Determination of Copper and Zinc

in Biological Material

Mary M. Parker, Fred L. Humoller, and Delmar J. Mahier

Copper and zinc are readily determined in biological material by atomic absorption
spectrophotometry. Two procedures are presented for the quantitative estimation of
these metals in serum. One of these involves simple dilution and aspiration into the
burner of the instrument. Suitably prepared standards must be used. The other
method involves trichloroacetic acid preparation of the proteins prior to aspiration.
With both methods satisfactory results in precision and recovery of added metals are
obtained. Urinary copper determinations require chelation of the metal with am-
monium pyrrolidino dithiocarbamate or some similar sequestering agent and con-
centration by extraction of the chelate into a suitable organic solvent. A similar pro-
cedure must be used in analyzing cow’s milk for copper. Tissue analysis requires
heating with boiling nitric acid and removal of the acid under reduced pressure prior
to aspiration into the burner. It has been shown that procedural errors in such
determinations are very much smaller than biological variation.

TITHE UTILIZATION of atomic absorption spectrophotometry in clinical

laboratory practice seems to be gaining increasing acceptance. While
perhaps most useful at the present time for the determination of alka-
line earth metals, it is capable, even in its present state of development,
of yielding valid results in the determination of some trace metals in
biological material. With this method it is possible to determine quickly
and quantitatively some metals in the range of a few micrograms per
gram or milliliter of tissue or serum respectively. A particular ad-
vantage of this method is that illterference by other elements is greatly
reduced, even when present in relatively large amounts. Then, too, com-
plete destruction of organic matter is not essential. If the material is a
liquid or can be liquefied by some simple procedure, it usually can be
aspirated directly into the burner of the instrument. The combined
effect of these advantages is that the chalIces of contaminating the

From the Biochemical Research Laboratory, Veterans Administration Hospital, Omaha, Neb.
68105.
Based on a paper presented at the 17th National Meeting of the American Association of
Clinical Chemists, Chicago, Ill., Aug. 30-Sept. 2, 1965.
Received for publication June 23, 1966; accepted for publication Sept. 9, 1966,

40
Vol. 13, No. I, 1967 DETERMINATION OF COPPER AND ZINC 41

sample are greatly reduced. Contamiimatiomi, be it positive or negative,

as Thiers (1) has pointed out, has been a very real obstacle in trace
metal analysis.
The present report describes convenient methods developed in this
laboratory for the quantitative determination of copper and zinc in
biological material. In these methods contamination is diminished by
reducing the number of steps and pieces of glassware required, and by
conducting the destruction of organic material, when needed, at a rela-
tively low temperature.
The method as modified for copper and zinc in serum should be suit-
able for routine work since only a few minutes are required for the
analysis and calculation of results.

Materials and Methods

This work was carried out with a commercially available double-beam
atomic absorption spectrophotometer (Perkin-Elmer Model 303)*,
equipped with a “high solids” burner head, with a slot width of 4 X
0.025 in., and using acetylene as fuel and air as oxidizer. Water for
dilution or rinsing glassware was distilled and then passed through a
mixed-bed deionizer. Nitric acid, reagent grade, was redistilled in an
all-glass still equipped with a Vigreux column.
Apparatus and Reagents
Flash Evaporaiort This all-glass instrument was found to be
effective in removing excess nitric acid under reduced pressure at a low
temperature without introducing metallic contaminants.
Methyl-isobutyl ketone (MIK) The commercially available sol-
vent is distilled in an all-glass still equipped with a Vigreux column.
A mmonium pyrrolidiu o dii hioca rba mate (A PDC) The commer-
cially available materialt was found to be free from detectable amounts
of copper and zinc. It was compared with a sample synthesized in this
laboratory by the method of Malissa and Sch#{246}ffmann(2), in nitric acid-
washed all-glass equipment. No difference in the results could be
detected in substituting this material for a commercial product.
Sodium Acetate-1M Since even the purest samples available to
us always seemed to contain heavy metal impurities, 100 ml. of the
solution of sodium acetate was treated with 2 ml. of 5% (w/v) APDC
then extracted with 10 ml. of MIK. The MIK layer was completely
removed after separation.
Trichioroacetic Acid (TCA) The certified reagent-grade acid was
*perkinElmer Instrument Corp., Norwalk, Conn.
ILaPine Scientific Company, Chicago, Ill.
Lot No. 53290L, K & K Laboratories, Inc., Plainview, N. Y.
42 PARKER ET AL. Clinical Chemistry

