Download Copper, Zinc, and Iron in Normal and Leukemic

[CANCER RESEARCH 46, 981 -984, February 1986]
Copper, Zinc, and Iron in Normal and Leukemic Lymphocytes from Children1
Ugo Carpentieri,2 Jerry Myers, Larry Thorpe, Charles W. Daeschner III, and Mary Ellen Haggard
Departmentsof Pediatrics [U. C., C. W. D., M. E. H.], Pathology [J. M.¡,and Human Biological Chemistry and Genetics [L T.], University of TexasMedical Branch,
Galveston, Texas77550
ABSTRACT
Copper, zinc, and iron were quantitated in the serum and
lymphoid cells of the peripheral blood of healthy children and
children with acute lymphocytic leukemia. Copper and iron con
centrations in serum and cells were significantly higher and zinc
concentration in the cells significantly lower in leukemic patients
than in healthy donors, whereas the increase of zinc in the serum
was not significant. The concentration of all minerals was higher
in T-cell enriched preparations. There was a significant correla
tion between copper and iron and between copper and zinc, but
not between iron and zinc in normal and leukemic lymphocytes.
No correlation was demonstrated among the three minerals in
the serum. There were no significant differences associated with
ethnicity, age, sex, type of leukemia, or number of leukemic
cells. However, a group of five children with non-B-, non-T-cell
acute lymphocytic leukemia, nonreactive skin tests, and low
serum immunoglobulins had high concentrations of copper and
iron and low concentration of zinc in their leukemic cells. Since
copper, zinc, and iron are associated with lymphocyte maturation
and regulation of immune function, these new data will provide
a tool for the study of the relationship between changes in
concentrations of these metals and the modification of the im
mune response often present in hématologiecancers.
INTRODUCTION
Copper, zinc, and iron have been associated with normal
lymphocyte maturation and regulation of immune function (1-8).
Low levels of these minerals have been demonstrated in a variety
of dysfunctions of the immune system; low serum concentrations
of zinc, in fact, may be present in patients with hypogammaglobulinemia and defective cell-mediated immunity (1, 2); impairment
of leukocyte function has been observed with copper deficiency
(7) and lymphoid atrophy with impaired cell-mediated immunity
has been identified in subjects with hypoferremia (8). Information
on high concentrations of copper, zinc, and iron and their effect
on lymphocyte function is also available (9,10).
Serum concentrations of copper, zinc, and iron are modified
in some cancers; serum copper concentrations may be increased
in some leukemias (11-13) and lymphomas (14), whereas a
decrease in iron and variations of zinc concentrations have been
demonstrated in leukemia (12-13). Abnormal immune response
has also been associated with these cancers; patients with
Hodgkin's disease are often anergic (15) and depression of
humoral and cellular immunity has been seen in untreated leu
kemia (16).
These studies suggest a possible causal relationship between
changes of copper, zinc, and iron concentrations and modifica-
RESEARCH
between changes in the cellular concentration of these minerals
and lymphocyte function in certain patients with leukemia.
PATIENTS AND METHODS
Twelve children, aged 2-12 yr (mean age, 5.7 yr) admitted consecu
tively to our institution with a diagnosis of ALL were studied before any
treatment was initiated. Five were black (two males and three females)
and seven were Caucasian (five males and two females).
Twenty-one healthy children in the same age range (siblings when
available), chosen at random (except for siblings) among those attending
our well child clinic were also studied. Nine were black (five males and
four females) and twelve were Caucasian (eight males and four females).
The study was approved by the Institutional Review Board for Human
Experimentation and informed consent was obtained in all cases.
History, physical examination, hemogram, and skin testing with Can
dida (1:250) and PPD were done on all subjects. Quantitation of serum
immunoglobulins was also obtained from all children as were serum
concentrations of copper, zinc, and iron. Diagnostic bone marrow aspi
ration was performed in children with leukemia.
Normal lymphocytes were isolated by Ficoll-Hypaque density gradient
centrifugation (19) from morning specimens of peripheral venous blood
of fasting healthy donors. Erythrocytes were removed by hypotonie
shock (17), platelets by discontinuous sucrose gradients (19), and mac
rophages by incubation in Retri dishes with inactivated calf serum (20,
21). Cell viability was tested by the trypan blue exclusion method,
contamination was tested by morphology, and macrophages by latex
particle ingestion. T-cell enriched aliquots of lymphocytes were then
obtained with mouse anti-human B-cell monoclonal antibody-coated
polystyrene Retri dishes (20, 21 ) and percentages of B- and T-cells were
calculated before and after enrichment with fluorescein-conjugated antihuman B- and T-cell antibodies. All antibodies were purchased from
Cappel-Worthington Biochemicals, Malvern, PA. The cells were then
diluted to 1 x 107 cells/ml in phosphate buffered saline, pH 7.4, and
digested overnight with an equal volume of concentrated nitric acid
(Ultrex; J. T. Baker Co.) at room temperature and for 4 additional h in a
water bath at 60°C (17). Blanks (phosphate buffered saline and nitric
acid in equal vol) were analyzed simultaneously. Standards were pre
pared as described, accounting for the matrix effect (17). The cell
specimens and sera were stored at -20°C until assayed.
Received5/21/85; revised 9/16/85; accepted 10/17/85.
1Supported in part by D. A. Rappoport Memorial Fund.
2To whom requests for reprints should be addressed, at Department of Pedi
atrics, Universityof Texas Medical Branch, Rt. C-61, Galveston, TX 77550.
CANCER
tions of the immune response associated with hématologiecan
cers. A proper evaluation of this relationship, however, may be
attempted only when data on serum concentrations of copper,
zinc, and iron are combined with data on their concentrations in
normal and malignant lymphoid cells. To the best of our knowl
edge, the only information presently available is limited to the
zinc concentrations in normal lymphocytes in adult subjects (17)
and patients with chronic lymphocytic leukemia (18).
In this study we report data on the quantitation of zinc, copper,
and iron in lymphoid cells of healthy children and children with
ALL.3 We also report observations on the potential relationship
Quantitation of metals in serum and cells was performed with an AA
spectrophotometer (ILI-Model 551 with microcup assembly and full scale
3The abbreviations used are: ALL, acute lymphocytic leukemia; PPD, purified
protein derivative (of tuberculin).
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1986
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TRACE MINERALS
expansion) (22). Plasticware was used during the entire procedure with
careful elimination of external contamination (17). Serum metals were
expressed in nQjm\ and cellular metals as M9/1010 cells. Intracellular
metals were also expressed in ng/mg protein.
For leukemic patients the same protocol was used with the following
modifications: identification of the type of leukemia (B-, T-, non-B, nonT-cell ALL) was obtained through the Pediatrie Oncology Group, the
percentage of B- and T-cells was not calculated, and T-cell-enrichment
was not done.
Potential loss of intracellular minerals due to the isolation procedure
was assessed in several randomly chosen healthy donors and patients
with leukemia, by quantitation of copper, zinc, and iron in the initial
specimen and in the several aliquots of cells and supernatant solutions
obtained during the procedure. Supernatant solutions were concentrated
in an oven at 135°C. Possible effects of the isolation procedure on T4/
T8 ratio of lymphocyte subsets in healthy children were also determined
with monoclonal antibodies (Ortho Diagnostic System, Raritan, NJ),
according to the manufacturer's indications.
Statistical evaluation included Student's f test and correlation coeffi
cient analysis.
RESULTS
Healthy Donors. Physical examination, hemogram, and serum
immunoglobulin concentrations were normal for age (23). The
skin test with Candida was positive after 48 h (>5 mm indura
tion), whereas the PPD skin test was nonreactive.
Lymphocytes were 98% pure and 96% viable. T-cell percent
ages were 65-75% before and 90-95% after enrichment. B-cell
percentages were 15-20% prior to and 3-4% after enrichment.
T4/T8 ratio was not significantly altered after cell isolation (range,
2.1-2.6 before isolation versus 2.0-2.3 postisolation).
The values for serum and intracellular concentrations of zinc,
copper, and iron in normal lymphocytes are summarized in Table
1. These values were 20-25% higher (P < 0.02) in T-cell-enriched
preparations (Table 1, footnote e). There were no significant
IN LEUKEMIA
differences associated with ethnicity, age, sex, or number of
cells.
