Magnesium, Zinc, and Copper in Dialysis Patients ;
DELMAR J. MAHLER, P H . D . , JOHN R. WALSH, M.D.,
Veterans Administration
AND GORDON D. HAYNIE,
Hospital and University of Oregon Medical
Portland, Oregon 97207
M.D.
School,
ABSTRACT
Mahler, Delmar J., Walsh, John R., and Haynie, Gordon D.: Magnesium,
zinc, and copper in dialysis patients. Amer. J. Clin. Path. 56: 17-23, 1971.
Magnesium, zinc, and copper of plasma and erythrocytes have been measured
in patients with renal disease using atomic absorption spectrophotometry.
The data revealed increased magnesium and copper but decreased zinc in the
plasma of dialysis patients compared with normal adult controls. Erythrocytic
magnesium was greatly elevated in the patients on dialysis. Plasma magnesium
was maintained at nearly normal levels when patients were dialyzed against
a bath which was essentially free of magnesium. Nondialyzed uremic patients
shared some, but not all, of the abnormalities of the dialysis group. The
etiology of these abnormalities remains obscure.
DETERMINATION of trace metals was
limited by methodology prior to the last
decade. This resulted in a limited knowledge of the concentrations of metals in
many diseases, including renal disease. Fortunately, this situation is being remedied
to a major extent by atomic absorption
spectrophotometry. Hence, studies of metal
concentrations in health and disease have
begun to appear. These have included evaluations of such elements as magnesium,
copper, and zinc in a variety of biologic
fluids and tissues.7- "•10-12-13>1B
phorylation. The biochemistry of copper in
ceruloplasmin has been studied * and the
importance of this has been investigated
with regard to Wilson's disease.1 We feel
that greater knowledge of the metabolism
of metals in persons with impaired renal
function will be helpful in understanding
the underlying pathophysiology and conceptually will lead to better management
of these patients. This may have special
significance for patients undergoing hemodialysis.
This report is concerned with the concentrations of magnesium, copper, and zinc
in the plasma and erythrocytes of patients
with renal disease. Each of these metals is
associated to some extent with enzymatic
function. Zinc is involved in the function
of alcohol dehydrogenase, alkaline phosphatase, malic dehydrogenase, glutamic dehydrogenase, and lactic acid dehydrogenase.11' " Magnesium is associated with phos-
Methods and Materials
THE
Magnesium, zinc, and copper determinations were performed on the plasma and
erythrocytes of 40 patients with renal disease. One patient group consisted of 13
men who were receiving regular hemodialysis therapy two or three times weekly. The
second group was 27 uremic patients on
whom dialysis had not been used. The
dialysis and uremic classifications were
based on reduced creatinine clearance rates.
Those in the dialysis groups had clearance
Received August 13, 1970; received revised manuscript October 5, 1970; accepted for publication
October 16, 1970
17
18
MAHLER ET
rates of 5 ml. per min. or less, whereas the
uremic group had clearances of between 5
and 12 ml. per min. These groups were
compared with 27 normal adult controls
consisting of male and female hospital personnel.
Fasting blood samples were collected
using a disposable plastic syringe and a
stainless steel needle. The specimen was
transferred to an acid washed centrifuge
tube containing heparin as an anticoagulant. The addition of heparin did not contribute any measurable contamination of
the metals under consideration in this
study. T h e plasma and erythrocytes were
separated by centrifugation and the cells
washed twice with 0.9% NaCl. The cells
were then hemolyzed by addition of an
equal volume of deionized water. The
plasma portion was centrifuged to separate
any contaminating erythrocytes. Plasma
that showed signs of hemolysis was discarded. Plasma and the hemolyzed cells
were stored in acid-cleaned polyethylene
vials at —5 C. until analyzed. The Travenol
twin-coil (Kolff) kidney 6 was used for
chronic hemodialysis during this study. The
composition of the bath used was: N a H C 0 3 ,
0.3%; glucose, 0.2%; NaCl, 0.57%; CaCl 2 2 H 2 0 , 0.0221%; KC1, 0.051%; MgCl26 H 2 0 , 0.0075%; 100 1. tap water. This
bath composition will be designated as bath
"with" Mg*\ In part of this study the magnesium salt was omitted from the above
bath and will be designated as bath "without" Mg++.