distilled under reduced pressure in an all-glass still, only the middle

third of the distillate was used in this work.
Standzrd copper and zinc solution Commercially available solu-
tions,* standardized for atomic absorption were used throughout. They
were checked against standards prepared from tile highest purity
metals obtainable and no differences in absorption could be detected.
Procedure
The procedure for determining the copper or zinc content of tissues
followed these general lines. An accurately weighed amount of fresh
tissue (0.2-5.0 gm.) is transferred to a 200-ml. round-bottomed flask,
having a standard taper joint. Then 10 ml. of concentrated redistilled
nitric acid is added to this tissue and the flask is covered with an in-
verted beaker. The flask is placed on a sand bath heated by a multiple-
unit hot plate. Heat is applied to cause a gentle boiling of the flask
content. This process is continued for 3 hr., irrespective of the amount
of tissue used. Tile 3-hr. period was chosen because preliminary studies
had shown that in this period as much as 5 gIn, of tissue is completely
liquefied by the boiling acid. Furthermore, heating for as long as 7 hr.
produced no detectable change in the amount of metal recovered. At
the end of the heating period the content of the flask is concentrated to
dryness in a flash evaporator at about 50#{176}. The residue is taken up in
5 ml. of water, carefully washing down the walls of the flask. Most
tissue residues are now ready for direct aspiration into the burner of
the spectrophotometer and are quantitated by using aqueous standards
to obtain a concentration-absorhance curve. In tissues that are rich in
lipid material the method should be slightly modified in order to prevent
clogging the capillary of the instrument. Tile tissue digest, as before,
is taken up in 5 ml. of water. Five milliliters of MIK is then added to
the flask and the content is thoroughly mixed. After the 2 layers have
completely separated, the capillary of the instrument is passed through
the ketone layer into the aqueous phase for aspiration. In this pro-
cedure the samples are quantitated with MTK-saturated aqueous stand-
ards. Recovery studies have shown that under these conditions no de-
tectable amounts of metals are taken up by the ketone layer.
The settings of tile spectrophotometer will, of course, vary from in-
strument to instrument, and optimum conditions have to be determined
by trial and error. In the present study they were: wavelength 3247 A
and 2138 A and scale 2 and 1 for copper and zinc respectively; range,
U.V.; air pressure, 30 psi and a flow rate of 7.0 on the flowmeter; and
acetylene, 8 psi and a setting of 6.5 on the flowmeter.
*HgrtmgjLeddon Company, Philadelphia, Pa.
Vol. 13, No. I, 1967 DETERMINATION OF COPPER AND ZINC 43

