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Ultra-structural Changes in Red Blood Cell Membranes and Morphology due to Cigarette Smoking

Ina-Adle Keyser & Prof Resia Pretorius

Department of Physiology School of Medicine Faculty of Health Sciences University of Pretoria

4 October 2013

CONTENTS

1. Abstract 2. Introduction a. Aim and Objectives 3. Methods and Materials a. Sample b. Blood Sample Collection c. Whole Blood Smear Preparation for Electron Microscopy d. Whole Blood Smear Preparation for Light Microscopy e. Statistics 4. Results 5. Discussion and Conclusion 6. References

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1. Abstract

The World Health Organization estimated in 2013 that 1.4 billion people smoke cigarettes and/or other tobacco products worldwide and 6 million people die due to smoking related diseases. Smoking is rapidly becoming an epidemic and holds major risk factors for diseases such as atherosclerosis, heart disease and stroke. Research has shown that smoking causes changes in erythrocyte (red blood cell) membrane fluidity. The aim of the current research is to determine if these changes in membrane fluidity are ultra-structurally visible. Sixty-five experimental and control subjects were selected for the study. Smokers had smoked on average 4 cigarettes per day for 230 years. Smears of whole blood were prepared for scanning electron microscopy and viewed with a Zeiss ULTRA plus FEG-SEM with InLens capabilities. Erythrocyte surface morphology was viewed at 1 kV and micrographs were taken at 30,000 150,000 machine magnification. It was also compared to light micrographs, taken with a Nikon Digital Camera DXM1200F using a Nikon OPTIPHOT light microscope, at 100x magnification. A difference in membrane surface as well as morphology was visible in smokers, as opposed to the smooth membrane surface and discoid shape in healthy individuals. Research has noted changed membrane fluidity and the preliminary results of the current study suggest that this is visible ultra-structurally. Therefore, changes in membrane fluidity are structurally visible and translate into a more irregular membrane surface and pointed cellular morphology.

KEYWORDS: Erythrocytes, red blood cells, scanning electron microscopy, smoking

2. Introduction

In 2013 The World Health Organization estimated that 1.4 billion people smoke cigarettes and/or other tobacco products worldwide and 6 million people die due to smoking related diseases. Smoking is rapidly becoming an epidemic and holds major risk factors for diseases such as atherosclerosis, heart disease, chronic obstructive pulmonary disease (COPD) and stroke. Recent research in humans focussed on the effect of toxins in cigarette smoke. It was estimated that smokers are exposed to excessive amounts of toxins and oxidants which generate up to 1017 free radicals per inhalation to the human body
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. Exposure to these radicals creates severe oxidative

stress and results in inflammation 1. Cell membranes are especially vulnerable to oxidative stress and play a key role in disease pathology and progression. The major constituent of membranes is phospholipids and therefore the properties and condition of these biological membranes is dependant of their lipid composition 4. Recent research has shown alterations in platelet membrane fluidity during smoking 5; and the reason for this was noted to be due to an increase in lipid peroxidation as well as carbonyl groups. It is these changes to the lipid bilayer and damage to polyunsaturated fatty acids that were suggested to cause the decreased fluidity 5. In 2012, Pretorius confirmed these changes by showing that changes in platelet membrane fluidity can be seen using scanning electron microscopy 6.

Researchers showed for the first time in 2012 that smoking not only affects blood platelets, but causes a decrease in membrane fluidity and possibly impair the functions of the plasma membranes of red blood cells (RBCs) in patients with COPD4. Because RBC membrane lipids are rich in

polyunsaturated fatty acids, research has therefor suggested that the exposure to toxins and peroxidants, when smoking, causes haemolyses as well as a decrease in membrane stability 7-9.

a. Aim and Objectives

The aim of the study is to determine if these changes in membrane fluidity are ultra-structurally visible using scanning electron microscopy (SEM) as well as light microscopy (LM).

Furthermore, we show the extent of the RBC membrane changes and argue that this has greater implications for general health than previously thought.

3. Methods and Materials a. Sample Sixty-five experimental and control subjects between the ages of 18 and 86 were recruited for the study. The experimental smoking group consisted of 30 males and females, who did not suffer from high blood pressure or heart conditions and who classified themselves as healthy individuals. None of the individuals used any medication or products (prescribed or recreational) other than smoking cigarettes. Smokers had smoked on average 4 cigarettes per day for 2 30 years. The control group consisted of 35 males and females; none of the individuals smoked or used any medication or in the case of the females, use contraception or hormone replacement therapy.