There was no statistically significant correlation between
serum metals and intracellular metals or between intracellular
iron and intracellular zinc. However, there was a significant
inverse correlation between intracellular copper and zinc (r =
0.6; P < 0.01 ) and a significant direct correlation between intra
cellular copper and iron (r = 0.8; P < 0.01).
Children with Leukemia. There were no significantdifferences
in socioeconomic status, diet, and previous medical history be
tween this group and the group of healthy donors. Physical
examination, hemogram, and bone marrow aspirate were con
sistent with the diagnosis of ALL. There were two pre-B-, two
T-, and eight non-B-, non-T-cell leukemias among this group of
patients. Serum immunoglobulin levels were normal for age and
Candida skin test was positive at 48 h in seven patients, whereas
serum immunoglobulins were low normal (between one and two
SDs below the mean for age) and Candida skin test was nonreactive in the remaining five patients (two Caucasian males, two
black females, and one Caucasian female), all with non-B-, nonT-cell leukemia. PPD skin test was negative in all patients.
Lymphoid cells were 85-90% leukemic lymphoblasts and lym
phocytes. The serum and intracellular concentrations of zinc,
copper, and iron are presented in Table 1. There were no
statistically significant differences in these concentrations asso
ciated with ethnicity, age, sex, type of leukemia, or number of
leukemic cells. Ten-12% of these metals were lost during the
procedure. This loss was significantly greater (P < 0.01) than
that observed in the processing of normal lymphocytes (data not
shown). All serum metals in patients with leukemia were higher
than in healthy children, but the increase was statistically signif
icant (P < 0.01) only for copper and iron. The increases in
intracellular copper and iron were significant (P < 0.01 ), whereas
the decrease in intracellular zinc was significant only when ex-
Table 1
Zinc, copper, and iron in serum and lymphoid cells of healthy children and children with acute lymphocytic leukemia
Serum (/ig/dl)
±22"
(66-1 52f
[70-150]"
Healthy children (n = 21
fChildren
with ALL (n = 12)
PHealthy
±29
(86-188)
[80-190]
328 ±74
(252-402)
<0.01Lymphoid
136 ±48
(64-290)
significantZinc^g/1010
not
±26
(43-155)
[40-120]
96 ±24
(63-180)
<0.01Ironi<g/1010
cellsCopper^g/1010
ng/mg
ng/mg
protein73
cells
21)Children
children6(n =
with ALL (n = 12)
PZinc103
±6
(57-96)
[104 ±3]'
54 ±9
(10-80)
not significant
protein15
cells
457 ±40
(380-610)
±4
(26-260)52
(4-40)
246 ±69
±16
(42-333)
(29-71)
<0.01Copper114 <0.01
98 ±21
207 ±43
(131-306)
<0.01Iron68
ng/mg
protein2.5
cells
+ 0.2
(5-27)5.5
(0.7-4)
17 ±3
±0.2
(1.9-8.9)
<0.01
23 ±7
(12-37)
<0.01
n, number of children tested.
6 Mean ±SD.
0 Numbers in parentheses, range.
" Numbers in brackets, range as reported in Refs. 17, 18, and 23.
8 Values significantly higher (P < 0.02) in T-cell-enrichedpreparations (90-95% T-cells)than in unfractionated lymphocytes: zinc, 84 ±7 /jg/10'°cells (75-103) or 560
±46ng/mg protein (500-686); copper, 20 ±5¿ig/1010
cells (17-45) or 158 ±33 ng/mg protein (113-250); iron, 3.3 ±0.4 Mg/1010cells (2.7-5.1 ) or 24 ±3 ng/mg protein
(8-37).
Numbers in brackets, mean ±SD as reported in Refs. 17 and 18.
CANCER
RESEARCH
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1986
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TRACE MINERALS
IN LEUKEMIA
pressed in ng/mg protein (Table 1).
A statistically significant direct correlation was found between
serum copper and intracellular copper (r = 0.7; P < 0.01),
between serum iron and intracellular iron (r = 0.7; P < 0.01), and
between copper and iron in the cells (r = 0.8; P < 0.001). A
statistically significant inverse correlation was found between
¡ntracellularzinc and intracellular copper (r = 0.6; P < 0.01). No
other significant correlation was demonstrated.
