CUN. CHEM. 2 1/4,
582-587
(1975)
Zinc Deficiency in Sickle Cell Disease
Ananda S. Prasad,1 Eric B. Schoomaker, Jesus Ortega, George J. Brewer,
Donald Oberleas, and Fred J. Oelshlegel, Jr.
Clinical
anemia
similarities
between
patients
with
sickle
cell
and zinc-deficient subjects suggested a secondary zinc deficiency in sickle cell anemia. Zinc was assayed in various biological fluids and tissues by atomic
absorption spectrophotometry.
Zinc in the plasma,
erythrocytes, and hair was decreased and irinary zinc
excretion was increased in anemia patients as compared to controls. Erythrocyte zinc and daily urinary zinc
excretion were inversely correlated in the anemia patients (r =
.63, p < 0.05), suggesting that hyperzincuna may have caused zinc deficiency in these patients.
Carbonic anhydrase, a zinc metalloenzyme, correlated
significantly with erythrocyte zinc (r = ±0.94, P <
0.001). Plasma RNase activity was significantly greater
in anemia subjects than in controls. We administered
zinc sulfate, 660 mg per day, orally, to seven men and
two women with sickle cell anemia. Two 17-year-old
males gained 5 cm and 7 cm in height during 49 and 42
weeks of zinc therapy, respectively. All but one patient
gained weight (0.5 kg to 4.1 kg). Five of the males
showed increased growth of pubic, axillary, facial, and
body hair, and in one a leg ulcer healed in six weeks on
zinc and in two others some benefit of zinc therapy on
healing of ulcers was noted.
-
Addftlonal Keyphrases:
treatment of side-effects of sickle
cell anemia #{149}trace elements #{149}effects of chronic hemolysis #{149}atomic absorption spectrophotometry
#{149}
Zn in
plasma, urine, hair and erythrocytes #{149}
carbonic anhydrase
Department
of Medicine,
Wayne
State
University,
Detroit
Gen-
eral Hospital, Grace Hospital, Detroit; the V. A. Hospital, Allen
Park; and the Department
of Human Genetics, University
of
Michigan, Ann Arbor, Mich..
Presented in part at the Plenary Session of the First National
Symposium
on Sickle Cell Disease,
sponsored
by the Sickle Cell
Disease
Branch
of the NHLI,
NIH, held in Washington,
D. C.,
June 27, 1974.
1 Address
communications
to A. S. P. at the Department
of
Medicine,
Wayne
State
University
School
of Medicine,
540 E.
Canfield
Ave., Detroit,
Mich. 48201.
Received
Oct. 2, 1974; accepted
Jan. 29, 1975.
582
CLINICAL
CHEMISTRY,
Vol.21,
No. 4, 1975
Zinc is essential
for man and its deficiency
causes
growth retardation,
loss of appetite,
and hypogonadism (1). Zinc is essential for the activity of a large
number
of enzymes
(2) and recently
its role in fibro-
plastic proliferation
and collagen synthesis has been
recognized (3). Studies by various investigators
indicate that zinc is required for deoxyribonucleic
acid
(DNA) synthesis
(4, 5) and DNA-dependent
RNA
polymerase (EC 2.7.7.6) has been demonstrated
to be
a zinc-dependent
enzyme (6). Our most recent study
has documented
that thymidine kinase (EC 2.7.1.75)
is a zinc-dependent
enzyme and that its activity is
adversely affected early by dietary lack of zinc (7),
thus further establishing
an enzymatic role of zinc in
DNA synthesis.
Certain clinical features are common to some sickle
cell anemia (SCA) patients
and zinc-deficient
patients, the latter as reported from the Middle East (1,
8). These symptoms include delayed onset of puberty
and hypogonadism
in the males, characterized
by decreased facial, pubic, and axillary hair, short stature
and low body weight, rough skin, and poor appetite.
Inasmuch
as zinc is an important
constituent
of
erythrocytes,
it appeared
possible that long-continued hemolysis in patients with SCA might lead to a
zinc-deficient
state, which could account for some of
the clinical manifestations
mentioned above. Delayed
healing of leg ulcers and the reported (9) beneficial
effect of zinc therapy on leg ulcers in SCA patients
would also appear to be consistent with the above hypothesis.
