**3. Results and discussion**

Morphological study using the smear reveals that there is deformation in RBC shape (Anisocytosis) in majority of patients. In Hypertension and patients with MI showed RBC deformation from spherical biconcave shape to tear shape which further continues to Rouleaux formation (**Figure 1a**–**f**). Also, it was observed that there is increased aggregation of Erythrocytes in patients suffering from Hypertension and Myocardial Infarction compared to normal volunteers.

SEM studies revealed that there are number of deformed erythrocytes observed in myocardial infarction patients (**Figure 2c**) in aggregated form. It was observed in patients with hypertension, Erythrocytes appears with uneven surface of cell membrane which indicates the oxidative damage in the cell membrane (**Figure 2b**) as compared to normal human erythrocyte as shown in (**Figure 2a**).

#### **Figure 1.**

*(a, b) Erythrocytes of normal human erythrocytes. (c, d) Erythrocytes of patient suffering from Hypertension, blood smear showing Rouleaux formation. (e, f) Erythrocytes from patient suffering from myocardial infarction, blood smear showing anisocytes.*

**99**

**Figure 3.**

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

Similarly, the result for erythrocyte membrane osmotic fragility in test samples

*Scanning electron microscopic images of normal human erythrocytes, patients suffering from hypertension and* 

By using a Zeta meter system 4.0, the zeta potential of human Erythrocytes was obtained. The results obtained showed ZP values ranged from −20.13 mV to −26.46 (+1.87) mV for the healthy individuals with a mean value of −23.39 mV. But the ZP values obtained from the Patients with MI is much lower and ranges from −2.58 mV to −22.76 (+3.57) mV compared to patients suffering from only Hypertension values ranges from −12.13 mV to −19.61(+1.20) mV. Also it was observed that Patients with MI who underwent CABG and angioplasty and receiving medications the zeta potential of such patients was ranging from −21.53 mV to −32.59 mV(+2.95)

It was specifically observed that the patients with Hypertension had a mean ZP value −16.06 mV and patients with MI mean ZP value was obtained as −9.938 mV (p < 0.001). Data obtained from the analysis of blood samples from patients with hypertension and healthy volunteers was subjected to one-way ANNOVA with the application of Tukey's multiple comparison tests, and results indicated that there is a significant lowering of zeta potential of Patients with Hypertension from that

*Comparison between mean erythrocyte fragility of erythrocytes in normal volunteers (CONTROL), patients* 

*with hypertension and myocardial infarction and myocardial infarction patients on treatment.*

reveals that erythrocytes of patients with MI and hypertension become more fragile compared to erythrocytes of control group (**Figure 3**). The erythrocytes are very much susceptible to oxidative cellular damage on exposure to the excessive

indicating higher stability of erythrocytes in blood vessels (**Figure 4**).

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

*myocardial infarction showing deformed erythrocytes.*

oxidative stress.

**Figure 2.**

#### **Figure 2.**

*Apolipoproteins, Triglycerides and Cholesterol*

meter-ZM4DAQ software.

**3. Results and discussion**

**2.6 Statistical analysis**

In this method, fresh blood samples were collected from volunteer and blood suspension was prepared as described in preparation of sample. Prior measurement of zeta potential temperature of and other parameters for ZP measurements were adjusted such as light intensity; focal plane and tracking duration were optimized for stable data collection with minimal error. The collected and prepared samples were then added to the previously cleaned zetameter cell, which were then connected to electrodes and placed under the lens over stage. The Erythrocytes were tracked by using remote by microscopicallyacquired video images, values were recorded 10 times for each sample and

average zeta-potential in mv was obtained standard deviation from software Zeta

The experimental results for ZP are expressed as mean ± standard (SD). Investigated groups were compared by the statistical one way analysis of variance (ANOVA) and evaluated by Tukey's multiple comparison tests using Graph Pad Prism version 5.00 for Windows. Experimental Results were considered significant at p < 0.05 for the different groups which indicates that the control and other

Morphological study using the smear reveals that there is deformation in RBC shape (Anisocytosis) in majority of patients. In Hypertension and patients with MI showed RBC deformation from spherical biconcave shape to tear shape which further continues to Rouleaux formation (**Figure 1a**–**f**). Also, it was observed that there is increased aggregation of Erythrocytes in patients suffering from Hypertension and Myocardial Infarction compared to normal volunteers.

SEM studies revealed that there are number of deformed erythrocytes observed in myocardial infarction patients (**Figure 2c**) in aggregated form. It was observed in patients with hypertension, Erythrocytes appears with uneven surface of cell membrane which indicates the oxidative damage in the cell membrane (**Figure 2b**)

*(a, b) Erythrocytes of normal human erythrocytes. (c, d) Erythrocytes of patient suffering from Hypertension, blood smear showing Rouleaux formation. (e, f) Erythrocytes from patient suffering from myocardial* 

patient groups differ significantly from one another in all situations.

as compared to normal human erythrocyte as shown in (**Figure 2a**).

