| VOLUME 20, NUMBER 1 |
JANUARY/FEBRUARY 2007 |
Short Reports 11
Antioxidant vitamin levels in
sickle cell disorders
Debes Ray, Pradeep
Deshmukh,
Kalyan Goswami, Neelam Garg
Abstract
Background.
Sickle cell disorder is a
haemoglobinopathy prevalent in the Vidharbha region of
Maharashtra, central India. With recent evidence of oxidative
stress in sickle haemoglobinopathy, a possible deficiency of
antioxidant vitamins was suspected.
Methods. We
measured plasma vitamin E, vitamin C and beta-carotene levels
in persons with heterozygous (n=80) and homozygous
sickle cell state (n=20), and suitable healthy controls
for these groups (n=100 and 66, respectively) in a
community-based study in the villages near our institution.
Results. Subjects with heterozygous sickle cell trait
had lower vitamin E levels than their respective controls
(p<0.05). Subjects with homozygous sickle cell disease had
lower levels of all three vitamins (p<0.05). Vitamins E and C
levels showed a significant positive correlation in both forms
of sickle cell disorder.
Conclusion. Our findings suggest that there is
depletion of the antioxidant vitamins, particularly in severe
forms of sickle cell disorder. A trial of administration of
therapeutic doses of vitamin E in this condition is warranted.
Natl Med J India 2007;20:11–13
Introduction
Sickle cell disorder is a haemoglobinopathy caused by a
point mutation in the b
chain of the globin gene.1
It has an autosomal recessive inheritance, and clinical
severity varies widely from the milder sickle cell trait
(heterozygous) to sickle cell anaemia (homozygous).2 The mutant haemoglobin undergoes aberrant polymerization on
deoxygenation, resulting in permanent distortion of the red
blood cells (RBCs) into characteristic irreversible sickled
cells.3
This disorder is the commonest inheritable haemoglobin-associated
disease affecting humans, and is predominantly seen in Africa
and Southeast Asia.2
In India, it is more common in the central and southern parts.4
This study was done in central India where the disease has a
higher prevalence than in the rest of India.5
Sickle cell anaemia is emerging as an important model of
oxidative stress.6 Since RBCs carry
oxygen to the body tissues, they are already rich in oxidative
fuel. Their distinctive structural features also make them
susceptible to an oxidant assault.7 In
disorders with abnormal haemoglobin, such as sickle cell
disorder, the haemoglobin stabilizing capacity is impaired,
making the RBCs even more vulnerable to oxidative stress. This
may overwhelm the antioxidant defence system.8
Since some vitamins play an essential role in the
antioxidant defence system,9-11 we
compared the levels of these antioxidant vitamins (ascorbic
acid, alpha-tocopherol and beta-carotene) in subjects with
heterozygous and homozygous sickle cell disorder, and age- and
sex-matched healthy controls from the same area with a similar
socioeconomic background.
Methods
We conducted this community-based case–control study in
villages located close to the Mahatma Gandhi Institute of
Medical Sciences (MGIMS) in Wardha district. In an initial
field survey, the population of these villages was screened
using the dithionite turbidity tube test for sickling.12
Those testing positive underwent haemoglobin electrophoresis
for confirmation of the diagnosis, and were classified into a
heterozygous or homozygous sickle cell state. A total of 80
heterozygous and 20 homozygous subjects were included in the
study. Apparently healthy individuals (without a history of
any major illness or blood transfusion) from a similar
socioeconomic background were selected from the same villages.
A total of 100 and 66 individuals consented to participate in
the study as controls for the heterozygous and homozygous
subject groups, respectively, after they were matched for age
and sex. About 5 ml of blood was collected in an EDTA vial
from the antecubital vein after taking informed consent.