Here, we present data on zinc metabolism
in patients with SCA and provide evidence of zinc deficiency in some patients
with SCA. Preliminary
results of the effect of zinc therapy in SCA patients are
also presented.
Methods
Results
Patients from the Detroit General Hospital Sickle
Of 18 men with SCA, 13 manifested
signs of hypogonadism (decreased or absent facial, axillary, chest
Cell Anemia Adult Clinic were investigated.
Eighteen
and pubic hair and delayed onset of puberty). In conmen and 18 women comprised the group. The diagnosis of SCA was established
by history, physical extrast, only four women showed decreased
gonadal
function, as suggested by late onset of menarche and
ainination,
and hematological
studies, which includmenstrual abnormalities.
This observation
is consised electrophoresis
of hemoglobin on cellulose acetate
tent with the known greater susceptibility
of male goand quantitative
determination
of A2, F, and S hemonads to a lack of zinc.
globins. Hemoglobin
electrophoresis
revealed the S-S
The height and weight of the patients with SCA
pattern; quantitation
of A2 hemoglobin gave normal
were compared
with data obtained
from the Ten
values
and F hemoglobin was less than 20% in all our
cases. The controls were healthy, nonanemic
Negro
State Nutrition
Survey in 1968-70 (13). Two men
with SCA were below the 10th height percentile,
14
men and women ranging in age from 23 to 57 years.
The ages of the SCA patients ranged from 15 to 69
were below the 15th weight percentile, and two were
in the 50th weight percentile
for their ages. Of 18
years.
We took the usual precautions
to avoid contaminawomen, two were below the 10th height percentile,
nine were below the 15th weight percentile, and four
tion with zinc during collection of samples throughwere
in the 50th weight percentile for their ages.
out the study (10). Zinc was assayed with an atomic
Table
1 shows results of measurements
of zinc in
absorption
spectrophotometer
(Model 303; Perkinerythrocytes
and
hair
in
SCA
patients
and
normal
Elmer Corp., Norwalk, Conn. 06856) according to
Negro
controls.
The
erythrocyte
zinc
concentration
methods published
earlier (10). Plasma was diluted
was significantly smaller in SCA patients than in the
fourfold and assayed directly by using standards
in
controls. Of the 27 SCA patients whose erythrocytes
glycerol:water
(5:95 by vol). Undiluted
samples of
were analyzed for zinc, six had very low values (more
urine were assayed directly by atomic absorption
than 2 SD below the control mean) and four older
spectrophotometry.
Phosphates
in the urine do not
interefere with zinc assay by atomic absorption specsubjects (over 30 years) had normal values. Erythrocyte zinc correlated
with age (r = +0.58, P < .01),
trophotometry
(10). Washed erythrocytes
were lysed
suggesting
that
older
subjects had higher zinc conwith de-ionized water and then digested with concentent in their erythrocytes.
The zinc concentration
in
trated HNO3 before assay. Hair samples were washed
with hexane and ethanol, dried overnight at 125 #{176}C,the hair of SCA patients was decreased as compared
to the controls. Mean plasma zinc concentrations
in
and then digested with concentrated
HNO3 before
SCA patients were significantly
less than those for
assay.
healthy
nonanemic
controls.
Plasma
RNase
activity
RNase (EC 3.1.4,22) activity
in plasma was assayed
was significantly
higher
in SCA patients
than in the
by a method reported previously (11). Red cell carcontrols. The mean urinary excretion
of zinc (per
bonic anhydrase (EC 4.2d.1) protein and its isozymes
I and II were measured
by a radioimmunosorbent
gram of creatinine) by SCA patients was greater than
for the controls.
technique (12).
Figure 1 shows results of carbonic anhydrase deterPlasma and erythrocyte
zinc were also assayed in
minations in relation to erythrocyte
zinc concentraother anemic subjects, including patients with megations in SCA patients. The values correlate closely (r
loblastic anemia, iron-deficiency
anemia, and anemia
=
0.94, P < .001). There
was a significant
negative
associated with chronic diseases.
correlation
(r = -0.71, P < 0.05) between 24-h uriSeven men and two women with SCA were orally
nary
zinc excretion
and erythrocyte
zinc values for
administered
zinc sulfate, 660 mg daily, for various
lengths of time. The clinical effects of this therapy
SCA patients (Figure 2).
