CIJN. Cl-EM.

21/4, 626-629(1975)

Determination of Cadmium in Blood and Urine by Graphite

Furnace Atomic Absorption Spectrophotometry

Elizabeth F. Perry, S. Roy Kolrtyohann, and H. Mitchell Perry, Jr.

We describe a method for measuring quantities of cad- The advent of the graphite furnace atomizer for
mium inblood,plasma, and urineby usinggraphitefur- atomic absorption allows cadmium to be measured
nace atomic absorptionpreceded by wet ashing. The with about 100 times greater sensitivity than is possi-
method appears to be extremelyaccurateand repro- ble with flame atomizers (7, 8). The graphite furnace
ducible (coefficient of variation is 7% for 2.8 jzg/llter methods do suffer from more severe problems of
concentration), witha detection limit of 2 pg. background absorption and matrix interference than
Additional Keyphrases: metallothionein #{149}
hypertension
are commonly seen in flame atomizers. In the method
#{149}
normal values described here, both of these problems are decreased
to manageable levels by preliminary digestion of the
Interest in cadmium concentration in human samples.
tissues was aroused with Tipton’s observation in 1952
that kidneys from American subjects without known Materials and Methods
exposure to cadmium contained a total of about 15 Apparatus: We used a Model 403 atomic absorp-
mg of that metal (1). After this Perry et a!. observed tion spectrophotometer with attached deuterium arc
considerable geographic variation in renal cadmium, for background correction, equipped with a Model
with kidneys from subjects from industrialized areas HGA 2000 graphite furnace. A Model 165 recorder
of the world having higher amounts than those from recorded the absorption peaks. All of this equipment
persons in underdeveloped nations (2). Recognition is supplied by Perkin-Elmer Corp., Norwalk, Conn.
that cadmium was concentrated in liver and kidney, 06856.
where it was bound to a peculiar low-molecular- Samples: Samples of whole blood, plasma, and
weight protein, named metallothionein (3, 4), and ex- urine were collected to test the applicability of the
perimental work indicating that long-term feeding of method to biological materials. The determination
small amounts of cadmium can cause hypertension in was done on samples of whole blood and plasma from
animals (5, 6) have spurred interest in measuring rats exposed to cadmium. For 18 months these rats
cadmium in living human subjects, particularly in had been maintained in a low cadmium environment
whole blood, plasma, and urine. To do this and to de- and had received a low cadmium diet; the drinking
lineate what biological effects, if any, cadmium may water of various groups had contained 0, 1, 2.5, 5, 10,
have in man, a method for measuring cadmium in na- 25, or 50 mg of Cd per liter (or parts per million). De-
nogram amounts is needed. Most recent analytical tails of cadmium exposure have been described pre-
methods for assaying nanogram quantities of cadmi- viously (6) for these animals.
um rely on multiple extraction procedures and flame By using animals with known and different expo-
atomic absorption spectrophotometry for detection. sures to cadmium, we hoped to be able to relate cad-
They are both cumbersome and time consuming, and mium concentrations in whole blood and plasma to
particularly subject to inaccuracies resulting from differing cadmium intake. Cadmium concentrations
loss or contamination during the many steps in the in plasma and urine were also determined for 16
procedures. mildly hypertensive human subjects.
The Medical Service, Veterans Administration Hospital, and the Standard and sample preparations: All water
Hypertension Division, Department of Medicine, Washington Uni- used for preparation of standards, dilution of sam-
versity School of Medicine, St. Louis, Mo. 63110 (E. F. P. and H. ples and washing of glassware was de-ionized and had
M. P.); and the Environmental Trace Substances Research Labo-
ratory, University of Missouri, Columbia, Mo. a minimum resistance of 5 M(L Redistilled nitric acid
Received Dec. 9, 1974; accepted Feb. 7, 1975. and perchioric acid (G. Frederick Smith Chemical

626 CLINICAL CHEMISTRY, Vol. 21, No.4, 1975

 Co., Columbus, Ohio) were used throughout. Hydro- .5
gen peroxide (30%) was purchased from Fisher Scien-
tific Co., St. Louis, Mo. 63032. Pyrex digestion tubes .4
were decontaminated by treating each tube with six
blank digestions with 0.5 ml of 70% perchloric acid
and rinsing six times with de-ionized water between 0

each digestion. (Decontaminated tubes are available .

from Environmental Science Associates, Burlington,
Mass. 01803).
Standard solutions of cadmium were freshly pre-
pared each week and carefully checked daily for con-
stancy of absorption in the concentration range of 1
to 5 ig/liter from a 1000 mg/liter reference standard
(Fisher Scientific Co.) in dilute (10 rnl/liter) nitric Cd Conc.ntrotion (pg/Iit.r)

acid. Fig. 1. Standard curve foraqueous cadmium standards (mean

Heparmnized rat blood was collected from anesthe- and standard deviation) for a two-month period (n = 16)
tized animals by percutaneous cardiac puncture, with
use of a 3-rn! disposable syringe and stainless-steel
disposable needle (Monoject), and placed into poly- omization step. Voltages (measured across the fur-
ethylene tubes. Human blood was obtained with sim- nace leads) were 0.6, 0.9, and 4.5 V for dry, char, and
ilar equipment from the antecubital vein, without use atomize, respectively. The absorption peak was re-
of a tourniquet. Immediately after collection, 0.5 ml corded at 228.8 nm, and the peak height used for
of blood was immediately transferred to digestion quantitative measurement. A series of cadmium stan-
tubes and 1 ml of concentrated nitric acid added. The dards in nitric acid (10 mi/liter) was analyzed in the
tubes were placed in a heating block and the blood same manner and a standard curve obtained. A 20 Ml
slowly digested for 3 h at a temperature just below volume of sample solution was used throughout, and
boiling. When the volume had been reduced to about all concentrations are expressed as micrograms of Cd
a third, 0.4 ml of 30% hydrogen peroxide was added, per liter of injected sample.
the sample evaporated at the same temperature, re-
moved from the heating block, and the residue dis- Results
solved in 5.0 ml of nitric acid (10 rnl/liter) to provide Figure 1 shows a representative calibration curve
a sample solution for analysis. Plasma was handled in for aqueous cadmium standards. The curve is linear
the same manner as blood after the cells were re- from 0 to 3 hg/liter with a diminution in signal at
moved by centrifugation. higher concentrations.
Twenty-four-hour urine samples were collected A representative recorder tracing (Figure 2) dem-
from the patients directly into disposable polyethyl- onstrates the importance of digestion, background
ene containers (cat. No. B7938; Scientific Products, correction, and atomization temperature. Intensifica-
McGraw Park, III. 60085) that contained 20 ml of hy- tion in signal is noted when digested samples are
drochloric acid (3 mol/liter). A 1.0-rn! aliquot of urine used with a 10-fold dilution factor and background
was placed in the digestion tube and 0.2 ml of con- correction (Figure 2D).
centrated nitric acid was added. The urine was di- Accuracy: Five aliquots of National Bureau of
gested as described above for blood. After digestion Standards bovine liver certified to contain 0.27 g ±
the dry sample was dissolved in 2.0 ml of nitric acid 0.04 ig of Cd per gram were assayed and found to
(10 ml/liter) for assay. contain 0.27 ± 0.01 jg of Cd per gram.
All reagents used in sample collection and prepara- Recovery: Standard was added to six samples of
tion were carried through in the blank. The reagent rat blood to check the effect of sample matrix on cad-
blank consistently corresponded to 50 ng/liter. Dis- mium determination. For four aliquots of each of the
posable urine containers were filled with 20 ml of hy- six samples of blood, recovery varied from 85 to
drochloric acid diluted to 1500 ml with de-ionized 110%, with an overall average recovery of 100.3 ±
water and an aliquot was monitored for cadmium. No 6.5% when 1, 2, or 3 g of cadmium were added
cadmium was detected. (Table 1).
Sample analysis: Twenty microliters of the sample Sensitivity: We found that 0.9 pg of cadmium is
solution was transferred to a standard graphite tube necessary for an absorption of 0.0044 A. Expressed as
with an Oxford pipet. Optimum temperature and concentration, this is 45 ng of cadmium per liter,
time for drying, charring, and atomizing, established when a 20 ILl sample volume is used. The detection
experimentally, were found to be 150 #{176}C for 30 s, 300 limit (2 X blank) for cadmium was considered to be 2
#{176}Cfor 60 s and 1950 #{176}C for 8 s, respectively. The pg, which corresponds to a concentration of 0.1 g of
chamber was purged with argon. The furnace was op- cadmium per liter of the digested and diluted sample.
erated in the “interrupt” mode, so that the flow of Reproducibility: Coefficients of variation for the
purge gas to the furnace was stopped during the at- measurement step, determined on 10 replicate injec-

CLINICAL CHEMISTRY, Vol. 21, No.4. 1975 627

 .9 -

Table 1. Recovery of Cd Added to Sample

.8 - Solution of Rat Plasma
% Recovery after addition of
Cd In sample
.7 -
,g/liter 1.0 Mg/liter 2.0 pg/liter 3.0 Mg/liter
0.30 110 98 109
.6 - 0.38 98 101 104
0.50 107 98 95
.5 - 0.68 97 85 102
0.67 99 105 110
1.28 97 98 92
Mean recove ry, 100.3± 6.59%.

Table 2. Blood and Serum Cadmium

Concentrations in Rats Ingesting Cadmium
for 18 Monthsa
Cd exposure Blood Cd Plasma Cd
(mg Cd/liter water) Mg/liter
Control <1.6 (1) <1.2 (3)
1 2.2 <1.19 (1)
FIg. 2. Representative recorder tracing for plasma from rat 3.7
2.5 <2.1 (1)
exposed to25 mg ofcadmium per liter of drinking water (See
text for exposure details)
5 8.6 3.8
Vertical scale representsabsorbance units.A, 20 I sample of undigested 10 26.6 9.2
plasma dilutedfivefoldwithde-lonizedwater and atomIzed in graphite fur- 25 94.0 14.0
nace withoutbackgroundcorrection. B, Same sample with backgroundcor-
rection.C. Same sample, digestedand diluted 10-fold with nitric acid (10
50 92.0 42.0
rni/Iter) and atomized without backgroundcorrection. D, Same as C, butato- Mean cadmium concentration in blood and plasma for seven
mized wIthbackground correction. Note thatthe sensitivityIsIncreasedal. groups of five rats each. The first group had no cadmium added
most twofoldwitha digestedsample. E, same as B, but usingselective vola-
tilization of Ross and Gonzalez to drinking water; the other six had increasing amounts added.
(See text for details of exposure). In obtaining means, samples
below detection limitswere calculated as being at the detection
limitfor the method (0.1 ,ig/liter
for the sample solution).The
tions of a single sample solution, were 7% at a cadmi- number of such samples is given in parentheses, with a less-
than (<)sign preceding the mean value.
um concentration of 2.8 g per liter, and 14% at 0.18
g per liter.
Sample results: Cadmium values for whole blood
and serum from a total of 35 rats with varying cadmi- ical separation. Polarography, particularly anodic
um exposure are given in Table 2. Cadmium concen- stripping voltammetry, is very sensitive, but time
trations in 16 plasma samples from untreated out- consuming (12). Although modification of the sam-
patients (with mild hypertension) averaged 10.6 ± pling system used in atomic absorption can increase
4.9 g per liter (range, 4.7 to 20.0 Lg). Cadmium sensitivity for cadmium (17), precision is affected.
values for 24 urines from the same patients averaged The carbon furnace attached to an atomic absorption
1.25 ± .84 g per liter or 1.63 ± 1.04 g per 24 h speci- spectrophotometer affords the possibility of detect-
men. ing cadmium in 10- to 100-fold smaller amounts than
was previously possible with atomic absorption spec-
Discussion trophotometry alone.
Many methods have been used to determine cad- The method gave values in good agreement with
mium in biological materials: colorimetry (9), emis- National Bureau of Standards certified values for bo-
sion spectroscopy (10), neutron activation analysis vine liver, analytical recovery of cadmium added to
(11), polarography, particularly anodic stripping vol- samples was good, and the results were in the accept-
tammetry (12), and atomic absorption spectropho- able range for human biological samples. Of course
tometry (13). Measurement of nanogram quantities the demonstration that cadmium can be accurately
of cadmium, particularly in blood and urine, has a!- determined in bovine liver suggests-but does not es-
ways been fraught with methodological problems. tablish-that similar determinations will be compa-
Unrecognized interference by sodium, particularly rably accurate in other biological materials with a
when atomic absorption spectrophotometry was used somewhat similar matrix. Because no certified serum
(14), has probably led to many falsely high values or urine standard is available, further proof of accu-
in the literature. Concentration, wet ashing, and nu- racy is difficult to obtain.
merous extractions with dithizone (9), methyl iso- Published values for whole blood and serum range
butyl ketone (15), or a combination of chelates from <2 to >40 Mg/liter (18). With the limited but
#{149}
(16)-with all of their potential for contamination- widely varying results reported from different labora-
characterized most previously used assay methods. tories, it is difficult to fix a range for the usual con-
Neutron activation analysis has often required chem- centrations of circulating cadmium. The data on hu-