Metal determination was made by atomic
absorption spectrophotometry. Our instrument (Perkin-Elmer Model 303) was
equipped with a Boling burner. The gas,
air settings, and instrument operations were
as recommended in the manufacturer's operations manual.
Plasma, zinc, and copper were determined by aspiration of a 1:1 dilution of
plasma with water. The concentration was
based on a comparison of sample absorbance with that of a standard curve dissolved
A.J.C.P.—Vol.
AL.
56
in a solution containing 3 % bovine albumin.* 9 The zinc and copper in the erythrocyte hemolysate were determined by combining 3 ml. of hemolysate with 2 ml. water
and 1 ml. 30% trichloracetic acid (TCA).
Analysis was made by aspiration of the
protein-free supernatant, similar to the
method of Rosner and associates.12 Comparison was made with aqueous standard
curves. In most cases it was necessary to
dilute 1 ml. of supernatant with 3 ml. water
for optimum conditions for the zinc analysis. In each case correction was made for
metal in an appropriately diluted TCA
bank.
Magnesium was determined in both
plasma and hemolysate by making a 1:100
dilution in 0.5% lanthanum solution, essentially as previously described.16 Concentration was based on comparison with a similarly composed standard curve.
Magnesium in the dialysis bath solution
was determined by aspiration of an appropriate dilution in 0.5% lanthanum, similar
to plasma analysis. Determinations of zinc
and copper in the dialysis bath solution
necessitated concentration of the metals in
the specimen. This was accomplished by
chelation with ammonium pyrrolidine dithiocarbamate (APDC) and extraction with
methyl isobutyl ketone (MIK).S>9 Comparison was made with a standard curve obtained by similar means.
The water used was demineralized distilled water. All glassware and polyethylene
vials were washed in 8 N H N 0 3 and rinsed
with demineralized water. Reagent TCA
was redistilled to minimize metal contamination. Reagent grade MIK was redistilled
before it was used.
Results
Magnesium, Zinc, and Copper in Dialysis
Patients. Magnesium was determined in the
plasma and erythrocytes of patients before
* Bovine albumin fraction V powder from Sigma
Chemical Company.
July 1971
19
MAGNESIUM, ZINC, AND COPPER IN DIALYSIS
Table 1. Magnesium, Zinc, and Copper in Patients with Renal Disease*
Plasma,
Bath with Mg,++
Plasma,
Bath without Mg"1"1'
Cu
Mg
70.6
7.1
11.0
2.273
175.1
36.5
11.0
8.23'
2.29
0.61
13.00
1.07
76.3
7.8
11.0
2.273
172.0
37.6
11.0
8.23'
1.46
0.20
13.00
14.9'
+5.64
5.4
11.0
3.453
-2.76
6.7
11.0
1.36
-0.83
0.2
13.0
15.0'
84.5
11.1
24.0
102.3
14.5
24.0
Mg
Zn
2.87
0.51
11.00
2.16s
2.35
0.44
11.00
2.16s
Erythrocytes,
Bath without Mg++
Cu
Mg
Zn
Cu
59.7
16.7
13.0
5.521
150.3
17.4
13.0
9.1'
6.35
0.51
13.00
8.161
1,541.0
150.0
13.0
1.3
81.1
10.9
13.0
0.35
61.4
12.7
11.0
5.521
148.9
18.9
13.0
8.141
6.24
0.38
13.00
9.061
1,557.00
103.00
13.00
1.75
83.8
13.4
13.0
0.86
-1.2
3.5
13.0
1.24
-0.096
0.19
13.0
1.83
+1.92
39.0
13.0
0.17
+2.25
6.38
13.0
1.27
Zn
Predialysis
Average
±
n
ISD
Postdialysis
Average
± 1 SD
n
<.t
Pre- to Postdialy:sis Change}:
Average
± 1 SD
n
-0.58
0.17
11.00
10.81
+1.26
4.4
10.0
0.9
Control (normal adults)
Average
± 1 SD
n
2.17
0.01
24.0
4.88
0.41
11.0
1,412.0
230.0
11.0
79.6
8.6
11.0
* Mg concentration given as mg. per 100 ml. Zn and Cu concentrations given as /ig. per 100 ml.
t ta = / value relative to control normal adults.
X Based on paired differences.
§ I value based on pre- to postdialysis change.
1
p < 0.001.