Table 2 shows results of zinc determinations
in
are summarized
below.
Table 1. Zinc in Plasma, Erythrocyte, Hair, and Urine, and RNase Activity in
Plasma of Sickle Cell Anemia Patients and Controls
ZInc in
Plasma,
Controls
Patients
P
± values are standard
subject.
112 ±
pg/di
2.5
(23)
102
±
2.7
(32)
<.025
error. Numbers
Erythrocytes,
g/g of Hb
41.7
±
(23)
35.2
±
1.2
1.6
(27)
<.01
in parentheses
are number
Halt,
Urine,
,.g/g creatlnln.
,tg/g
193 ± 4.3
(17)
149
±
495
±
35
(10)
10.3
739 ± 60
Plasma RNase,
A/mIn
per ml
0.310
.001
±
(16)
0.435
.002
±
(21)
(12)
(31)
<.01
<.01
<.001
of subjects.
At least three 24-h urines were collected
from each
CLINICAL CHEMISTRY, Vol. 21, No.4, 1975
583
30
Table 2. Plasma and Erythrocyte Zinc in Six
Subjects with Anemias Other Than
Sickle Cell Anemia
ra.94
P <0.001
125
a’
E
0
320
Li
Zinc In
Erythrocytes,
Hb,
a,
Plasma, pg/dl
68
128
55.7
Fe and folate
58
122
41.2
47.0
Fe deficiency
chr. urinary tract
infection
megaloblastic
anemia
folic acid
deficiency
Cl)
4
g/g
Diagnosis
liter
S
Hb
deficiency
‘5
I
z
61
96
0
66
104
44.1
90
112
50.6
90
106
57.0
4
I
z
.
Fe deficiency
4
0.
I
I
20
30
ERYTHROCYTE
I
I
40
50
I
60
Zn (pg/gHb)
Controls
FIg. 1. CarbonIc anhydrase protein and zinc content of erythrocytes In sIckle cell disease patients
Mean
112 ± 11.8
41.7
±
5.6
SD
±
Shaded areas kdcate mean ± SD for eiythrocyte zinc (vertloa4and erythrocyte carbonic anhyhase protein (horizontal)
CflWTN
Of laTh
FIlM
S
U flAtS
1100
#{149}
1000
#{149}
:
r = -0.7!
900
S
800
Ri 700
Li
z
a”
Ill’.,
600
“C
#{149}
I
#{149}
Fig. 3. Gain in height of two subjects on zinc therapy
For comparison, standard growth curve of boys between 13 to 18 years is
shown (Watson, E. H., and Lowrey, G. H., Growth and Development of Chit-
soo
c’J
cken, 4th ed., ChIcago: Yearbool Medical PublicatIons, Inc., 1962). Numbers
on top of the columns Indicatetotal number of weeks on zinc therapy
400
300
L
22
24
26
28
30 32
ERYTHROCYTE
34
36
38
ZINC
40
p.g/g
42
44
46
48
Mb
FIg. 2. Twenty-four-hour urinary zinc excretion compared with
erythrocyte zinc content of sickle cell disease patients
plasma and erythrocytes
of persons with other anemias. In patients with untreated
inegaloblastic
anemia, erythrocyte
zinc was increased; in other anemic
subjects the zinc values were unremarkable.
Figure
3 shows the change, during zinc therapy,
in
the height of two young men with SCA, both 17 years
old at the time treatment
was begun. The height of
one increased 5 cm in 49 weeks and the other 7 cm in
42 weeks.2
The normal
growth
pattern
of boys between 13 and 18 years is also shown in this figure.
Normally, by age 17 the height curve shows a plateau.
2 Since the submission
of this paper, both of these subjects
gained an additional 3 cm in height as of Nov. 1, 1974.
584
CLINICAL CHEMISTRY, Vol. 21, No.4, 1975
have
Figure 4 shows the growth and development
in the
first of the patients referred to above. This patient
grew pubic hair, and his external genitalia became
more adult-like during 49 weeks of zinc therapy. His
initial bone age was 11 years; after zinc therapy for 49
weeks, his bone age was 12.6 years. Before zinc therapy was begun, his skin appeared rough and a biopsy
revealed mild parakeratosis.
These skin abnormalities could not be detected after zinc administration.
Figure 5 shows the change in weight after zinc
therapy, for all nine subjects. Except for one female
who lost 0.5 kg during the observation period, all others gained weight.
Table 3 summarizes
our results of zinc therapy
with respect to growth of body hair in males. Five of
seven males have shown a positive effect on growth of
body hair; one male has been on zinc for only four
weeks, rather too soon to show such an effect, and the
other has not shown any effect of zinc on body hair
growth so far.
Two males and one female in this group have had
leg ulcers
for many years. In one male
the ulcers
healed within six weeks of zinc
therapy and in the cases of the other two, some benefit of zinc therapy on healing of ulcers has been
noted.
chronic
subject,
Table
4 shows
the
changes
in plasma
and
erythro-
zinc values, and values for carbonic anhydrase
and plasma RNase activity after zinc therapy. Plasma zinc increased in all subjects and a trend towards
higher values for zinc in erythrocyte
and carbonic
anhydrase
protein were observed in three subjects.
Plasma RNase activity definitely decreased in five of
seven subjects in which this was measured before and
after zinc therapy.
cyte
Fig. 4. A sickle cell anemia patient, before and after
zinc ther-
apy
Changes in external genitalia and growth of pubic har as a resub of therapy
are evident
Discussion
4,
42
+4
4
+i
0
z
+2
I
I)
SO
S
flllnfln
]]
+1
27
#{149}1
WEIGHT PERCENTILES
5
RY AGE
10 5
5
5
15
5
15
50
Fig. 5. Changes in body weight of sickle cell anemia patients
during zinc therapy
Number of weeks each patient had been on zinc therapy
columns
is given at
top
of
Table 3. Growth of Body Hair in Seven Men
with Sickle Cell Anemia, on Zinc Therapyo
Wks.
Pubic
0
Axillary
Facial
dominal
Chest
0
1+
0
0
0
0
1+
0
0
0
0
0
0
0
0
49
1+
,,
therapy
3+
2+
3+
3+
2+
2+
3+
3+
0
3+
0
3+
60
4+
3+
3+
2+
3+
0
2+
0
2+
0
1+
10
3+
3+
2+
2+
1+
1+
0
2+
0
1+
7
3+
2+
1+
0
0
43
3+
3+
3+
2+
2+
2+
1+
0
0
1
0
0
4
0
1
0
0, none;
1+,
Our data show that some SCA patients are zinc deficient, although
the severity varied considerably
from one patient to another in this group. This conclusion is supported by our clinical and biochemical
data. The older subjects showed higher erythrocyte
zinc concentrations,
the significance of which is not
clear at present. Possible erythrocyte
zinc concentrations may have some prognostic significance, but this
must await future longitudinal
clinical studies in a
large group of subjects. A good correlation
between
erythrocyte zinc and carbonic anhydrase activity further supports our conclusion that tissue zinc was depleted in our subjects.
Plasma zinc alone is not a good indicator of a zincdeficient state in patients such as these who continuously hemolyze; because the zinc content of erythrocytes is 14-fold that of plasma, values for plasma zinc
would tend to be falsely elevated in such cases. In
spite of this, the mean plasma zinc for our SCA patients was significantly
lower than for the controls.
Activity of RNase is inhibited by zinc and in zincdeficient tissues its activity is increased (11). Thus an
mcreased RNase activity in plasma of SCA patients
may also indicate a zinc-deficient
state.
Changes in the plasma and erythrocyte
zinc
concentrations
similar to those observed in SCA patients
were not seen in other anemic subjects in this study.
Our data with respect to plasma and erythrocyte zinc
in patients with megaloblastic
anemia and iron deficiency anemia are similar to those previously
re-
fine
data
is after the indicated
hair;
2+,
definite;
3+,
definite
in each set is for pre.treatment,
number
of weeks
of therapy.
42
ported
and
creased,
in the
bushy;
bottom
line
by other
investigators
(14, 15).
Previously, one of us (16) reported results of plasma and erythrocyte
zinc determinations
in thalassemia patients from Egypt. Although both were dethe
widespread
nutritional
deficiency
of zinc
Middle East makes it impossible
to relate
these changes to the chronic hemolytic state per se.
Zinc metabolism
should be studied in patients with
other
chronic
hemolytic
disorders,
to establish
whether or not continued hemolysis may lead to a
zinc-deficient
state in such patients.