The determination of copper and zinc in serum is much simpler than

the tissue analysis. Two approaches are available for serum analyses.
The first of these is similar to that of Sprague and Slavin (3) with sonic
modifications, the serum is simply diluted with an equal volume of
water and then aspirated directly into the burner. However, tile ill-
strument will have to be equipped with a ‘‘high solids’’ burner head,
because the proteins in the solution will lead to frequent clogging of
tile standard burlier. Tile advantage of the method is that it requires
a minimum of handling of the sample and of glassware. Tile chances
for contamination are therefore correspondingly reduced. Tile second
method has tile advantage in that no special burner head is required.
In this procedure equal volumes of 8% (w/v) TCA and serum are
thoroughly mixed and then allowed to stand for 5 mii. before being
centrifuged. Tile superiiatant is then directly aspirated from the centri-
fuge tube into tile burner. The disadvantages of this method are that it
is more time-consuming and that it is a good deal more laborious to
purify the TCA than to purify water. Both methods, however, give
acceptable recovery of added metal. The settings of the instrument are
identical to those used in tissue analyses. However, to construct a cali-
bration curve it is necessary for the standards to contain approxi-
mately the same amount of protein as the diluted serum or tile same
concentration of TCA as the saniples because tile viscosity of the test
solution modifies the response of tile instrument. in tile present study
the standards for serum w’ork were made up in 3% bovine albumin or
in 4% TCA. Since it is quite difficult to obtain metal-free albumin, tile
amount present must be determined by tile method of additions (4). In
this simple procedure 3 aliquots of tile albumin solution are used. Two
of these are fortified with different, but known, amounts of the metal
to be determined. The absorbamice of all 3 samples is determined, and
an absorbance versus concentration line is plotted. Tile intercept of
this straight line on the concentration axis gives the concentration of
the metal in the albumin solution used to make up the standards.
The determination of zinc in uriie is carried out by aspirating the
undiluted or diluted urine into tile burner vitii a high solid head, using
the same settings of the instrument as are used in tile tissue zinc
determination. The copper content of normal urine is so low that chela-
tion and concentration int.o an organic solvent must be resorted to if
significant results are to be obtained. To this end a 50-mi. sample of
urine is adjusted to pH 5.5 with either metal-free sodium acetate or
nitric acid. Then 1 ml. of a 5% solution of APDC is added to tile sample
with thorough mixing. This is followed by 10 ml. of MIK. The mixture
is shaken for 3 mm. and is then centrifuged to break any emulsion that
44 PARKER El AL. Clinkal hemis+ry

may form. The M1K layer represents a five-fold concentration of the

copper in urine and can be aspirated directly into the burner. The cali-
bration curve is prepared from a series of known amounts of copper
carried through the whole procedure. The operating conditions of the
spectrophotometer are the same as previously stated except that the
flow rates of the fuel and air are reduced to compensate for the presence
of the organic solvent.
The copper content of milk varies tremendously among different
species. hence different procedures for the determination of this metal
have to be used. In the case of cow’s milk, which is low in copper, the
metal has to be chelated and concentrated by extraction into an organic
solvent. Rat’s milk (5), which contains a relatively large amount of
copper, requires only simple dilution (up to 20-fold) prior to aspiration
into the burner.

Results
Serum copper values were determined on a group of 28 employees of
this hospital. All of these were in the fasting state and presumably
normal individuals. The method employed was the TCA precipitation
procedure. Tile mean value for the group, which consisted of 18 male
and 10 female subjects was 105.1 g. of copper per 100 ml. of serum
with a standard deviation (S.D.) of ± 15.9. The mean value for the 18
men in this group was 106.5 g. Cu per 100 ml. ± 15.9 S.D., while the
10 women of the group showed a value of 102.6 g. Cu per 100 ml.
± 16.4. Serum zinc values were obtained on 23 individuals, again using
the TCA precipitation procedure. The mean value found was 89.9 g.
Zn per 100 ml. of serum ± 10.3 (S.D.).
In order to determine the precision and reproducibility of the meth-
ods for serum copper and zinc, a study was carried out, the results of
which are shown in Table 1. These values were obtained on 2 pools of
the remnants of sera, drawn at different times for diagnostic purposes.
They neither represent normal sera nor samples collected with all the
precautions necessary for trace metal analysis. However, the results
show that both methods give satisfactory precision on replicate samples
in the copper and zinc methods. Recovery of added copper is very close
to 100%, but the recovery of zinc is slightly less.
The determination of the zinc content of urine presents no particular
difficulties, since usually it is sufficiently high to permit direct aspiration
into the burner of the instrument with or without dilution. In the case
of urinary copper, which in normal individuals is very low, the more
tedious chelation and extraction method must be used. This method
involves several extra steps, each of which could lead to contamination
Vol. 13, No. I. 1967 DETERMINATION OF COPPER AND ZINC 45

Table 1. RECoVERY OF COPPER AND ZINC ADDED TO SERUM (po./100 ML.)