3
P < 0.05.
and after dialysis to determine net changes
as a result of the dialysis treatment. Plasma
magnesium was significantly elevated at
2.87 mg. per 100 ml. before dialysis against
the bath with Mg++ added. At the end of
dialysis the plasma magnesium was reduced
by 0.58 mg. per 100 ml. This was approximately a 20% reduction. The resultant
level was essentially the normal adult control level. We were interested in the possibility of maintaining the patients at a more
nearly normal serum magnesium. Therefore, in a subsequent series of analyses the
usual magnesium salt was not added to the
dialysis bath. After only two or three dialysis sessions against baths without magnesium added, the serum magnesium was noticeably lower. As can be seen in Table 1,
this form of dialysis treatment resulted in
a predialysis plasma magnesium level of
2.29 mg. per 100 ml., which by the end
of dialysis was lowered by 0.83 mg. per
100 ml. to a value of 1.46 mg. per 100 ml.
We have followed seven of these patients
for as long as 2 years since they were shifted
to the bath without magnesium added.
This form of dialysis bath has stabilized
their serum magnesium at essentially normal levels. The predialysis serum magnesium for these patients during this 2 year
interval averaged 2.27 ± 0.46 mg. per 100
ml., with only 10% of the 42 values exceeding 2.40 mg. per 100 ml. (Table 3).
Erythrocytic magnesium was measured only
under conditions in which Mg*+ was not
added to the bath. T h e predialysis concen-
20
MAHLER ET
A.J.C.P.—Vol. 56
AL.
Table 2. Magnesium, Zinc, and Copper in Patients with Renal Disease
Erythrocytes
Plasma
Mg
(mg. per
100 ml.)
Zn
fog- per
100 ml.)
Cu
(Mg- per
100 ml.)
Mg
(mg. per
100 ml.)
Zn
(Mg. per
100 ml.)
Cu
(Mg- per
100 nil.)
2.42
0.82
25.0
0.69
63.4
18.1
25.0
4.98*
110.4
19.4
25.0
2.25f
6.21
1.94
25.0
2.25f
1,688.0
318.0
25.0
2.60f
91.6
29.1
25.0
1.32
2.17
0.01
24.0
84.5
11.1
24.0
102.3
14.5
24.0
4.88
0.41
11.0
1,412.0
230.0
11.0
79.6
8.6
11.0
Uremic patients,
(nondialyzed)
Average
± 1 SD
n
a
Controls (normal adults)
Average
± 1 SD
n
*p < 0.001.
t p < 0.05.
%t0 = I value relative to control normal adults.
Table 3. Predialysis Plasma Magnesium of Patients Maintained on a Bath
without Magnesium Added*
Patient
Patient
Patient
Patient
Patient
Patient
Patient
1
2
3
4
5
6
7
0
5
12
2.03
2.54
2.42
1.82
3.07
1.74
2.25
2.75
2.30
1.78
1.70
2.97
1.67
2.85
3.95
2.23
2.30
2.14
2.22
2.86
2.40
Months
14
16
19
Concentration (mg. per 100 ml.)
21
22
25
2.27
1.87
1.96
2.86
2.32
2.22
2.16
2.16
1.73
2.11
1.94
2.76
1.91
2.34
1.93
2.34
1.89
1.88
1.75
2.23
1.80
* Average = 2.27 ± 1 SD = 0.46.
Table 4. Predialysis Metal Concentrations in Dialysis Bath and Plasma
Bath with Mg++
]Bath without M g
++
Mg
(mg. per
100 ml.)
Zn
(Mg. per
100 ml.)
Cu
(Mg. per
100 ml.)
Mg
(mg. per
100 ml.)
Zn
(Mg. per
100 ml.)
Cu
(Mg- per
100 ml.)
1.03
0.17
24.0
8.6
3.4
36.0
11.5
3.4
36.0
0.067
0.007
39.0
2.2
1.2
29.0
7.2
4.9
39.0
2.87
0.51
11.0
70.6
7.1
11.0
175.1
36.5
11.0
2.29
0.61
13.0
7.5
0.001
59.7
16.7
13.0
0.85
Bath
Average
± 1 SD
n
Plasma
Average
± 1 si>
II
1
P
150.1
17.6
13.0
0.078
July IV71
21
MAGNESIUM, ZINC, AND COrPER IN DIALYSIS
Tabic 5. Serial Determinations of Plasma Copper [jig. per 100 ml.] in Right Uremic Patients
Prior to and after Starting Hemodialysis Therapy
Patient 1
Patient 2
Patient 3
Patient 4
Patient 5
Patient 6
Patient 7
Patient 8
128
82
93
90
91
82
Jieforc hemodialysis
14
12
10
8
6
4
2
0
months
months
months
months
months
months
months
months
Overall average
109
138
100
123
134
109
127
103 ± 18
108
89
81
116
100
92
106
118
102
165
103
99
113
105
79
102
95
87
Hemodialysis
2 months
4 months
6 months
8 months
10 months
12 months
14 months
16 months
18 months
20 months
22 months
24 months
26 months
96
78
102
97
95
116
147
83
118
156
67
105
116
91
91
71
94
94
94
96
125
116
103
110
73
Overall average 99 ± 28
tration of magnesium in erythrocytes was
6.35 mg. per 100 ml., significantly higher
than normal aclult control values, and was
reduced by only 0.096 mg. per 100 ml. at
the end of dialysis, indicating that dialysis
had very little effect on erythrocytic magnesium (Table 1).
Plasma zinc and copper were determined
under the same conditions. The predialysis
zinc was 70.6 mg. per 100 ml., and zinc
amounted to 59.7 mg. per 100 ml. when
dialyzed against baths with and without
Mg**, respectively. These values are significantly below normal adult control values
(Table 1). Erythrocyte zinc did not differ
from control values (Table 1). Neither
plasma nor erythrocytic zinc levels were
significantly changed by dialysis conditions
used here (Table 1).
Plasma copper concentrations were 175.1
nig. per 100 ml. and 150.3 mg. per 100 ml.
prior to dialysis against baths with Mg t+
and without Mg++, respectively. These values are considerably elevated over the normal adult control value of 102.3 mg. per
100 ml. (Table 1). Erythrocytic copper in
the dialysis patient was not different from
the control value (Table 1). T h e copper
concentrations of both plasma and erythrocytes were not significantly affected by dialysis (Table 1).
Predialysis plasma magnesium was greatly
influenced by the removal of Mg++ from the
dialysis bath (Table 1). It is noteworthy
that predialysis plasma zinc and copper
were lower when patients were dialyzed
against the bath without added Mg**. This
reduction in plasma zinc and copper was
not significant, however (Table 4). On the
other hand, the predialysis plasma magnesium for patients dialyzed against a bath
without Mg*+ added was significantly lower
22
MAHLER ET AL.
than that of those dialyzed against the
bath fortified with Mg++. The corresponding predialysis concentrations of magnesium, zinc, and copper in the bath are
also shown in Table 4.
The copper in plasma of dialysis patients
has been reported to be subject to contamination from the dialysis system.2 Therefore, we compared the plasma copper of
eight patients before and after they were
put into the dialysis program. In this way
we could determine the extent to which our
dialysis system contributed to the increase
in serum copper seen above. Our results
indicated that dialysis did not cause increased plasma copper in these eight patients (Table 5), observed before beginning
hemodialysis twice weekly and for as long
as 26 months afterwards.
Magnesium, Zinc, and Copper in Uremic
Patients. The results of the analyses of
plasma and erythrocytes of nondialyzed
uremic patients are also shown in Table 2.
The plasma magnesium of this group was
slightly greater than normal adult control
values. This was in contrast to the group's
erythrocytic magnesium level, which was
significantly elevated relative to control.
Plasma zinc was significantly depressed
(similar to dialysis patients), whereas plasma
copper was within control limits of normal
adults. Zinc of the erythrocytes was above
control levels, but erythrocytic copper was
within control limits. These results are in
contrast to the erythrocytic zinc of the dialysis patients, in whom no significant deviation from control was seen.
Discussion
The influence of magnesium concentration in the dialysis bath should be emphasized, since in this study it had a definite
effect on plasma magnesium (Table 2). A
sustained lowering of plasma magnesium
resulted when the bath was not supplemented with magnesium. This may be the
result of the dialysis technic used at this
A.J.C.P.—Vol.
56
hospital (Kolff type of dialyzer). Others,
using the Kiil type of dialyzer, have reported no lowering of serum magnesium at
the end of dialysis.6 This difference in dialyzing technic, along with differences in
bath composition, may be important, because some dialysis centers have reported
hard water syndrome in patients when magnesium in the bath had inadvertently become abnormally elevated.8
If plasma magnesium is a primary concern, then regulation of dietary intake and
loss through dialysis is a means of control.