In spiteof tissuezincdepletionin SCA patients,
the mean urinary excretion of zinc was higher than in
CLINICAL CHEMISTRY, Vol. 21, No.4, 1975
585
Table 4. Effect of Zinc Therapy on Plasma and Erythrocyte Zinc, Plasma RNase
Activity, and Erythrocyte Carbonic Anhydrase Content in Nine SCA Patients’
Carbonic
Plasma
Zn, g/dl
Zn, ,.g/g Hb
Eryth.
RNase,
per ml
Wks.
anhydrase
on Zn
II
116
223
25.3
27.3
.41
6.50
.12
7.55
104
201
39.7
43.7
1.5
.41
15.3
100
32.2
34.6
212b
Mean ±
Before
Plasma
bA/mm
therapy
49
19.0
1.27
1.85
2.55
2.94
.50
12.0
2.15
4
.31b
10.1E
2.21
94
144
27.2
.37
32.7
.28
60
9.07
1.88
11.05
2.28
13.1
17.1
2.09
2.14
10
10
98
41.4
-
167
40.0
.44
133
28.2
.41
184
37.0
.24
7.8
15.3
2.12
2.30
112
37.2
333
.38
.32
13.5
200
11.3
119
30.2
.30
8.06
2.75
2.69
1.58
124
29.9
.32
9.26
2.19
85
167
33.9
33.9
.49
9.63
-
42
7
43
1.76
11.10
27
2.15
SE
106.8
After
190.2
P
±
±
4.9
8.8
32.8
±
±
34.7
1.9
1.7
0.552
0.325
NS
<.001
0.158
0.036
±
±
10.55
±
±
12.42
NS
1.01
2.02
1.28
2.31
NS
±
±
0.15
0.11
NS
Controls
Mean
±
SE
112
±
2.5
41.7
±
1.2
0.310
16.6
.001
±
±
0.53
1.88
±
.005
Means of three separate zinc determinations
in plasma and erythrocytes, before (top line of each set of data) and three after
(bottom line of each set of data) zinc therapy. For plasma RNase activity and carbonic anhydrase (protein), means of at least two
separate determinations
before and two after zinc therapy are presented
in this table.
a
Single
determinations.
the controls. This may have been directly a result of
increased filtration of zinc by the glomeruli,
owing to
continued hemolysis, or there may have been a defect
in tubular re-absorption
of zinc somehow related to
SCA-.a possibility that cannot be excluded at present. Continued hyperzincuria
may have been responsible for tissue depletion of zinc as suggested by a significant negative correlation
between values for 24-h
urinary zinc excretion and erythrocyte zinc. At this
stage, however, one cannot rule out additional factors
such as predominant
dietary use of cereal protein and
other nutritional
factors that affect zinc availability
adversely, thus accounting
for zinc deficiency (1, 8).
Further work is warranted
for proper elucidation
of
pathogenesis
of zinc deficiency in SCA.
It is interesting
to note that in SCA patients, the
zinc concentration
in the erythrocytes
correlated well
with the carbonic anhydrase
content. We measured
carbonic anhydrase
protein rather than its activity in
the erythrocytes;
our data suggest that the synthesis
of specific carbonic anhydrase
apoenzyme
depends
on the availability
of zinc and that this apoenzyme
does not accumulate
if zinc is lacking. This is consistent with our previous studies with respect to other
zinc-dependent
586
CLINICAL
enzymes,
CHEMISTRY,
in which
we were
Vol. 21, No.4,
1975
unable
to
in vitro stimulation
of enzyme activities
zinc in zinc-deficient
tissues (17). The mechanism by which zinc influences
specific apoenzyme
demonstrate
with
synthesis
Our
is unclear.
a potential
process, in
vitro, mediated
by its effect on the oxygen dissociation curve (18, 19) and the erythrocyte
membrane
(2O). Thus it is suggested that SCA patients may derive potential benefits from zinc therapy
in more
ways than one. The results of our limited clinical trial
with zinc therapy revealed beneficial effects of zinc
on growth, development
of secondary sexual characteristics in male subjects, and healing of chronic leg
ulcers. These observations
support our biochemical
data that deficiency of zinc was a complicating
factor
in our SCA patients. Zinc therapy in such patients
therefore
should be considered. In addition to the
above, we also noted apparent symptomatic
improvement, presumably
related to the anti-sickling
effect
of zinc, in our patients (2O). Double-blind
studies,
recent
studies
have demonstrated
beneficial effect of zinc on the sickling
‘ Brewer, G. J., Oelshlegel, F. J., Jr., Prasad, A. S., Knutsen, C.
