A medical research paper on sickle cell disease.Erythrocytes from normal subjects and from cases of sickle cell disease were exposed to hydrogen peroxide and the extent of membrane lipid peroxidation studied. Significantly increased peroxidation was observed in intact erythrocytes, isolated lipids from intact erythrocytes or their ghosts compared with normal controls. However, the extent of lipid peroxidation was less for isolated lipids of intact erythrocytes or their ghosts. This difference may be due to loss of factors enhancing peroxidation in intact erythrocytes in the extraction procedure. It is likely that damage to erythrocyte membranes caused by increased susceptibility to lipid peroxidation may be a factor in some of the haematological features of sickle cell disease

Essien, E.U.

Central African Journal of Medicine

English

IDS OpenDocs

Vol. 40, No. 8 CONTENTS August, 1994
ORIGINAL ARTICLES
Neoplasms involving the spinal cord in 
Zimbabweans: an analysis of 262 cases..............
Termination of pregnancy using extra-amniotic 
prostaglandin F2a-  experience in a peripheral 
hospital..................................................................
Epidemiological study of drug use in urban and 
rural secondary schools in Zimbabwe..................
A comparison of three types of “M” traps for 
sampling tsetse fly (Diptera: Glossinidae) popu­
lations at South Luangwa Game Management 
Area, Zambia..........................................................
Increased susceptibility of erythrocyte membrane 
lipids to peroxidation in sickle cell disease..........
CASE REPORTS
PI Garrido, S Laher-Mooncey, NL Murphree,
N Jonker, LF Levy, S Makarawo..........................201
A Krolikowski, KA Janowski, JV Larsen............ 204
SW Acuda, AH E ide............................................. 207
MM Ziba, M Mukunyandela, S Siziya, JS Chipipa,
L Mwanza...............................................................212
EU Essien...............................................................217
Intracranial Burkitt’s lymphoma with extension 
into orbital spaces resulting in bilateral blindness
Munchausen syndrome by proxy -  a case report 
and review of literature.........................................
Hypothyroxinemia in Acquired Immune 
Deficiency Syndrome (AIDS)..............................
K Ramesh, LD Gahukamble, DB Gahukamble ...220
OA Oyelami, AM Alhaj, IK Airede.....................222
CC Ezeala, E Chukwurah..................................... 226
REVIEW ARTICLES
Advances in electrodiagnosis in Zimbabwe.
Part II: “Evoked Potentials” .................................  O Parry, LF Levy, J M ielke................................. 229
SUPPORTED BY CIMAS MEDICAL AID SOCIETY
CENTRAL AFRICAN
JOURNAL OF MEDICINE
Vol. 40, No.8,1994
Increased susceptibility of 
erythrocyte membrane 
lipids to peroxidation in 
sickle cell disease
EU ESSIEN 
SUMMARY
Erythrocytes from normal subjects and from cases of 
sickle cell disease were exposed to hydrogen peroxide 
and the extent of membrane lipid peroxidation studied. 
Significantly increased peroxidation was observed in 
intact erythrocytes, isolated lipids from intact erythro­
cytes or their ghosts compared with normal controls. 
However, the extent of lipid peroxidation was less for 
isolated lipids of intact erythrocytes or their ghosts. 
This difference may be due to loss of factors enhancing 
peroxidation in intact erythrocytes in the extraction 
procedure. It is likely that damage to erythrocyte mem­
branes caused by increased susceptibility to lipid 
peroxidation may be a factor in some of the 
haematological features of sickle cell disease.
INTRODUCTION
Although sickle cell disease (SCD) is well understood 
as an abnormality of the haemoglobin molecule, the 
complete mechanism by which this defect is expressed 
at the clinical level is not fully understood12 even 
though secondary alterations in red cell metabolism 
and membrane structure and function have been de­
scribed.3 A considerable body of evidence suggests that 
defective function of the sickle cell membrane may 
contribute to some of the manifestations of the disease. 
The membrane defect is demonstrated in the perma­
nently deformed morphology of the irreversibly sick- 
led cell, binding of haemoglobin to the erythrocyte
Department of biochemistry 
College o f Medical Sciences 
University o f Calabar 
Calabar, Nigeria
z!7
CENTRAL AFRICAN
JOURNAL OF MEDICINE
Vol. 40, No. 8,1994
membrane, easy rupturing of the sickle cell, decreased 
filterability of sickle erythrocytes and increased cation 
leak from sickle cells when they are deoxygenated.24 
Equally, it has been established that the circulation of 
membrane damaged, rigid and irregularly shaped eryth­
rocytes resulting in vascular occlusion are involved in 
this disease.2
However, the genesis of the membrane defect re­
mains not fully known and relatively few examinations 
of the factors precipitating the collapse of the mem­
brane have been made. Increased susceptibility of the 
membrane to peroxidant threats may be one of those 
factors. It is known that the erythrocyte membrane is 
rich in polyunsaturated fatty acids and that these mol­
ecules are themselves highly susceptible to peroxidation.
During its life span in the circulation the erythrocyte 
is exposed to small amounts of hydrogen peroxide 
(H202) generated as a result of normal metabolic pro­
cess to greater amounts resulting from interactions of 
certain oxidant drugs and their metabolites with 
oxyhaemoglobin.5 Intracellular H2()2 may cause 
peroxidation of membrane lipids with the formation of 
breakdown products, including oxygen radicals and 
m alonyldialdehyde (M DA).6 M em brane lipid 
peroxidation has been recognized as a deleterious pro­
cess leading to cell damage in various tissues.7 MDA 
itself has been found capable of reacting with 
aminophospholipids and proteins of membrane to pro­
duce cross linking of membrane constituents which can 
cause rigidity and decreased deformability of red blood 
cells.6
The present study was undertaken to examine the 
susceptibility of sickle cell membrane to lipid peroxi­
dation and the possible role of lipid peroxidation in the 
damage to the membrane and reduced erythrocyte 
survival in sickle cell disease.
MATERIALS AND METHODS
Blood was drawn from healthy adult male volunteers or 
sickle cell patients who were in a steady state uncom­
plicated by infection, clinical orhaematological crises, 
into ETDA vials and the plasma separated. Red blood 
cells were routinely washed in cold normal saline and 
suspended in phosphate-saline medium (1 vol. of po­
tassium phosphate buffer, 0,1 M, pH 7,3 mixed with 9 
vol. of 0,15 M NaCl). Saline washed, packed red cells 
were lysed in four times their volume of cold distilled 
water.
An aliquot of the lysate was used to determine the 
haemoglobin (Hb) content by the cynmethaemoglobin 
method described by Dacie and Lewis.' The lipids in 
the remainder of the lysate were extracted according to 
the method of Rose and Oklander.9 Peroxidation of 
intact erythrocytes, isolated lipids of intact erythro­
cytes or their ghosts was carried out by exposure to 
hydrogen peroxide which was added directly to the 
medium as described by Kurien and Iyer.'® 
Erythrocyte ghosts were prepared according to Dodge 
et al". The extent of peroxidation of the erythrocyte 
and its membrane lipids was measured by estimating 
the MDA formed using the thiobarbituric acid (TBA) 
reaction described by Stocks and Dormandy.12 Data 
were analyzed by using student’s t-test.
RESULTS
As seen in Table I, a significantly higher level of MDA 
formation was observed when intact red cells from 
SCD patients were exposed to H202 added directly to 
the red cell suspension, compared to that of erythro­
cytes from normal subjects, (p < 0,001). The same was 
true of isolated lipids from intact red cells or their 
ghosts, but the amount of MDA formed was less. The 
formation of MDA, a secondary fragmentation product 
of polyunsaturated fatty acid peroxides, is a specific, 
sensitive and extensively used measure of lipid 
peroxidation.12
Table I: Lipid peroxidation in normal and sickle 
erythrocytes (n moles ofMDA/g Hb).1
Lipid Source n moles M D A  (13)* P
Intact red cell normal 
Normal 198,6 + 24
Sickle cell 408 ,7  + 33 < 0 ,0 1
Isolated lipids from 
intact red cells 
Normal 78 ,5  + 11
Sickle cell 1 4 5 ,8 + 1 2 < 0 ,01
Isolated lipids from  
red cell ghosts 
Normal 63 ,9  + 10
Sickle c e l 187,8 + 14 < 0 ,0 1
