Ultra-structural Changes in Red Blood Cell Membranes and Morphology due to Cigarette Smoking
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Transcript of Ultra-structural Changes in Red Blood Cell Membranes and Morphology due to Cigarette Smoking
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.
4
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.
10
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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6. References
1. Assinger A, Schmid W, Volf I. Decreased VASP phosphorylation in
platelets of male and female smokers of young age. Platelets.
2010;21(8):596-603.
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:
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Correlation with in vivo reduced glutathione concentration and membrane
protein oxidation (blood (1994) 83, 4 (1117-1123)). Blood. 2012;120(22):4447.
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 21-
aminosteroid 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):79-
84.
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