Ethical clearance was obtained for this study from the University of Pretoria Human Ethics Committee and each test subject was required to complete a consent form before they were included in the sample.

b. Blood Sample Collection

Great care was taken to keep the participants and sample collectors safe and prevent exposure to blood. Blood specimens from each participant were obtained using a simple lancet to prick the participants finger after the area was disinfected and sterilized with an ethanol swab. The blood was then quickly collected using a pipette and transferred to Eppendorf tubes containing 12 l citrate to prevent
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coagulation and agglutination. The samples were immediately analysed to prevent degradation thereof.

c. Whole Blood Smear Preparation for Electron Microscopy

Whole blood smears to study RBCs from participants were prepared by making a smear on a glass cover slip and left to air-dry in a wellventilated and sterile environment. The glass cover slips were placed in a Petri dish and washed with phosphate buffered saline (PBS) for 20 minutes after which the samples were fixed using 25% gluteraldehyde for 30 minutes. The samples were rinsed 3x in PBS for three minutes before undergoing a second fixation for 15 minutes with 1% osmium tetra-oxide (OsO4.) This was followed by another 3x rinsing with PBS and a serial dehydration, in 30%, 50%, 70%, 90% and three times with 100% ethanol (all three minutes each). The material was then dried using hexamethyldisilazane (HMDS) for 30 minutes, mounted and coated with carbon. Each sample was viewed with a Zeiss ULTRA plus FEG-SEM with InLens capabilities. Erythrocyte surface

morphology was viewed at 1 kV and micrographs were taken at 30,000150,000 machine magnification.

d. Whole Blood Smear Preparation for Light Microscopy

The blood sample of each participant was prepared for electron microscopy as well as optical/ light microscopy. An adapted method of the hematoxylin and eosin stain (H&E) was followed to create glass slides of each specimen. A blood smear of each specimen was made and left to air-dry on a slide warmer. The smears were fixed with methanol for 5 minutes and again left to dry on the slide warmer until completely dry. Each slide was exposed to hematoxylin for 4 minutes, rinsed with clean running water and left to dry on the warmer. After drying completely, the slides were exposed to eosin for 30 seconds and again rinsed and air-dried, when each sample was completely dried, they were covered with a glass coverslip using a hypoxy resin. Light micrographs of each sample was obtained with a Nikon Digital Camera DXM1200F using a Nikon OPTIPHOT light microscope, at 100x magnification.

All the instrumentation is located in the Microscopy and Microanalysis Unit of the University of Pretoria, Pretoria, South Africa.

e. Statistics An Excel-based calculator designed by Vertex42 was used to obtain the actual ratios of the RBCs from each light micrograph and to

conduct a t-test and generate a p-value to correlate the control and experimental groups.

4. Results

The membranes of healthy RBCs have a typical globular architecture when observing the SEM micrographs (Fig. 1A and B). A general change in morphology is visible in smoking, where the RBCs deform from the typical discoid shape to form pointed extensions (Fig. 1C). Pretorius and co-workers have recently reported similar shape changes in inflammatory conditions such as thrombo-embolic ischemic stroke, diabetes and in iron overload
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. This

structural change, however, is not always visible under low magnifications that can be obtained by LM (Fig. 2B) and appears to be similar to RBCs of healthy individuals (Fig. 2A). The actual size ratios of RBCs in smokers are similar to those of healthy individuals (Fig. 3) with a correlating p-value of 0.0941. Also, surface membrane changes in smoking have been noted here for the first time at machine magnification of 100 000x (Fig. 1D).

Figure 1: A) Red blood cell from a healthy individual. Scale = 1m. B) Red blood cell from a healthy individual (150 000x machine magnification). Scale = 100nm. C) Red blood cell from a smoker individual showing membrane and shape changes. Scale = 1m. D) Red blood cell from a smoker individual (100 000x machine magnification). Scale = 200nm.

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Figure 2: A) Red blood cells from a healthy individual (100x machine magnification). B) Red blood cells from a smoking individual (100x machine magnification).

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Figure 3: BoxPlot graph to compare the actual size ratios of the RBCs of the control group and the experimental smoking group.