**98**

**Figure 1.**

*infarction, blood smear showing anisocytes.*

*Scanning electron microscopic images of normal human erythrocytes, patients suffering from hypertension and myocardial infarction showing deformed erythrocytes.*

Similarly, the result for erythrocyte membrane osmotic fragility in test samples reveals that erythrocytes of patients with MI and hypertension become more fragile compared to erythrocytes of control group (**Figure 3**). The erythrocytes are very much susceptible to oxidative cellular damage on exposure to the excessive oxidative stress.

By using a Zeta meter system 4.0, the zeta potential of human Erythrocytes was obtained. The results obtained showed ZP values ranged from −20.13 mV to −26.46 (+1.87) mV for the healthy individuals with a mean value of −23.39 mV. But the ZP values obtained from the Patients with MI is much lower and ranges from −2.58 mV to −22.76 (+3.57) mV compared to patients suffering from only Hypertension values ranges from −12.13 mV to −19.61(+1.20) mV. Also it was observed that Patients with MI who underwent CABG and angioplasty and receiving medications the zeta potential of such patients was ranging from −21.53 mV to −32.59 mV(+2.95) indicating higher stability of erythrocytes in blood vessels (**Figure 4**).

It was specifically observed that the patients with Hypertension had a mean ZP value −16.06 mV and patients with MI mean ZP value was obtained as −9.938 mV (p < 0.001). Data obtained from the analysis of blood samples from patients with hypertension and healthy volunteers was subjected to one-way ANNOVA with the application of Tukey's multiple comparison tests, and results indicated that there is a significant lowering of zeta potential of Patients with Hypertension from that

#### **Figure 3.**

*Comparison between mean erythrocyte fragility of erythrocytes in normal volunteers (CONTROL), patients with hypertension and myocardial infarction and myocardial infarction patients on treatment.*

#### **Figure 4.**

*Comparison between mean ZP values of normal volunteers, patients with hypertension and myocardial infarction patient and myocardial infarction patients on treatment.*

of the healthy volunteers. Also, there is greater decrease in ZP values of patients with Hypertension suffering from MI indicate the exacerbation in RBC deformity in patients. This may be due to hypertension induced complications. Higher ZP in treated patients indicates the increased stability of erythrocytes due to reduced oxidative stress.

#### **4. Conclusion**

The exact mechanism of development of CVD is complex and is not yet fully understood. But from the literature survey it was clear that ROS plays an important role in the progression and development of CVD. Also, it was known that there is a strong relation between ROS and the pathophysiology of CVD. From the present research work it can be concluded that due ROS in Patients with Hypertension the erythrocytes are affected, their membrane gets oxidized resulting in various types of morphological deformity (Anisocytosis). Also, membrane potential (ZP) which is a characteristic property of RBC responsible for free flowing of RBC in the blood stream without aggregation, get affected. These conditions get exacerbated in erythrocytes of patients suffering from myocardial infarction. Development of membrane deformity directly reduces the membrane potential of RBC. Due to oxidation of RBC membrane by ROS, the membrane becomes fragile and therefore the fragility of erythrocytes increases in Patients with Hypertension and MI compared to healthy volunteers.

Results obtained in our lab suggest that variations in erythrocyte morphology, zeta potential, lipid peroxidation and erythrocyte Fragility can act as a key indicator to determine the risk factor of myocardial infarction in hypertensive patients. In the present study, we have developed a greater understanding of effect of ROS on morphology of RBCs, effect on its membrane potential (ZP) due to deformity in the membrane and its erythrocyte fragility. This could be a new way to realize a better treatment in hypertensive patients and a prevention of cardiovascular complications (i.e.: myocardial infarction, TIA, etc.). However, more works in the near future are necessary to improve the detection and treatment of the ROS mediated dysfunction.

**101**

**Author details**

Swati S. Gaikwad1

\*, Jasmine G. Avari<sup>2</sup>

1 Nagpur College of Pharmacy, Wanadongri, Nagpur, India

\*Address all correspondence to: swati.gaikwad05@gmail.com

provided the original work is properly cited.

2 Department of Pharmaceutical Sciences, RTMNU, Nagpur, India

3 Dr. D. Y. Patil Institute of Pharmaceutical Sciences and Research, Pune, India

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

and Mansi Liladhar Patil3

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

We are grateful to Dr. Varma Pathology Laboratory, Platina heart hospital, Nagpur and healthy voluntary donors of the Department of Pharmaceutical

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

Sciences, Nagpur University, Nagpur.

**Acknowledgements**