Copper Zne

Serum Serum + Serum + Serum + Serum Serum + Serum + Serum +

only 100 pg. Cu 25 pg. Cu 50 pg. Cu only 100 pg. Zn 25 pg. Zn 50 pg. Zn

VGA METHOD

148 245 - - 80 174 - -

146 245 - - 74 170 - -

148 248 - 73 170 - -

146 247 - - 74 172 - -

145 247 - - 72 169 - -

MEAN 147 246 75 171

% Rscovsav 99.8 96.5

SIMPLE DILUTION METHOD

138 - 165 186 70 - 94 114

139 - 162 188 71 - 94 113
137 - 163 190 71 - 92 115
136 - 162 188 72 - 93 115
137 - 161 180 72 - 90 115
140 - - - 71 - - -

141 - - - 72 - - -

139 - - - 71 - - -

139 - - - 72 - - -

140 - - - 72 - - -

MEAN 139 163 187 72 93 114

S.D. ±0.02 ±0.02
% RECOVERY 99.3 98.8 95.8 94

of the sample, either positive or negative. Furthermore, it had to be

ascertained that the extraction step would remove all the copper I)resent
in the urine sample. In agreement with Mahissa and Sch#{246}ffmann(2) it
was found that the extraction of the APDC chelate of copper illto MIK
could be carried out over a wide range of pH values, so long as one dealt
only with pure aqueous solutions. In the case of extraction from urine,
only at a pH 5-6 were satisfactory recoveries obtained.
In order to determine the precision and the recovery of added copper
by the method, amounts of pooled urine ranging from 20-50 ml. were
chelated and extracted with 10 ml. of MTK each. Duplicate samples
were fortified with 0.05 mg. of copper per liter and carried through
the procedure. The average recovery in 8 samples was found to be
102%. The results show that satisfactory precision and recovery can
be obtained with urine containing only fractional numbers of micrograms
per milliliter of copper. The values found in the 8 samples differed only
by a few micrograms per liter. Similarly satisfactory recoveries were
obtained when zinc was added to pooled urine samples as shown in
46 PARKER El’ AL. Clinical Chemistry

Table 2. In this case, however, the results were obtained by direct analy-
sis without resorting to chelation and extraction into an organic solvent.
The analysis of tissues for copper and zinc requires heating the
sample for 3 hr. with nitric acid. In order to determine the accuracy of
the method, a series of aqueous standards were carried through the
whole procedure for tissues, including chelation and extraction. The
results listed in Table 3 show that the proposed procedure yields ex-
cellent recovery values for copper. Iii the case of zinc the recoveries
are satisfactory, but not nearly so good as those obtained with copper.
It seems that in this laboratory positive contamination by zinc is more
difficult to control than is the case with copper. These results prove that
the proposed method itself can give acceptable recovery in spite of
the several additions and manipulations involved. These findings are
confirmed by the results of recovery studies of metals added to tissue
homogenates (Table 4). It does not mean, however, that the same
degree of precision can be obtained in the actual analysis of tissues. It
is difficult to obtain samples from one organ that contain identical
amounts of blood, fat, and connective tissue. Since these different tis-
sues may have different metal contents, wider variations must be ex-
pected. This is particularly true if the analyses are carried out on
samples obtained by needle biopsies. Here the difficulties are magnified

Table 2. RECOVERY OF ZINC ADDED TO URINE (MO/LITER)

Un ne + Zinc

Urine alone 0.25 mg. 0.5 mg.

