Determination of Serum Zinc Concentrations in
Normal Adult Subjects by Atomic
Absorption Spectrophotometry
ROBERT S. PEKAREK, P H . D . , WILLIAM R. BEISEL, M.D.,
AND KAREN A. BOSTIAN,
PETER J. BARTELLONI, LTC,
MC,
B.S.
U. S. Army Medical Research Institute of Infectious Diseases,
Frederick, Maryland 21701
ABSTRACT
Pekarek, Robert S., Beisel, William R., Bartelloni, Peter J., and Bostian,
Karen A.: Determination of serum zinc concentrations in normal adult subjects by atomic absorption spectrophotometry. Am. J. Clin. Pathol. 57: BOGSI 0, 1972. Serum zinc concentrations were determined repeatedly in 99
healthy young men by a simple automated atomic absorption spectrophotometric method. The mean serum zinc concentration of these normal subjects
was 102 ng. per 100 ml. (SD = ±!L7), with a calculated range of 68 to 136
/jg. per 100 ml. When determined on a day-to-day basis, 95% of values from
a given individual were within 18 fig. per 100 ml. of his own mean value.
IT IS NOW well recognized that zinc is an
essential nutrient, necessary for normal
growth and general health.10-13>15 Zinc has
been shown to be an integral constituent
and cofactor of a number of enzymes,8 and
is receiving widespread attention as a possible limiting factor in normal wound healing. 11 More recent evidence indicates that
zinc may have a fundamental role in DNA,
RNA, and protein synthesis.17 Since zinc is
essential to such an important variety of
metabolic processes in man, it is important
to gain further understanding of this metal
in both health and disease.
Under normal conditions zinc maintains
a relatively stable consistency in the fluids
and tissues of the body despite its continual and rapid turnover. However, alterations in zinc metabolism, notably significant decreases in serum values, have been
reported to occur in response to the stress
Received May 13, 1971; accepted for publication
July 9, 1971.
of a variety of diseases.*' '• 14 - 18 Such alterations in zinc metabolism may provide potential diagnostic, prognostic, and even
therapeutic usefulness during various disease states. Before such changes in zinc
concentration within the various compartments of the body can be evaluated meaningfully, however, a "normal" concentration or range must be established both for
an individual and for a given population.
Both serum and plasma zinc concentrations have been measured over the past 20
years by numerous investigators employing
a variety of methods. There has been considerable variation in the "normal" mean
concentrations and ranges being reported.
As pointed out by Davies and his co-workers,2 the dithizone method used by earlier
investigators was tedious, with numerous
sources of potential contamination and
error. The advent of atomic absorption
spectrophotometry has offered a more accurate and sensitive analytical method for
506
April
1972
NORMAL SERUM ZINC CONCENTRATION IN MAN
the determination of trace metals in biologic fluids and tissues. The present communication reports the results of serum
zinc concentrations in more than 800 serial
observations in 99 healthy subjects as determined by a simple atomic absorption
spectrophotometric method.
Materials and Methods
Subjects. Ninety-nine healthy 20 to 26year-old male soldiers participated in the
study. They were completely informed as
to the purpose and nature of the study
before volunteering. 1 All subjects were on
normal diets, and none was a vegetarian.
Collection of Specimens. Venous blood
samples were obtained from fasting individuals at 8 AM using stainless steel needles
and zinc-free plastic disposable syringes.
T h e blood was allowed to clot in acidcleaned glass test tubes and centrifuged.
T o further minimize the possibility of zinc
contamination from exogenous sources,
zinc-free polyethylene test tubes and pipettes were used exclusively for the handling and preparation of the serum
samples.
From 6 to 23 daily specimens were obtained from each normal subject. Since preliminary studies demonstrated that freezing
had no effect on the zinc content of the
serum, all serum specimens were held
frozen at —16 C. until the collection period
was completed.
Analytical Method. Individual aliquots
of serum (0.3 ml.) were diluted 1:4 with
deionized water and serum zinc concentrations were measured with a Perkin-Elmer
Model 303 Atomic Absorption Spectrophotometer equipped with a three-slot Boiling
burner head, a Perkin-Elmer intensitron
hollow-cathode lamp for zinc, and an automatic null-recorder readout. The readout
chart was set at a speed of 0.75 in. per min.
with a X3 scale expansion for zinc.
The system was automated by employing
a Technicon Sampler II with the aspirat-
507
Tdble 1. Reproducibility of Analytical Method as
Determined from Independent Measurements
of a Single Serum Pool
Aliquot
Zn
No.
(^g./lOOml.)
1
2
3
4
5
6
7
8
9
10
11
12
13
92
88
92
88
92
92
92
92
92
92
92
92
88
Aliquot
No.
Zn
Gig./100ml.)
14
92
15
96
16
92
17
92
18
92
19
92
20
88
21
92
22
92
23
92
24
92
25
92
Mean = 91.5 Mg-/100 ml,
ing sample probe being directly connected
to the burner capillary with polyethylene
tubing. The sampler could aspirate 60 samples per hour with an aspiration sequence
of 15 sec. of sample or standard, 15 sec. of
air, 15 sec. of deionized water, and 15 sec.
of air. This aspiration sequence provided
enough sampling time to produce, maintain, and record a satisfactory absorption
peak with enough air and water to keep
the line clean between samples.
Working standards of 0.1, 0.3, and 0.5
p.p.m. of Zn in deionized water were run
after every 8 samples. The reproducibility
of this analytical method and the experimental error involved were determined by
independently measuring sample aliquots
from a single serum pool.
Results
As shown in Table 1, the atomic absorption spectrophotometric method yielded
highly reproducible results. The coefficient
of variability of 2% for 25 determinations
on a single pooled sample was well within
the range of acceptability and includes the
combined effects of any dilutional or technical error.
Figure 1 illustrates the results of 827
508
PEKAREK. ET AL.
A.J.CP.—Vol.
57
pg / iOOml
Fie. 1. Normal serum zinc concentrations: 827 observations in 99 healthy young men.
serum zinc measurements as determined
for 99 healthy adult men. The mean serum
zinc value established by this study was
102 pg. per 100 ml. (SD = ± 17). Since individual assay values assumed a normal distribution, an acceptable normal "range"
for serum zinc should be 68 to 136 p.g. per
100 ml. Therefore, fewer than 4% of the
values for normal subjects were either
below 68 fig. per 100 ml. or above 136 ^g.
per 100 ml.
Although Figure 1 illustrates the normal
mean concentration and range for a given
population, individual means and variabilities must be taken into consideration
when evaluating such data. In this respect,
individual variability of normal zinc values
and the distribution of differences from
the mean value of an individual were cal-
culated for 25 normal men. Each man had
14 or more determinations, giving a total
of 495 observations. Figure 2 demonstrates
the distribution of differences about the
mean after individual means were adjusted
to zero. When determined on a day-to-day
basis as shown here, 95% of values from
a given individual will fall within ± 1 8 ^g.
per 100 ml. of his own mean serum zinc
concentration.
Discussion
The present study describes a simple,
sensitive, and highly reproducible atomic
absorption spectrophotometric method for
the determination of serum zinc. The simple dilution of the serum sample with deionized water and direct aspiration into
the burner eliminated pretreatment of the
April
1972
NORMAL SERUM ZINC CONCENTRATION IN MAN
509
ID 3 0
-40
-35
-30
-25
•5
+10
+15
+20
+25
+30
+35
+40
•+45.•
+50
|ig / 100ml
FIG. 2. Variability of normal serum zinc values. Distribution of differences from the mean value
for each man: 495 observations in 25 normal men (14 to 23 determinations per man).
serum and thus minimized the handling
and chance of contamination. Not only
did this study establish a normal mean
value of 102 /xg. per 100 ml., with a calculated range of 68 to 136 iig. per 100 ml.,
but it also established a calculated individual range of ± 18 iig. per 100 ml. about
the individual's mean. Table 2 compares
the results of the present study with others
reported in the literature.
The slight differences between normal
mean values and ranges in this and other
studies appear to be mainly due to methodology and source: plasma or serum. As
stated earlier, the dithizone method has
more sources of potential contamination,
which probably accounts for the slightly
higher values reported in the literature.
Further, the results also appear to depend
on whether the samples are plasma or
serum. Recently, Foley and associates8 reported that serum zinc values were both
slightly and consistently higher than those
for plasma, due to the zinc content of platelets. This would account for the slightly
lower mean plasma zinc values reported by
some investigators also employing atomic
absorption spectrophotometric methods.2-*
Although this study was restricted to male
subjects in a particular age group, studies
of normal healthy adults have shown no
statistical differences in plasma or serum
zinc concentrations due to either sex or
age in adults. 2 ' *•"
The fact that alterations in metabolism
of this trace metal occur during a variety
of diseases and stressful conditions may
make the routine examination of serum or
plasma zinc a potentially valuable diagnostic and prognostic tool. However, since it
has been shown that the individual range
is much smaller than the overall range of
the population, significant alterations in
the individual may be difficult to assess
when comparing the individual's level with
that of the population. An accurate interpretation of an individual serum zinc measurement during illness or stress may be
difficult unless the value can be compared
with the control or normal level of that
individual. With the increased use of
lower mean plasma zinc values reported by
510
A.J.CP.—Vol. 57
PEKAREK ET AL.
Table Z. Comparison of Results with Other Published Values
Zn (Gm./100 ml.)
Subjects
Source
Mean ± SD ± Range*
Method
Reference
99 men
Serum
102 ± 17
68-136
32 men,
8 women
14 men
Serum
120 ± 19
82-158
A.A.S.f
Dithizone
Plasma
103 ±
104 ±
108 ±
94 ±
9
14
15
7
85-121
76-132
Dithizone
A.A.S.
Prasad et al.'
78-138
80-108
Fluorometric
Mahanand and Houck6
95 ± 1 3
96 ± 1 1
96 ± 1 2
97 ± 1 1
121 ± 18
118 ± 2 1
69-121
74-118
A.A.S.
Davies et al.2
72-120
75-119
A.A.S.
Halsted and Smith*
85-157
76-160
A.A.S.
Sinha and Gabrieli"
43 children,
7 adults
Plasma
36 men,
31 women
Plasma
62 men,
27 women
Plasma
100 men,
100 women
Serum
Present study
Vallee et al.u
* Range based on ± 2 standard deviations,
t Atomic absorption spectrophotometry.
many laboratories, serum zinc, as well as
iron and copper, can be easily determined
from 1 ml. of serum.
Acknowledgments. These studies were conducted
in conjunction with a continuing program for development and testing of vaccines against acute
infections and were supervised by the Commission
on Epidemiological Survey of the Armed Forces
Epidemiological Board. The cooperation of the
National Service Organization of the Seventh-day
Adventist Church is gratefully acknowledged. Mr.
Roger Simpson, Mr. Walter Nixon, and Mr. Raymond Ramirez gave technical assistance and Mrs.
Russell Clabaugh aided in the preparation of this
manuscript.
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