628 CLINICAL CHEMISTRY, Vol. 21, No.4. 1975

mans presented here are somewhat higher than nor- References
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literature. Such a consensus suggests that there is no human tissues. Physiol. Rev. 87,179(1952).
obvious difference between whole blood and serum, 2. Perry, H. M., Jr., Tipton, I. H., Schroeder, H. A., et al., Varia-
in both of which cadmium appears to range from 2 to tion in the concentration of cadmium in human kidney as a func-
tion of age and geographic origin. J. ChronicDis. 14,259(1961).
5 ag/liter in control (no specific exposure) subjects 3. Kagi, J. H. R., and Vallee, B. L., Metallothionein: A cadmium
and from 10 to 30 pg/liter in workers exposed to cad- and zinc-containing protein from renal cortex, II. Physiochemical
mium. The data on rat blood show a strong relation- properties. J. Biol. Chem. 236,2435(1961).
ship between exposure and whole blood and plasma 4. Pulido, P., Kagi, J. H. it, and Vallee, B. R., Isolation and some
properties of human metallothionein. Biochemistry 5, 1968 (1966).
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5. Schroeder, H. A., Cadmium hypertension in rats. Amer. J.
than the plasma values, particularly with heavy expo- Physiol. 207,62 (1964).
sure. This pattern is reminiscent of the impression 6. Perry, H. M., Jr., and Erlanger, M. W., Metal-induced hyper-
that cadmium tends to be bound to erythrocytic pro- tension following chronic feeding of low doses of cadmium and
mercury. J. Lab. Clin. Med. 83,541 (1974).
teins in chronic human exposure (18). Until the re-
7. Analytical Methods for Atomic Absorption Spectrophotome-
cent observation in Japan suggesting that urinary try, Perkin-Elmer, Norwalk, Conn., 1971.
cadmium was a function of age and therefore pre- 8. Analytical Methods for Atomic Absorption Spectroscopy
sumably of exposure (19), renal excretion of cadmi- Using the HGA Graphite Furnace, Perkin-Elmer, Norwalk, Conn.
um in man has generally been considered to be low 1973,p 34.
and nonreflective of exposure unless there was 9. Saltzman, B. E., Colorimetric micro-determination of cadmium
with dithizone. Anal. Chem. 25,493 (1953).
marked kidney damage. The urinary values that we 10, Tipton, I. H., Cook, M. J., Steiner, R. L., et al., Trace elements
obtained are within the range of values obtained by in human tissue, I. Methods. Health Phys. 9,89 (1963).
several other workers (19, 20). 11. Westermark, T., and Sjostrand, B., Activation analysis of cad-
As with other methods, the carbon furnace also has mium in small biopsy samples. mt. J. Appl. Radioisot. 9, 78
(1960).
methodological problems. Pretreatment of blood,
12. Matson, W. R., Roe, D. K., and Garritt, D. E., A composite
plasma, and urine by wet ashing before analysis was mercury graphite electrode for anodic stripping voltammetry.
essential to minimize background. Higher charring Anal. Chem. 37, 1598 (1965).
temperatures decreased the background for undigest- 13. Slavin, W., Atomic Absorption Spectroscopy, Interscience,
New York, N. Y., 1968, pp 86,87.
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14. Pulido, P., Fuwa, K., and Vallee, B. L., Determination of cad-
Gonzalez controlled temperature at various stages to mium in biological materials by atomic absorption spectrophotom-
avoid pretreatment of plasma and urine samples etry. Anal. Biochem. 14,393 (1966).
(21). We observed cadmium losses at temperatures 15. Lehnert, F., Schaller, K. H., and Haas, T., Atom absorptions-
below the temperature (450 #{176}C) that they used for spektrotnetrische Cadmiumbestimmung in Serum und Ham. Z.
KIm. Chem. Kim. Biochem. 6, 174 (1968).
charring, while their atomization at 1300 #{176}C
resulted 16. Perry, H. M., Jr., and Perry, E. F., Normal concentrations
in incomplete volatilization, although it lessened the of some trace metals in human urine: Changes produced by ethyl.
problem with background. In our hands, their meth- enediaminetetraacetate.J. Clin. Invest. 38, 1452 (1959).
od was less than half as sensitive as the one described 17. Delves, H. T., A micro sampling method for the rapid determi-
nation of lead in blood by atomic absorption spectrophotometry.
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temperature. Although we used relatively large Cadmium in the Environment, CRC Press, Cleveland, Ohio, 1974,
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19. Katagiri, Y., Tati, M., Suata, H., and Kauai, M., Concentration
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This work was supported by the Veterans Administration graphite tube reservoir atomic absorption spectrometry. Anal.
(6680-01). Chim. Acta 70, 443 (1974).

CLINICAL CHEMISTRY, Vol. 21. No. 4, 1975 629