The dialysis bath without magnesium favors greater diffusion of magnesium from
the blood stream. This would account for
better control of plasma magnesium (Table
The abnormal metal concentrations in
renal patients is not clearly understood. It
is hoped correlation of these findings with
possible abnormal physiologic functions
will be made. Magnesium in both plasma
and erythrocytes was found to be abnormally elevated (Table 1), while plasma zinc
(but not erythrocytic zinc) was low in the
dialysis patients. The fact that each of these
metals is present predominately as intracellular elements causes us to wonder why
they are not similarly affected. A depression
of plasma zinc has been observed in chronic
lymphocytic leukemia and lymphomas, 12 as
well as in patients with various debilitating
chronic diseases.8
Zinc was not effectively lost through dialysis. This confirms the report of others. 8
In addition, Blomfield and associates2 reported a measurable uptake of zinc and
copper by plasma and erythrocytes during
priming of the dialysis system. If this uptake during priming were significant, one
might anticipate accumulation and toxicity
at some storage site in the body. Our findings would not indicate such to be the case
for zinc, which we found to be low in
plasma and normal in erythrocytes.
July 1971
MAGNESIUM, ZINC, AND COPPER IN DIALYSIS
On the other hand, we did find increased
plasma copper but normal erythrocytic copper in the dialysis patients. The cause of
this increased plasma copper should be further investigated, inasmuch as the nondialysis uremic group did not have increased
plasma copper. The increased plasma copper found in dialysis patients might be explained according to the report by Blomfield and colleagues 2 as a result of increased
absorption of copper during dialysis. However, the erythrocytic copper in these patients was not elevated, and the finding
that they did not have progressive increases
in plasma copper during their first 26
months on hemodialysis (Table 5) does not
support this explanation. Hence, further
studies regarding the control of copper in
patients on hemodialysis are planned.
References
1. Beam AG, Kunkel HG: Biochemical abnormalities in Wilson's disease. J Clin Invest 31:
616-617, 1952
2. Blomfield J, McPhcrson J, George CRP: Active
uptake o£ copper and zinc during hemodialysis. Brit Med J 2:141-145, 1969
3. Freeman RM, Lawson RL, Chamberlain MA:
Hard water syndrome. New Eng J Med 276:
1113-1118, 1967
23
4. Holmberg CG, Laurell CB: Investigations in
serum copper. II. Isolations of the copper
containing protein and a description of some
of its properties. Acta Chem Scand 2:550-556,
1948
5. Kolff WJ: T h e artificial kidney—past, present,
and future. Circulation 15:285-294, 1957
6. Mansouri K, Halsted JA, Gombos EA: Zinc,
copper, magnesium, and calcium in dialyzed
and nondialyzed uremic patients. Arch Intern Med 125:83-88, 1970
7. McCall J T , Goldstein NP, Randall RV: Comparative metabolism of copper and zinc in patients with Wilson's disease (hepatolenticular
degeneration). Amer J Med Sci 254:13-23, 1967
8. Mulford CE: Solvent extraction techniques for
atomic absorption spectroscopy. Atomic Absorption Newsletter 5:88-90, 1966
9. Parker MM, Humoller FL, Mahler DJ: Determination of copper and zinc in biological material. Clin Chem 13:40-48, 1967
10. Prasad AS, Oberleas D, Halsted JA: Determination of zinc in biological fluids by atomic absorption spectrophotometry. Zinc Metabolism.
Edited by AS Prasad. Springfield, 111, Charles
C Thomas, 1966, pp 27-37
11. Prasad AS: Nutritional metabolic role of zinc.
Fed Proc 26:172-185, 1967
12. Rosner F, Gorfien PC: Erythrocyte and plasma
zinc and magnesium levels in health and disease. J Lab Clin Med 72:213-218, 1968
13. Sullivan JF, Lankford HG: Zinc metabolism
and chronic alcoholism. Amer J Clin Nutr
17:57-63, 1965
14. Vallee BL: Biochemistry, physiology, and pathology of zinc. Physiol Rev 39:443-481, 1959
15. Wallach S, Cahill LN, Rogan FH, et al: Plasma
and erythrocyte magnesium in health and disease. J Lab Clin Med 59:195-210, 1962
16. Willis JB: Magnesium in blood serum. Spectrochimica Acta 16:273-278, 1960