TIIK AMERICAN JOURNAL OF CLINICAL PATHOLOGY
Vol.44, No. 2
Copyright © 1005 by The Williams & Wilkins Co.
Printed in
U.S.A.
MEASUREMENTS OF NICKEL IN BIOLOGICAL MATERIALS BY
ATOMIC ABSORPTION SPECTROMETRY
F. WILLIAM SUNDERMAN, J R . , M.D.
Pathology Department, College of Medicine, University of Florida, Gainesville, Florida 32603
Quantitative determinations of trace
metals by atomic absorption spectrometry
were first described by Walsh in 1955.21 The
theory and analytical applications of atomic
absorption spectrometry have been summarized by Allan,3 David, 4 Elwell and
Gidley,5 Mahnstadt and Chambers,10 Robinson,14 Russell and co-workers,15 Willis,22 and
Zettner.23 The limits of sensitivity for the
detection of nickel by atomic absorption
spectrometry have been estimated by
Allan,1'2 Euwa and Vallee,0 Ivinson and
Belcher,9 Menzies,11 Robinson,14 and Willis.22
In the present paper, an atomic absorption
procedure is described which facilitates
quantitative measurements of nickel in
biological materials. The method has been
employed for determinations of nickel in
urine, ribonucleic acids, and serum proteins.
METHOD
Principle
After organic material has been destroyed
by wet digestion, nickel ion is converted to
nickel dimethylglyoxime, and extracted
into chloroform at alkaline pH. Nickel is
recovered from chloroform by extraction
with dilute hydrochloric acid. Aspiration
of the extract into the burners of the atomic
absorption spectrometer produces thermal
molecular dissociation and dispersion of
nickel atoms throughout the flames. A
small proportion of the nickel atoms becomes excited to emit light, but most of the
Received, December 21, 1964.
These studies were initiated while Dr. Sunderman was a member of the Division of Metabolic
Research, Jefferson Medical College, Philadelphia, Pennsylvania. The research was supported
by grants to Jefferson Medical College from the
U. S. Atomic Energy Commission, from the Rohm
and Haas Company, and by a grant to the University of Florida from the American Cancer
Society.
atoms remain in the ground state and are
capable of absorbing specific wave lengths
of incident light. These discrete wave lengths
are provided by a lamp with a hollow
cathode constructed of metallic nickel. The
beam of light from the hollow cathode lamp
is passed several times through the flames
and then is focused upon the entrance slit
of a diffraction grating monochromator.
The absorption of light is proportional to
the concentration of nickel in the sample.
Precautions
Metal-free water, which is employed
throughout the procedure, is prepared by
ion-exchange and by distillation in an allglass still. Glassware is cleaned with nitric
acid immediately before use. Analyses are
performed in duplicate or triplicate. A
calibration curve is prepared with each
group of a.nalyses.
Reagents
1. Nitric acid, reagent grade, sp. gr. 1.5.
2. Sulfuric acid, reagent grade, sp. gr.
1.84.
3. Hydrochloric acid, 0.5 N.
4. Ammonium hydroxide, reagent grade,
29 per cent (w/v).
5. Chloroform, reagent grade.
6. Dimethylglyoxime solution. Dimethylglyoxime, 0.25 Gm., is dissolved in 50 ml.
of absolute ethanol and diluted to 250 ml.
with water.
7. Citrate buffer. Diammonium citrate,
200 Gm., is dissolved in 600 ml. of water
and adjusted to pH 9.0 to 9.5 with concentrated ammonium hydroxide. The solution is transferred to a 1-liter separatory
funnel and 10 ml. of dimethylglyoxime
solution are added. The mixture is extracted
3 times with 30 ml. of chloroform. The
solution is passed through a retentive filter
paper, and diluted to 1 liter with water.
8. Phenolphthalein indicator solution. Phe182
Aug. 1965
N I C K E L IN BIOLOGICAL
nolphlhalein, 1 per cent (w/v) is dissolved
in ethanol, 70 per cent (v/v).
9. Nickel stock standard solution. Metallic nickel, 500 mg., is transferred to a 1-liter
volumetric flask, dissolved in 20 ml. of
nitric acid, and diluted to volume with water.
10. Nickel working standard solutions.
Nickel stock standard solution, 1, 2, 3, 4,
and 5 ml., is transferred to 500 ml.
volumetric flasks and diluted to volume
with water. These working standard solutions contain 1, 2, 3, 4, and 5 ng. of nickel
per ml.
A tomic A bsorption A pparatus
The principles and operation of the atomic
absorption spectrometer* have previously
been described by Sunderman and Carroll.18
For measurements of nickel, 3 Beckman
oxygen-acetylene burners are mounted in
series upon the burner manifold. The cannulas
of the burners are attached to 6-cm. lengths
of polyethylene tubing,f in order that the
sample may be aspirated from a test tube.
The quartz mirrors of the optical system
are focused so that the beam of light from
the nickel hollow cathode lamp passes 5
times through the flames, providing an
absorption path length of approximately 30
cm. The output of the photomultiplier tube
amplifier circuit is recorded graphically by
means of a strip-chart recorder.:): The amplification of the recorder is increased to provide full-scale deflection with a 5-mv.
signal.
Procedure
Digestion of urine. Fifty-milliliter samples
of urine are transferred to 100-ml. Kjeldahl
flasks. Twenty milliliters of nitric acid and a
few Pyrex beads are added to each flask and
the contents of the flask are digested to a
volume of approximately 10 ml. The flask is
cooled and 3 ml. of sulfuric acid are added.
* Atomic absorption spectrometer, Model 82-362,
Jiirrcll-Ash Company, Waltham, Massachusetts.
t Polyethylene " I n t r a m e d i c " tubing, No. P E 100, Clay-Adams Company, New York, New
York.
% Strip-Chart recorder, Model No. E15-I, BrownHoneywell Regulator Company, Philadelphia,
Pennsylvania.
MATERIALS
183
Digestion is continued until the appearance
of white fumes of sulfur trioxide. If charring
occurs, nitric acid is added as needed to
clarify the digestion mixture. