Journal of Radioanalytical and Nuclear Chemistry Articles, Vol. 168, No. 1 (1993) 233-242
TRACE ELEMENTS IN SICKLE CELL DISEASE M. A. DUROSINMI,* J. O. OJO,** A. K OLUWOLE,** O. A. AKANLE, ~ W. ARSHED,*** N. M. SPYROU***
*Department of Haematology andImmunology, ObafemiAwolowo University, lle-lfe (Nigeria) **Departmentof Physics, ObafemiAwolowo University, lle-lfe, (Nigeria) **~ of Physics, UniversiO,of Surrey, Guldford (UK)
(Received September 22, 1992)
Instrumental Neutron Activation Analysis (INAA) and Proton-Induced X-ray Emission (PIXE) analysis (employed as a complementary technique) have been used to determine the concentration of 11 elements in blood samples and its components erythrocytes and plasma obtained from three groups of subjects in Nigeria viz: sickle cell anaemia (SCA) subjects, subjets with sickle cell trait and normal control subjects. The results suggest that SCA subjects have significantly higher concentrations of Na, CI, Ca and Cu in their whole blood and erythrocytes and a higher concentration of CI and Cu in their plasma relative to the control subjects. Furthermore, a significantly lower concentration of K, be, Zn, Se, Br and Rb were found in the whole blood and erythrocytes of the SCA subjects as compared to the controls while the concentration of K and Fe in the plasma of the SCA subjects were however, found to be significantly higher than that of the control group. The study also shows that there was no significant differences between the concentration of these 11 elements in the group with sickle cell trait and the normal control group.
Introduction Sickle cell disease is an inherited disorder of haemoglobin formation of which the most c o m m o n and severe type is sickle cell anaemia (SCA). There are about 300 different types of haemoglobin and the most c o m m o n type is haemoglobin A (HbA).
Most people
inherit HbA from both parents but sickle cell anaemia occurs when most of the haemoglobin in the red cell (over fifty percent) is of sickle haemoglobin (HbS) type. It is given this name because it causes the red blood cell to become sickle or crescent shape when it gives up oxygen. Although SCA is present from birth, symptoms are rare before the ages of 3 to 6 months since a large percentage of the erythrocyte haemoglobin is of the fetal type (HbF). As more HbS replaces HbF in the subject, the main symptoms, episodes of anaemia, pain and infection, called crises, become manifest due to irreversible sickling of the erythrocytes when the HhS molecules polymerize, invariably leading to vaso-occlusion in the smaller capillaries.
Elsevier Sequoia S. A,, Lausanne A kad(miai Kiad6, Badapest
M. A. DUROSINMI et al.: TRACE ELEMENTS IN SICKLE CELL
The disease is most common in Iropical Africa but with a lower incidence in the Mediterranean region, Southern Arabia and India. It also occurs in emigr6 populations such as the American and West Indian negroes. It is reported that the disease is responsible for about 80,000 deaths annually throughout the world[I]. The incidence of sickle cell in West Africa ranges from 20% to as high as 30% and in Nigeria, about 2% of the population are believed to be affected. The disease has therefore, been a matter of concern to clinicians and scientists for over a century. Attempts have been made over a number of years, to treat the disease by modifying the haemoglobin S molecule so as to suppress the sickling process, but in recent years, trace element metabolism have been receiving increasing attention in sickle cell anaemia research and attempts have been made to relate the concentration of the elements to the sickling process. Significant differences have been reported in the concentration of such elements as Na, K, Ca, Zn and Cu by various authors and Zn which has been the most widely studied element in sickle cell disease has been reported to improve filterability of sickled cells and has anticalcium as well as anti-sickling effects. When given as oral supplement, a beneficial role on growth and development has been reported. The aim of this study is therefore, to determine elemental levels as well as to study the distribution of these elements in whole blood, erythrocyte and plasma collected from sickle cell subjects and normal control subjects in a Nigerian population. Blood samples were also collected from subjects diagnosed to have the sickle cell trait which occurs when one of the parents passes on the gene for the usual adult haemoglobin (HbA) and the other passes on that for the sickle haemoglobin (HbS) resulting in the formation of the HbAS molecules. Initial findings of this on-going study are presented.
