretic cell and electrodes are connected to the cell and electric field at specific voltage is applied (200 V). Charged particles move towards oppositely charged electrode and their velocity is measured and expressed in terms of electro kinetic potential/zeta potential which indicates the mobility of particle under applied electric field. Recently this method is widely used for determining the membrane potential of biological membranes. In this experiment, fresh capillary blood samples were obtained from volunteer by puncturing the skin with a syringe and blood suspension was prepared as described in above procedure. Prior to zeta potential measurement temperature of the RBC suspensions was measured and parameters for ZP measurements were detected such as light intensity, focal plane and tracking duration were optimized for stable data collection. The RBC suspensions were then added to the previously cleaned zeta-meter cell placed under the zeta-meter stage and the mobility of individual RBCs was tracked by equipped Zeta meter-ZM4DAQ software using microscopically-acquired video images, and data were recorded 10 times for each sample and average zeta-potential in mv was determined using a standard Helmholtz– Smoluchowski formula.
Calibration of Zeta meter using Min-U-Sil
A suspension of 100 mg/L of Min-U-Sil in a 100 mg/L sodium chloride (NaCl) solution is easy to prepare and gives reasonably consistent results:A stock suspension was prepared by weighing out 1 g of Min-U-Sil and 1 g of NaCl and combined with 100 mL of distilled (not deionized) water. The Min-U-Sil test suspen-sion was prepared by adding 10 ml of the stock suspension to one liter of distilled (not deionized) water and stored this in a glass bottle. If the test suspension is used within 6 hours, the average zeta potential of the Min-U- Sil test suspension will be about -56 +/- 3 millivolts, with a standard deviation between 3 and 5 millivolts and if more than 24 hours old, the average zeta potential will be about - 54 +/- 4 millivolts, with a standard deviation between 3 and 6 millivolts.