Haemoglobin electrophoresis was done on cellulose acetate
membrane at an alkaline pH after preparation of the
haemolysate from 2 ml of this EDTA-blood.13
Plasma was separated by centrifuging the blood at
3000 rpm for 10 minutes. It was stored at –20 °C and used to
estimate the levels of beta-carotene and vitamin E. Vitamin C
was estimated immediately. To exclude any obvious difference
in nutritional status between the subjects and controls, the
albumin level was measured by the standard method.14
Estimation of ascorbic acid
Ascorbic acid in plasma is oxidized by Cu2+
to form dehydroascorbic acid that reacts with acidic
2,4-dinitrophenyl hydrazine to form red bis-hydrazone, which
was measured spectrophotometrically.15
Briefly, 0.5 ml plasma was added to 2 ml of freshly prepared
metaphosphoric acid (6 g/dl in water), mixed well and
centrifuged at 2500 g for 10 minutes. To 1.2 ml of the
supernatant or similar volume of metaphosphoric acid blank,
0.4 ml of a reagent containing thiourea (5 g/dl), copper
sulphate (0.6 g/dl) and 2,4-dinitrophenyl hydrazine (2 g/dl in
4.5 mol/L sulphuric acid) solutions mixed in a ratio of 1:1:20
was added and incubated in a 37 °C water bath for 3 hours,
followed by cooling for 10 minutes in an ice bath. To all
tubes, 2 ml of cold sulphuric acid (concentrate) was then
added and the optical density recorded at 520 nm. The
concentration of ascorbic acid in plasma was obtained using a
standard curve.
Estimation of beta-carotene
Plasma beta-carotene level was assayed
using a spectrophotometric method after extraction into
petroleum ether.16 In brief, 1 ml of
plasma was mixed with 1 ml of alcohol in a capped centrifuge
tube to precipitate the proteins. Then, 3 ml of petroleum
ether was added, and the tube shaken vigorously for 5 minutes
followed by centrifugation at 2500 rpm for 10 minutes. The
optical density of 2 ml of petroleum ether extract was then
measured at 450 nm using petroleum ether as blank. The
concentration of plasma beta-carotene was obtained using a
standard curve.
Estimation of alpha tocopherol (vitamin E)
Plasma vitamin E level was estimated using
the spectrophotometric method of Bieri et al.17
in which tocopherol is oxidized to tocopheryl quinone by
ferric chloride and the resultant ferrous ion forms a red-coloured
complex with dipyridyl reagent. Briefly, 1.5 ml of petroleum
ether extract of plasma (prepared as described for
beta-carotene estimation) was dried in a water bath at 50 °C
and the residue was dissolved in 1 ml of ethanol. To this, 1
ml of 0.2% 2,2'-dipyridyl and 100
ml of 0.1% ferric chloride hexahydrate were added. The
optical density was measured after 2 minutes at 520 nm. The
concentration of vitamin E in plasma was obtained using a
standard curve.
Statistical analysis
Intergroup comparisons were done using the t test and
p<0.05 was considered significant. Pearson correlation
analysis was done to study the relationship between the levels
of vitamin E and the other two vitamins.
Results
Eighty heterozygous subjects (median age: 23 [range 16–35]
years; 61% men) and 100 controls (median age: 26 [range 16–34]
years; 62% men), and 20 homozygous subjects (median age: 9.5
[range 7–12] years; 60% men) along with 66 controls (median
age: 10 [range 5–14] years; 59% men) were enrolled. Hence,
both these comparison groups were considered to be age- and
sex-matched. Mean (SD) albumin levels were similar in the
heterozygous subjects and their controls (4.1 [0.4] and
4.1 [0.4] g/dl, respectively; p=0.93), and in homozygous
subjects and their controls (4.0 [0.5] and 4.1 [0.4] g/dl,
respectively; p=0.60).
Among subjects with the sickle cell trait, the vitamin E
level was significantly lower than that in their respective
controls (p<0.05; Table I), whereas those of the other two
vitamins showed no difference. In subjects with homozygous
sickle cell anaemia, levels of all the vitamins were lower
than those in their respective controls (Table II).
The correlation coefficient between vitamin E and vitamin C
was 0.53 and 0.77 in heterozygous and homozygous subjects,
respectively (p<0.001 for both) against lower
correlation coefficient values of 0.41 and 0.46 in the
corresponding control groups (p<0.001 for both). Levels
of beta-carotene did not show significant
Table I. Mean (SD) levels of plasma antioxidant
vitamins in heterozygous sickle cell cases (HbAS) and controls
(HbAA)
| Parameters |
Controls (n=100) |
Heterozygous (n=80) |
p value |
| Vitamin C
(mg/dl) |
0.87 (0.17)
|
0.83 (0.11) |
0.069 |
| Vitamin E
(mg/dl) |
0.87 (0.11)
|
0.61 (0.06) |
<0.001 |
|
Beta-carotene (mg/dl) |
73.07
(5.67) |
73.04
(5.63) |
<0.974 |
correlation with those of either vitamin E
or vitamin C in any group.