1.700 Min Outlier 1.600 Max Outlier 1.500 1.400

1.300
1.200 1.100 1.000 0.900 Controles Smoking

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5. Discussion and Conclusion

RBCs in COPD patients have decreased membrane fluidity and research has also reported changes in membrane fluidity of the blood platelets confirmed by Pretorius using ultra-structural SEM analysis 6. This study illustrated that with high magnification SEM technology, RBC membranes of smokers have a changed morphological ultra-structure. Areas that balloon outwards as well as fine bubble-like extensions are present on the membranes. The ballooning areas are particularly smooth, as seen in Fig. 1C, and present on all RBC membrane surfaces in the smokers sample. We suggest that these changes may impact membrane fluidity and cause conformational changes in the RBC. Higher magnifications (Fig. 1D) show these abrupt changes in membrane fluidity clearly. These structural changes, however, are not always visible under low magnifications that can be obtained by LM (Fig. 2B) and appears to be similar to RBCs of healthy individuals (Fig. 2A). The actual size ratios of RBCs in smokers are similar to those of healthy individuals (Fig. 3) with a insignificant p-value of 0.094. In a few instances some RBCs also seem to lose their ability to maintain the typical discoid shape and develop a pointed extension. This however is not the norm, but agrees with the theory where similar changes were previously noted in inflammatory conditions like diabetes and iron overload
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. This was

. It is known that cigarette smoke contains metals, including iron,

and therefore the metal composition of cigarette smoke may possibly be the cause of the changed RBC membrane surface ultra-structure and the reason for the pointed extensions.
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We suggest that these ultra-structural membrane changes of the surface are due the vast amount of toxins obtained per single inhalation. In the lungs these toxins cross a single layer epithelium in the alveolar sac causing RBCs to immediately and constantly be engulfed when passing through the pulmonary circulatory system. The limitations of the study are that SEM technology and equipment is not widely available, however, it creates the opportunity to utilise a simple modest whole blood smear made on a glass cover slip to investigate the general health status of RBCs.

These SEM observations are novel and have not previously been noted, as light microscopy (being the most frequent method to study RBC structure) does not provide adequate detection of these morphological changes.

Using an old technique in a novel application may provide new insights on the negative effects of smoking and provide new avenues for future improvements in clinical medicine pertaining to conditions like COPD and stroke. Due to the vast adaptability of RBCs, their general state of health may be a major indication for the general health status of the individual

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6. References

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2. Pryor WA, Stone K, Cross CE, Machlin L, Packer L. Oxidants in cigarette smoke: Radicals, hydrogen peroxide, peroxynitrate, and peroxynitrite. Ann N Y Acad Sci. 1993;686:12-28.

3. Smith CJ, Fischer TH. Particulate and vapor phase constituents of cigarette mainstream smoke and risk of myocardial infarction. Atherosclerosis. 2001;158(2):257-267.

4. Gangopadhyay S, Vijayan VK, Bansal SK. Lipids of erythrocyte membranes of COPD patients: A quantitative and qualitative study. COPD: Journal of Chronic Obstructive Pulmonary Disease. 2012;9(4):322-331.

5. Padmavathi P, Reddy VD, Maturu P, Varadacharyulu N. Smoking-induced alterations in platelet membrane fluidity and na+/K+-ATPase activity in chronic cigarette smokers. J Atheroscler Thromb. 2010;17(6):619-627.

6. Pretorius E. Ultrastructural changes in platelet membranes due to cigarette smoking. Ultrastruct Pathol. 2012;36(4):239-243.

7. Johnson RM, Ravindranath Y, El-Alfy M, Goyette Jr. G. Oxidant damage to erythrocyte membrane in glucose-6-phosphate dehydrogenase deficiency: Correlation with in vivo reduced glutathione concentration and membrane protein oxidation (blood (1994) 83, 4 (1117-1123)). Blood. 2012;120(22):4447.
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8. Asgary S, Naderi GH, Ghannady A. Effects of cigarette smoke, nicotine and cotinine on red blood cell hemolysis and their -SH capacity. Experimental and Clinical Cardiology. 2005;10(2):116-119.

9. Fernandes AC, Filipe PM, Manso CF. Protective effects of a 21aminosteroid against copper-induced erythrocyte and plasma lipid peroxidation. Eur J Pharmacol. 1992;220(2-3):211-216.

10. Pretorius E, Lipinski B. Iron alters red blood cell morphology. Blood. 2013;121(1):9.

11. Lipinski B, Pretorius E, Oberholzer HM, Van Der Spuy WJ. Interaction of fibrin with red blood cells: The role of iron. Ultrastruct Pathol. 2012;36(2):7984.

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