Experimental
Method
and Analysis
Blood samples were collected from three groups of subjects in a Nigerian population viz: those diagnosed to be suffering from sickle cell anaemia (HbSS) which consisted of 17 females and 2 males with their ages ranging from 15-32 years; those with sickle cell trait (HbAS) consisting of 11 females and 7 males and with ages ranging from 13 - 50 years and the normal control group (HbAA) which also consists of 13 females and 14 males, with ages ranging from 17 - 60 years. The diagnosis of sickling condition was established by history, physical examination and haematological studies including electrophoresis of haemoglobin.
234
M. A. DUROS1NMI et al.: TRACE ELEMENTS IN SICKLE CELL
The normal control group and those with sickle cell trait were healthy and non-anaemic. Weight and height of each of the subjects -were also noted. About 10ml of blood were collected into polyethylene containers from each subject using scalp vein set with polyethylene catheter but after 1-2 ml of blood had flowed through and discarded, in order to reduce contamination. Immediately after collection, 20 gl of heparin was added to prevent coagulation and to allow for subsequent separation of the samples.
Samples of the heparin used were also
analyzed for its trace element content. About 2-3 ml of the blood was retained in a separate polytheylene containers as whole blood and the rest separated into blood components: erythrocytes and plasma by centrifugation at 3000 rpm for 15 rains.
Thus three samples
(whole blood, erythrocytes and plasma) were available for analysis from each subject.
The
samples were kept in a low temperature freezer and then freeze-dried for 24 hours in their individual containers using an Edward Vacuum freeze dryer, noting the weights of the samples before and after freeze-drying. The freeze-dried samples were crushed with polyethylene spatula into a fine powder in their individual containers and pellets were made from the fine powder to give reproducible irradiation and counting geometries with sample masses ranging between 50-100rag dry weight. Clean pre washed polyethylene containers were used throughout the procedures. Two techniques were used for trace element analysis of the samples: (i) (ii)
Instrumental Neutron Activation Analysis (INAA) and Proton-Induced X-ray Emission analysis which was used as a complementary technique to INAA.
Instrumental Neutron Activation Analysis (INAA) Powdered samples were made into pellets and placed in a small polyethylene capsules. These were double contained in a bigger polyethylene container and heat-sealed, ready for irradiation. The samples were irradiated using the Core Tube irradiation facility at the Imperial College Reactor Centre, Ascot, UK. The samples were irradiated for a period of .65 hours and counted for 1800s and 5000s on a high resolution Ge(li) detector at the University of Surrey after waiting periods of 3 and 10 days respectively in order to determine the activity of the long lived isotopes induced in blood. To determine the elemental concentrations, the relative method was used, hence two multi elemental standards, Bowen's Kale and IAEA Animal blood were also prepared and 235
M. A. DUROS|NMI el al.:T R A C E ELEMEN'I"S IN SICKLE CELL
~md~ated ~al~g w~h ~
szm~le~,. Neulron flux cone~fions were made by irradiating with
~he m m # e s an~ s,mmLards a fifth ~
m
wire (0.t27mm dfick). The modified eompmer
code S A M t ~ , was ~ e d f~r ~ e c ~ a a~alys~s.
Prot0n-l~duced
X-ray emissior~ CPIXE) Analysis
Samp~e~ and starrdm-ds were a l ~ pellefi.~l and ,/hese were ~x~ek side by ~de on a 3nma ~ c k ~ m ~ n
~rget plate ~
~he help of d~mble-s~ded ~he~ive tape aud then c o , ted
~fi~h a ~ n ( 2 0 - 3 ~ ) ) a , a ~ of ~ Anzty~L~ :of ~
~#e~
L~c~z~It ~ me ~ e ~ a~g co~c~e~ ~ h
~r
carried oat u.~hag a 2 MeV proton b~am, under vacuum,
'z~ ~
the U r m v e ~ ~
~ make ~he sarap~es conducting.
Io~ e ~
~k~eri ~ 501) ~
of 3urmy's Van de Gmaf accelerator. ph~
~aqrpre.~ fine $~nal from ~ow e n ~ r Shnulmneous RBS s ~ c ~
~ameter spo~ ~ze on ~ e sample
$~(Li) delec~tor f i ~
PIXE specwa were
w~th a 350 p m mylar filter m
x-rays in order to reduce dead ~
problems.
were also coUec~d ~,Ath a surface barrier demcmr. Analys~s of
PIXE s p e c ~ for de~ernfinafion of peak areas was carded out by the PIXAN computer code.
Results and Discussion
"I'ne elemental concenn-ations delennined in whole blood, crythrocytes and plasma using both I~chniques (INAA & PIXE) for 11 elements are presented in Tables 1,2 and 3 for the sickle cell anaemia (SCA) subjects, the normal control group and subjects with sickle cell traits.
This is presented in t e r m s of their ranges of concentrations, the arithmetic and
geometric means. Both techniques allow the determination of the concentration of K, Fe, Zn and Br. The ranges of concentrations obtained for these common elements were found to be the same. However, in addition to these four elements, INAA allows the determination of the concentration of Na, Cr, Se and Rb while PIXE analysis allows the determination of the concentration of (21, Ca and Cu. Moreover, PIXE technique has a lower detection limit than INAA for K and Fe in plasma, hence the values obtained from PIXE analysis were used in these cases. Significant differences in concentration was observed for the 11 elements detected in the whole blood, erythrocytes and plasma of the three groups but with no significant differences in the elemental concentration obtained between the group with sickle cell traits
236
M. A. DUROSINMI el al.: TRACE ELEMENTS IN SICKLE CELL
Table
1:
Range
of
concentrations,
geometric (S$),
means
controls
arithmetic
SS
Element
means and standard deviations, sickle cell anaemia subjects sickle cell trait (AS)
in the whole blood of and subjects with
(AA)
AA
AS
NO
Range
Arith mean ~D
Geom mean
No
Range
Arith mean ISD
Geom mean
No
Range
A. mean +SD
Geom mean
Na
29
10344170
2341 --+ 746
2233
27
14512468
1807+ 298
1782
18
14912441
1891 +273
1873
K
18
5971528
1234+-322
1189
13
12223373
1849+572
1781
Ii
11672230
1697 +347
1664
Cr
11
8.020.07
0.05--+ 0.02
0.04
12
0.03 -0.18
0.09+0.06
0.08
9
0.030.27
0.I0 + 0.09
0.07
Fe
19
183516
255-+ 82
245
27
318660
471+75
465
18
106945
445+ 159
416
Zn
18
1.404.34
2.89+0.73
2.80
27
2.4913.56
5.27+1.97
5.03
18
2.2615.89
5.21 -+ 2.94
4.74
Se
18
8.030.12
0.87+0.82
0.07
27
0.040.19
0.09+ 0.04
0.08
17
0.050.18
0.I0 --+ 0.04
Br
17
0.963.36
2.12_+ 0.74
1.97
26
1.235.47
2.45+1.09
2.25
18
1,074.13
2.45 +0.82
2.32
1.295.29
2.27_+ 1.02
2.08
2.596.23
3.97+0.87
3.88
1.157.11
3.82 + 1.33
3,58
66-
86-+ 16
27
38101
69+13
67
119 1.12.9
1.9_+ 0.5
27
0.752.12
14643243
2164_+ 512
27
10252117
Rb
Ca Cu
C1
19
19 19 19
85
27
1.9 2108
18
0.09
68
18
51-84
68+9
1.27+ 0.30
1.23
18
0.792.27
1.23 +-0.4
1.18
1549+260
1526
18
8692602
1554 -+434
1499
and the normal control group tonfirming that this group of subjects are normal healthy individuals. A higher mean concentration was observed for the elements Na and Ca in the whole blood and erythrocytes of the SCA subjects as compared with the normal control groups and this was found to be very significant when statistically tested using the students' t-test. Table 4 lists the calculated t-test and their level of significance for the 11 elements. In the plasma however, a decrease in the concentration of Na was observed in the SCA subjects as compared to the controls even though it was not statistically significant, while there was no difference in the concentration of Ca in the two groups.
It was also observed that the
concentration of K in the whole blood and erythrocytes of the SCA subjects was significantly less than that of the normal controls while the concentration of this element in plasma was significantly higher than that of the normal control subjects.
Elevated Ca level in the
erythrocytes have been reported by a number of other workers [2,3,4,5,6], while Ney et al and
237
M. A. DUROSINMI et ah: TRACE ELEMENTS IN SICKLE CELL
Table 2: Range of concentrations, arithmetic means and standard deviations, geometric means in the erythrocytes of the sickle cell anaemia subjects (SS), controls (AA) and subjects with sickle cell trait (AS) I
SS
)
AA
AS
i
Element NO
Range
Arith mean •
Geom mean
No
Range
Arith mean• SD
Geom mean
No
Range
A. mead •
Geom mean
Na
19
2392948
1276• 759
1057
26
3331540
865• 283
818
15
4111710
939• 395
863
K
15
8783641
22191 813
2070
17
26843790
3155• 333
3139
14
17154577
2817 •
7
0.05 0.25
0.16 • 0.08
0.04 0.67
0.16• 0.16
0.04 0.19
0.14 0.19
0.89
1941041
683• 215
6611458
917• 136
6661302
870• 167
856
Cr
0.14
19
644
27
6.63
27
4,52 15.35
9.49• 2.46
27
0.08 0.28
0.14 • 0.06
1.90
26
1.18 20.13
5.02 9 6.06
Fe
19
Zn
19
2.77 13.41
7.19 9 2.87
18
0.05 0.19
0.10 • 0.04
Br
16
0.38 i1.41
2.43 • 2.51
2.49
4.64
4.56
7.50
Rb
19
7- . 4 7
1~ . 3 4
4.46
27
-
•
Ca
19
21116
51•
4.6
27
14-51
29• i0
Cu
19
0.98 2.51
1.68 • 0.48
1,61
27
0.52 1.65
1.0 • 0.3
C1
19
2542078
882• 473
771
27
295952
639• 182
Se
0.10
10.54
1.58
2735
0.ii
i0
987
18
9.18
18
5.75 12.93
9.18 • 2.08
8.95
18
0.06 0.26
0.1~ • 0.05
0.13
2.79
16
0.81 16.54
4.71 • 5.19
5.11
7.30
7.33
18
-
•
1.68
7.14
28
18
21-66
35• ii
34
0.96
18
0.48 1.45
1.02 • 0.24
0.26
609
18
2271128
742• 214
703
0.13
12.04
2.92
Johnson et al [7,8] demonstrated in a controlled laboratory experiment that shear induced red blood cell deformation into elliptical shaped causes a leakage of K from the cell which however results in the influx of Na into the cell. The increased concentration of Na and Ca as well as the decreased concentration of K in the erythrocytes was therefore, thought to be a result of an increased permeability of the red cell membrane during sickling of the red cell when it gives up oxygen. Normal red cell has low permeability for Ca++ ions but sickling of the red cell result in the opening of membrane pathways which allow an influx of Ca into the cell. The effects of calcium in increasing the rigidity of the red cell membrane has been wellreported. Tosteson et al [9,10] also observed that if sickling is prevented, there was neither K loss nor Na gain into the cell. Moreover, our findings of an increased concentration of K and a decreased concentration of Na in the plasma of the sickle cell subjects tend to support this hypothesis, but as noted earlier however, no significant differences was observed in the plasma Ca concentrations of the two groups, though Adeyefa et al [11] found a significantly
238
M. A. DUROSINMI et al.: TRACE ELEMENTS IN SICKLE CELL
Table 3: R a n g e o f concentrations, arithmetic means and standard deviations, geometric means in the p l a s m a of the sickle cell anaemia subjects (SS), controls (AA) a n d subjects w i t h sickle cell trait (AS) ss
AS
AA
Element No
Range
Arith mean• SD
Geom mean
No
Range
Arith mean• SD
Geom mean
No
Range
A mean •
Na
18
20534478
2685• 602
2631
27
18443895
2804• 501
2760
17
20883389
2793 •
K
18
133465
238• 92
223
27
94 344
172 •
165
18
344195
166 •
0.02 0.05
0.03 • 0.01
0.007 0.08
0.03 • 0,02
0.02
4
0.02 0.05
0.03 • 0.03
0.03
1.074.8
0.482.37
1.31• 0.5
1.21
18
0.~4-0
1.53 i 0.76
1.41
0.282.67
0.74• 0.44
0.67
0.433.26
0.86 • 0.64
0.76
0.020.09
0.06• 0.02
0.05
0.030-09
0.06 ! 0.02
0.05
1.006.38
2.66• 1.27
2.43
1.354.48
2.52 ~ 0.82
2.40
0.211.31
0.40• 0.21
0.37
0.271.45
0.43 • 0.28
0.38
Cr
7
Fe
18
Zn
Se
Br
Rb
19
19
39
18
0.03
8
2.7• 0.96
2.43
27
0.221.86
0.54• 0.38
0.44
0.030.24
0.06• 0.04
0.05
O.925.39
2.18 • 1.05
1.88
0.040.82
0.38• 8.18
0.33
Ca
19
77132
98•
Cu
39
8.982.88 20083248
C1
19
27
27
27
27
17
17
17
36
G. mean
2767 166
97
27
54131
95i 23
92
18
74128
97i 12
1.6 • 0.5
1.52
27
8.362.39
].0 • 0.4
0.97
18
0.58i_71
1.05 • 0.26
1.01
2523• 318
2503
12663000
2272i 460
2218
19372974
2462 • 293
2444
27
18
96
Table 4: t-test of differences between arithmetic means of elemental concentrations in whole blood, erythrooytes and plasma of sickle cell anaemia and control subjects with their significance levels
Element
I ~'~
Whole Bloodl
!
Erythrocyte
1.v.~ cp~
Plasma
1o~,z (pl
z*vol 4pJ
Na
44
3,37
<0. 001
43
2.54
<0.01
43
-0,72
<0.2
K
21
-3.04
<0. 005
30
-4.36
<0. 001
44
3.08
<0. 005
Cr
21
-2.10
<0. 025
24
NSD
Fe
44
-9.25
<0. 081
44
-4.52
<0. 001
41
5.8
<0. 001
Zn
43
-4.89
<0. 001
44
-2.91
<0. 005
44
-i. 6
<0.1
5e
44
-0.83
<0.2
43
-2.48
<0.01
44
NSD
Br
41
-1 .09
<0.2
40
-i. 62
<0.1
44
-I. 35
<0.1
44
-6.08
<0.001
44
-6.43
<0.001
I 43
-0.33
<0.2
Ca
44
3.92
<0. 001
44
3.98
<0. 001
44
0.51
<0.2
Cu
44
5.13
<0.001
44
6.24
<0.001
44
4.28
<0.001
C1
44
5.36
<0. 001
44
2.44
<0.1
44
2.05
Rb
13
NSD
D.F. = Degree o~ freedom
239
M. A. DUROSINMI et al.: TRACE ELEMENTS IN SICKLE CELL
lower plasma Ca concentrations in the SCA subjects as compared to the controls. A significant increase in the C1 concentration in whole blood, erythrocytes and plasma of the SCA subjects as compared to the normal controls was also observed. This shows the elements increased uptake in the incidence of sickle cell disease. The only published work with respect to sickle cell disease was that of HelIerstein et al [12].
They studied red blood cell
composition in children with SCA and found no difference in the concentration of this element when compared with the normal controls. A decrease in the mean Fe concentration which was found to be statistically significant in the whole blood and erythrocytes of the SCA subjects as compared to the controls was observed. Plasma Fe level in the sicklers, was however, found to be significantly higher than that of the controls.
This increased Fe level
may be due to the haemolysis of the red ceil thereby releasing Fe into the plasma. A higher plasma Fe in the sicklers has also been reported by other workers Adeyefa et al [11], Oluboyede et al [13]and Serjeant et al [14] . Elevated and statistically significant Cu level was observed in the whole blood, erythrocytes and plasma of the SCA subjects as compared to the controls. The increased plasma level is consistent with reports of Olatubosun et al [15] and Prasad et al [16] and Adedeji et al [17]. It is known that about 90% of plasma Cu is associated with the plasma protein cerulosplasmin which is known to be involved in mobilisation and utilisation of Fe in haemoglobin synthesis, hence low level of Fe in SCA may trigger off a feedback mechanism to produce more cemloplasmin for haemoglobin manufacture which can result in an increased Cu level in the plasma. Adeyefa et al [1 I] reported that the concentration of Cu in the sicklers were found not to be age related but sex related. They found that male sicklers had higher levels of Cu than the females. Our finding shows that despite the fact the majority of the SCA samples analyzed are female subjects, the Cu concentration is still higher in the SCA subjects as compared to the controls. A significant higher Cu level was also observed in the hair of Nigerian sicklers by Oluwole et al [18]. As mentioned earlier, Zn is the most widely studied element in sickle cell disease. A statistically significant decrease in the concentration of Zn was found in the whole blood, erythrocytes and plasma of the SCA subjects as compared to the controls. This is found to be consistent with the reports of other workers [11,16,19]. Prasad et al [16] also found that the excretion of Zn in the urine of the SCA subjects was higher than that of the controls suggesting that increased loss of Zn may be one of the mechanisms by which the SCA subjects become Zn deficient.
It is also believed that
haemolysis of the red cell may contribute to the loss of Zn by increasing glomcrular filtration
240
M. A. DUROSINMI et ai.: TRACE ELEMENTS IN SICKLE CELL
of Zn since more Zn is being released into the plasma by the red cell and being made available for filtration. A significant decrease in Se concentration in the erythroeyte of the SCA subjects as compared to the Controls was observed. A decrease in Se concentration was also observed in the whole blood of the SCA subjects but which was not statistically significant while there was no change in the plasma Se concentration of the two groups. There has been no reported evidence of the Se status of the SCA subject other than the one reported by Natta et al [20] that observed a significant decrease in the Se concentration of whole blood and plasma of SCA subjects as compared to the controls.
It is however,
important to study the Se status of the SCA subjects as deficiency of the element can result in biochemical and functional abnormalities of the erythrocyte.
Our result also show a
significant decrease in the concentration of Br and Rb in the whole blood, erythrocytes and plasma of the SCA subjects as compared to the controls. There has been no published results of Br and Rb concentration in sickle cell anaemia. Both elements are reported to be essential elements but little has been said about their biochemical functions.
Conclusions It has been shown from the elemental analysis of whole blood, erythrocytes and plasma collected from sickle cell aaaemia and normal control subjects that the concentration of K, Fe, Zn, Se., Br and Rb are significantly lower in the whole blood and erythrocytes of the SCA subjects as compared to the controls while the level of K and Fe in the plasma of the sicklers were found to be significantly higher than that of the controls but a significant decrease in the concentration of Fe, Z, Br and Rb in the plasma of the SCA subjects was however, observed. Again, the result also shows that there was a significant increase in the concentration of Na, CI, Ca and Cu in the whole blood and erythrocytes of the SCA subjects as compared to the controls as well as a significant increase in the concentration of C1 and Cu and a decrease in the concentration of Na in the plasma of the SCA subjects as compared to the controls. The decrease in the concentration of K and an increase in the concentration of Na and Ca in the erythrocytes of the SCA subjects confirms the findings of other workers of an increase in pemleability of the red cell of the SCA subjects during the sickling period. The results atso shows clearly that the SCA subjects are both ICe and Zn deficient due to the decrease in the concentration of these elements in the three blood components.
241
M. A. DUROSINMI et al.: TRACE ELEMENTS IN SICKLE CELL
Moreover, an increased concentration of Cu in the three blood components o f the SCA subjects might also be due to the deficiency of Zn since Zn and Cu tend to compete for the same biochemical binding sites. Our findings also show that there are no significant differences between the normal control subjects and subjects with sickle cell trait confirming that these group of subjects are normal and healthy. No attempt has been made at this stage to separate the male and female subjects but these initial f'mdings are sufficient encouragement for further investigations into the role o f trace elements in the sickle cell disease. ,g
This work would not have been possible without the EEC Linkage Programme under the Lome-III agreement which fostered exchange and collaboration between our universities.
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