The five patients with non-B-, non-T-cell leukemia (Table 2),
copper, zinc, and iron and between the serum level of each of
these metals and their content ¡nnormal lymphocytes demon
strated in this study suggests that their blood concentrations
may not be interdependent at variance with other reports (28,
29), and that an active regulation of their intracellular level,
possibly at the membrane level, may exist (29-31). In this con
text, the higher mineral content of T-cell-enriched preparation
may also be due to membrane regulation, perhaps related to the
greater diversity and number of functions of T-cells as compared
low serum immunoglobulins, and negative skin test did not differ
significantly from the other seven in age range, diet, socioeco-
to other types of lymphocytes (32).
The changes in the mineral concentrations of ALL cells iden
tified in our patients have not been described before; only de
crease of intracellular zinc in chronic lymphocytic leukemia has
been reported (18). We speculate that the changes in ALL may
represent either a characteristic of an abnormal clone population
of lymphocytes or a phase of the maturation process of normal
lymphoid elements. The high turnover of these cells (33) may
explain the increased concentrations of minerals in the serum,
although anemia and poor general nutrition, conditions frequent
in acute lymphocytic leukemia, may be additional factors (34).
Changes in metallothioneins should also be considered (29).
The causes for a greater loss of metals from leukemic cells
than from normal lymphocytes during the isolation process are
not readily apparent. Modification of the cell membrane may be
one factor which, in turn, may explain the direct correlation
between copper and iron concentrations in the sera and lymphoid
elements of children with ALL which was not present in healthy
donors.
Few children with ALL and low serum immunoglobulins (35)
and, in one case, with potent suppressor activity of neoplastic
T-cells (36), have been identified in the past, but a study on the
nomic status, and medical history, but revealed high intracellular
concentrations of copper and iron and low concentrations of
intracellular zinc. Serum concentrations were in the high ranges
for all three metals.
DISCUSSION
Serum concentrations of zinc, copper, and iron in healthy
children reported in this study are similar to previously published
values (23). In addition the variations of serum copper and iron
in patients with acute lymphocytic leukemia have been described
before (11-13). However, the changes in serum zinc concentra
tion found in some leukemic patients (12-13) were not observed
in ours. This discrepancy is possibly due to differences in diet
and time of blood sampling since zinc is subject to a arcadian
rhythm and its concentration is sensitive to dietary intake (24).
No mention of these factors is made in the other study (13).
The values for lymphocyte content of zinc in our healthy
children were lower than those reported previously (17, 18).
Since our donors were healthy and on a balanced diet, the
discrepancy may be due to the differences in age (pediatrie
donors in our study versus adult subjects in the others). It may
also reflect, however, the careful and complete removal of mac
rophages and platelets (which contain discrete amounts of zinc)
(22) from our samples before the assay; when in fact platelets
were removed results similar to ours were obtained (22).
We were unable to compare the values for iron concentration
in the lymphocytes of our healthy children with those reported
by others (25) since those investigators studied normal lympho
cytes in culture and made no mention of the iron content of the
media and possible loss of metal from the cells. To the best of
our knowledge, no data are available for freshly isolated lympho
cytes. Similarly, no comparison can be made between our results
for copper and zinc content of normal lymphocytes and those
reported previously (26, 27) since the latter results were deter
mined on mixed populations of WBC without separation of
lymphocytes from neutrophils.
The lack of correlationamongthe serum concentrationsof
mineral content of their cells was not done. The identification of
some leukemic children among our patients with the simultane
ous presence of low concentration of serum immunoglobulins,
absent cutaneous reactivity, and rather extreme variations of
intracellular metals (low zinc, high copper and iron) is provocative.
In the absence of other identifiable causes it is tempting to
conclude that a relationship may indeed exist between fluctua
tions of mineral concentrations and cell function and/or matura
tion in some patients. Since this conclusion may have therapeutic
implications (37, 38) more stringent criteria of evaluation and a
study on a larger number of patients are recommended.
ACKNOWLEDGMENTS
We wish to thank Dr. D. Rassin for critical review of the manuscript. Jeanne
Koehlerfor the technical work, and Mary Caldara for secretarial assistance.
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Copper, Zinc, and Iron in Normal and Leukemic Lymphocytes
from Children
Ugo Carpentieri, Jerry Myers, Larry Thorpe, et al.
Cancer Res 1986;46:981-984.
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