A., and Meyers, N., Antisickling effects of zinc. First National
Symposium
on Sickle Cell Disease, NIH, Bethesda, Md., June
27-29, 1974 (Abstract).
however, must be done before any conclusion can be
reached regarding symptomatic
improvement.
We have observed no serious toxic side effects
caused by zinc administration
so far. Occasionally
some patients
have complained of nausea after taking
zinc while they are in a fasting state. In general, zinc
administration
has been tolerated
well by our patients. However, inasmuch
as long-term
effects of
zinc administration
in doses used in this study are
not known, one must be cautious in treating SCA patients with zinc on a prolonged basis.
We gratefully acknowledge the technical assistance of Ray Collins, Dana Koniuch,
and Conrad Knutsen.
We thank Dr. D. Rucknagel
for the hemoglobin
electrophoresis,
Dr. R. E. Tashian
for the carbonic
anhydrase determinations,
Dr.
A. Poznanski
for the interpretation
of bone ages, and Dr. J.
Voorhees for reviewing the skin biopsies.
References
1. Prasad, A. S., Metabolism
of zinc and its deficiency in human
subjects. In Zinc Metabolism,
A. S. Prasad, Ed., Charles C Thomas, Springfield, III., 1966, pp 250-293.
2. Vallee, B. L., Biochemistry,
Physiol. Rev. 39, 443 (1959).
physiology
and
pathology
of zinc.
3. Fernandez-Madrid,
F., Prasad, A. S., and Oherleas, D., Effect of
zinc deficiency on nucleic acids, collagen and non.collagenous
protein of the connective tissue. J. Lab. Clin. Med. 82, 951 (1973).
4. Sandstead, H. H., and Rinaldi, R. A., Impairment
of deoxyribonucleic acid synthesis by dietary zinc deficiency in the rat. J.
Cell Physiol. 73, 81 (1969).
5. Grey, P. C., and Dreosti,
I. E., Deoxyribonucleic
acid and protein metabolism
in zinc deficient
rats. J. Comp. Pathol. 82, 223
(1972).
6. Terhune, M. W. and Sandstead, H. H., Decreased RNA polymerase activity in mammalian
zinc deficiency.
Science
177, 68
(1972).
7. Prasad, A. S., and Oberleas, D., Thymidine kinase activity and
incorporation
of thymidine into DNA in zinc-deficient
tissue. J.
Lab. Clin. Med. 83,634 (1974).
8. Prasad,
A. S., and Oberleas, D., Zinc: Human nutrition
and
metabolic effects. Ann. Intern. Med. 73, 631 (1970).
9. Sargeant, G. R., Galloway, R. E., and Gueri, M. C., Oral zinc
sulphate
in sickle cell ulcers. Lancet ii, 891 (1970).
10. Prasad,
A. S., Oberleas,
D., and Haisted, J. A., Determination
of zinc in biological fluids by atomic absorption
spectrophotometry
in normal and cirrhotic subjects. J. Lab. Clin. Med. 66, 508 (1965).
11. Prasad, A. S., and Oberleas, D., Ribonuclease
and deoxyribonuclease
activities
in zinc-deficient
tissues. J. Lab. Clin. Med.
82, 461 (1973).
12. Magid, E., Desimone, J., and Tashian, R. E., Genetic variation
in the carbonic
anhydrase.isoenzymes
radioimmunosorbent
assay. Biochem.
of macaque
monkeys.
I. The
Genet. 8, 157 (1973).
13. Ten-State
Nutrition
Survey,
1968-1970,
U.S. Department
of
Health, Education and Welfare, Ill-Clinical, Anthropometry,
Dental, D.H.E.W.
Publ. No. 72, 8131,111-48 and 111-54.
14. Talbot, T. R., Jr., and Ross, J. F., The zinc content of plasma
and erythrocytes
of patients
with pernicious
anemia,
sickle cell
anemia, polycythemia vera, leukemia, and neoplastic
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