Ultra-structural Changes in Red Blood Cell

Membranes and Morphology due to Cigarette

Smoking

Ina-Adéle Keyser & Prof Resia Pretorius

Department of Physiology

School of Medicine

Faculty of Health Sciences

University of Pretoria

4 October 2013

CONTENTS

1. Abstract 3

2. Introduction 4

a. Aim and Objectives 5

3. Methods and Materials

a. Sample 6

b. Blood Sample Collection 6

c. Whole Blood Smear Preparation for Electron Microscopy 7

d. Whole Blood Smear Preparation for Light Microscopy 8

e. Statistics 8

4. Results 9

5. Discussion and Conclusion 13

6. References 15

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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 2–30 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

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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 1-3. 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.

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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.

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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 participant’s

finger after the area was disinfected and sterilized with an ethanol

swab. The blood was then quickly collected using a pipette and

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transferred to Eppendorf tubes containing 12μl citrate to prevent

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 well-

ventilated 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

30,000–150,000× machine magnification.

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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

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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 10. 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).

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Figure 1: A) Red blood cell from a healthy individual. Scale = 1μm. 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 = 1μm. 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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B

A

Figure 3: BoxPlot graph to compare the actual size ratios of the RBCs of the

control group and the experimental smoking group.

Controles Smoking0.900

1.000

1.100

1.200

1.300

1.400

1.500

1.600

1.700

Min Outlier

Max Outlier

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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 3,5. This was

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 10,11. 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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