0.200 0.450 0.660

0.190 0.442 0.660
0.190 0.425 0.665
0.190 0.435 0.665
0.195 0.430 0.670
MEAN 0.193 0.436 0.664
% RECOVERY 98.4 95.8

Table 3. ACCURACY OF THE PROPOSED METHOD OF TISSUE ANALYSIS (io./GM. op TISSUE)

Amount added Amount found % recovery

Cu Zn (u Zn Cu Zn

0.50 0.25 0.50 0.27 100 106

0.50 0.25 0.50 0.25 100 100
1.00 0.50 1.02 0.53 102 105
1.00 0.50 1.02 0.52 102 108
2.00 1.00 2.00 1.12 100 112
2.00 1.00 2.00 1.05 100 105
Vol. 13, No. I, 1967 DETERMINATION OF COPPER AND ZINC 47

because the small amounts of tissue available for analysis may not be
representative of the whole organ. lii Table 5 copper determinations
were carried out on the livers of 7 rats of the same age. in each in-
stance pieces of tissue were snipped from several lobes of each liver
and combined for analysis. It will be noticed that tile variations among
the different determinations carried out on tile same liver are indeed
larger than the variatiolls showii in Table 2. Hence tile analytical errors
are small compared to biological variations.
In our studies on the effect of aging on trace metal metabolism (5)
it became necessary to analyze milk for its copper content. Cow’s milk
obtained from the dining room of this hospital contained very little of
this element (0.02-0.03 g./ml.), while it has been reported (6) to be
about 10-fold this value in milk obtaine(l earlier Ill the lactation period.
These low values necessitate that tile nietais be chelated and concen-
trated in MIK. Rat’s milk, which contauls several ilulidred times as
much copper as does cow’s milk, at least in tue early stages of lacta-
tioii, can be determined by simple dilution and aspiration. However,
the high values obtained must be considered in the light of the fact that
it is difficult to obtain milk from an unanesthetized rat without intro-
ducing some contamination (7).

Table 4. RECOVERY STUDY OF ADDED METALS WITH TISSUE PROCEDURE

(/LG./iaL. OF HOMOGENATE)

Cu Zn
-

Brain homogenate only 0.505 1.837

0.525 0.504 1.825 1.824
0.482 J 1.810

Brain homogenate + 0.5 og./ml. Cu ZII 0.945

1.088 J 1 016 2.2051
2.185 J -.

% RECOVEHY 101 95

Table 5. BIOLOGICAL VARIATION IN THE COPPER CONTENT OF RAT LIVERS (a./aM. OF TISSUE)

Rat No. No. determinations* Average Range

1 3 3.8 3.3-4.2
2 4 4.1 3.9-4.3
3 4 3.8 3.7-3.9
4 4 4.1 3.9-4.3
5 3 4.2 3.9-4.4
6 4 4.4 4.1-4.6
7 4 4.1 3.9-4.4
GROUP MEAN 4.1
S.D. ±0.2

*Number of complete replicate determinations carried out on pooled segments of each liver.
48 PARKER El AL. Clinical Chemis+ry

References
1. Thiers, R. E., “Contamination in Trace Element Analysis and its ControL” In Methods of
Biochemical Analysis (Vol. 5) Glick, D., Ed. Interscience, New York, 1957, p. 273.
2. Maliasa, H., and Sch#{246}fimann,E., Uber die Verwendung von substitutieren Dithiocarbamaten
in the Mikroanalyse. III. Mikroehim. Acta 1, 187 (1955).
3. Sprague, S., and Slavin, W., Determination of iron, copper and zinc in blood serum by an
atomic absorption method requiring only dilution. Atomic Absorption Newsletter 4, 228
(1965). Perkin-Elmer Corp., Norwalk, Conn.
4. David, D. J., Determination of strontium in biological material and exchangeable strontium
in soil by atomic-absorption spectrophotometry. Analyst 87, 576 (1962).
5. Parker, M., Humoller, F. L., and Mahler, D. J., Age influenced changes in the tissue
copper and zinc contents of rats. In preparation.
6. King, B. L., and Dunkley, W. L., Relation of natural copper in milk to incidence of
spontaneous oxidized flavor. J. Dairy &i. 42, 420 (1959).
7. MeBurney, J. J., Meier, H., and Hoag, W. G., Device for milking mice. J. Lab. 4 Clin.
Med. 64, 485 (1964).