’ ■ Values are expressed as means ±  SEM.
*  Number o f eases studied given in parenthesis.
218
CENTRAL AFRICAN
JOURNAL OF MEDICINE
Vol. 40, No.8,1994
DISCUSSION
Certain abnormalities that occur in SCD are difficult to 
relate directly to the presence of HbS or the sickling 
phenomenon.1 It is now known that a combination of 
intrinsic erythrocyte abnormalities and other factors 
are likely to be epiphenomenologic. In this study it is 
shown that the susceptibility to peroxidation of intact 
erythrocyte and isolated lipids of intact ertlirocytes or 
their ghosts is appreciably increased in SCD compared 
to normal subjects. This is suggestive that in SCD the 
erythrocyte membrane is more vulnerable to lipid 
peroxidation and that this increased vulnerability may 
be a feature which contributes to the easy collapse of 
the erythrocyte membrane in SCD.
The increased vulnerability may be due to disorgani­
zation of the Hb molecule, which as a haem-protein, is 
apowerful catalyst of lipid peroxidation,13 distortion of 
normal Hb-membrane and membrane-membrane rela­
tions, and plasma and erythrocyte vitamin E deple­
tion.14 In fact, increased TBA -  reactive materials have 
been demonstrated to precede haemolysis of sickle 
erthrocytes15 and abnormal cadon leaks that develop in 
SCD damaged membranes.1617
It appears from the above consideration that in SCD 
11,02 may be generated in increased amounts and not 
physiologically well managed. If its behaviour in this 
study is a guide, 11,0, is a potent oxidant initiating the 
production of a steady supply of free radicals which 
degrade membrane phospholipids, followed by the 
collapse of the membrane.
The disparity in the extent of peroxidation between 
lipids of intact erthrocytes compared to isolated lipids 
of intact erythrocytes or their ghosts as measured by 
MDA formation (Table I) may be related to loss of 
unsaturated lipids in the extraction procedure. This 
observation that the extent of peroxidation was signifi­
cantly higher for intact erythrocytes than for lipids 
isolated from intact erythrocytes or their gtyosts is 
consistent with the report of Stocks and Dormandy12 
that isolated lipids yield much less MDA compared to 
lipids of intact red cells. However, MDA formation 
was still higher for isolated lipids from sickle erythro­
cytes compared to normal erythrocytes.
From the above consideration it is plausible that in 
SCD, the primary event of crystallization of HbS is 
aided by damage to the cell membrane caused by 
increased peroxidation of membrane constituents junong 
other factors. Excessive and symmetric fragmentation
219
and loss of cell membrane due to peroxidation may 
precipitate irreversible sickling of erythrocytes con­
taining HbS. These cells will not return to a normal 
configuration even upon oxygenation. In fact, these 
cells can be lysed and the solubilised Hb removed, and 
the membrane will remain in the sickled configuration2 
indicating permanent damage to the membrane.
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Increased susceptibility of erythrocyte membrane lipids to peroxidation in sickle cell disease

Erythrocytes from normal subjects and from cases of sickle cell disease were exposed to hydrogen peroxide and the extent of membrane lipid peroxidation studied. Significantly increased peroxidation was observed in intact erythrocytes, isolated lipids from intact erythrocytes or their ghosts compared with normal controls. However, the extent of lipid peroxidation was less for isolated lipids of intact erythrocytes or their ghosts. This difference may be due to loss of factors enhancing peroxidation in intact erythrocytes in the extraction procedure. It is likely that damage to erythrocyte membranes caused by increased susceptibility to lipid peroxidation may be a factor in some of the haematological features of sickle cell disease

Essien,  E.U.

National Research Database of Zimbabwe

https://opendocs.ids.ac.uk/opendocs/bitstream/20.500.12413/7049/1/Eissien%2c%20%20%20%20%20%20%20%20%20EU%20%20The%20%20CAJM%20%20vol.%2040%2c%20no.%208.pdf

Increased susceptibility of erythrocyte membrane lipids to peroxidation in sickle cell disease

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