Discussion
In sickle cell disease, iron decompartmentalization due to
unstable haemoglobin has been thought to be a cause of
oxidative stress.18
One study has reported redox imbalance in this condition.19
Moreover, there is some evidence that supplementation with
antioxidants leads to a short term improvement in clinical
parameters.20
The reported normal value of vitamin C is 0.4–1.5 mg/dl,
vitamin E 0.5–1.8 mg/dl and beta-carotene 10–85 µg/dl.15 The observed values of all the vitamins
for both the control groups against heterozygous and
homozygous subjects were within this range. In sickle cell
disorder, particularly in homozygous subjects, the nutritional
status may be affected leading to depletion of antioxidant
vitamins. We therefore measured the albumin level as a
surrogate marker of nutritional status and found no difference
between the subjects and controls.
Both vitamins E and C have a protective role against
oxidative membrane attack, while carotenoids act at a low
oxygen tension.21
However, vitamin E, carotene and vitamin C possibly have an
interrelationship.22
Our study revealed low levels of all antioxidant vitamins in
subjects with the sickle cell trait and sickle cell anaemia
compared with controls. However, only the vitamin E level was
significantly lower in the heterozygous subjects compared with
controls. In homozygous subjects levels of all three vitamins
were significantly lower compared with controls. This finding
probably indicates the relative severity of oxidative stress
in relation to the relative severity of the sickle cell
disorder.
Vitamin E is a chain-breaking antioxidant with a
membrane-protective role in almost all cells.10
Thus, in view of the evidence of multiple membrane defects in
this condition,23
it is possible that membrane damage might be a critical factor
in this disorder. Interestingly, oxidatively modified
membrane-associated proteins are currently implicated in the
formation of irreversible sickle cells, which is leading to a
paradigm shift from the older cross-linking theory.2
In a small trial of patients with sickle haemoglobinopathy,
vitamin E supplementation was shown to reduce sickling.24
On closer observation of the data it becomes evident that the
level of difference in vitamin E in heterozygous cases as
compared with the respective controls was less than the
corresponding difference obtained in homozygous cases. Hence,
it is possible that in the presence of oxidative stress,
vitamin E gets depleted in proportion to the severity of
oxidative stress. However, this needs further studies.
In the heterozygous condition (supposedly a milder form of
the disease), the vitamin C level was low but not
significantly different from that of controls. However, in the
homozygous state (more severe form of the disease), the
difference in vitamin C level was almost similar to that of
vitamin E. This suggests that while vitamin C is not involved
in the first line of the antioxidant system, it might play a
role as a replenishing agent for vitamin E.25
This is further supported by the significant positive
correlation between
Table II. Mean (SD) levels of plasma antioxidant vitamins
in homozygous sickle cell cases (HbSS) and controls (HbAA)
| Parameters |
Controls (n=100) |
Heterozygous (n=80) |
p
value |
| Vitamin C (mg/dl) |
0.82
(0.18) |
0.51
(0.12) |
<0.001 |
| Vitamin E (mg/dl) |
0.83
(0.07) |
0.47
(0.04) |
<0.001 |
|
Beta-carotene (mg/dl) |
77.24
(5.58) |
55.61
(6.36) |
<0.001 |
the levels of vitamins C and E, with a
step-wise increase in these coefficients from the heterozygous
to the homozygous state. In both the homozygous and
heterozygous states, the difference in the beta-carotene
levels was the least, indicating that it has a less direct
role.
Our study suggests that levels of antioxidant vitamins are
low in subjects with sickle cell disorder. We feel this
warrants the use of antioxidant vitamins as a therapeutic
measure in subjects with this disorder.
Acknowledgements
We thank Mr D. Mehta, President, Kasturba
Health Society and
Dr (Mrs) P. Narang, Dean, Mahatma Gandhi Institute of Medical
Sciences for their encouragement. We also thank Professor
M.V.R. Reddy, Department of Biochemistry and Professor B.S.
Garg, Department of Community Medicine at our institution for
their active support.
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Mahatma Gandhi Institute of Medical
Sciences, Sevagram,
Wardha 442102, Maharashtra
Debes Ray, Kalyan Goswami, Neelam Garg Department of
Biochemistry
Pradeep Deshmukh Department of Community Medicine
Correspondence to Neelam Garg; jbtdrc_wda2@sancharnet.in
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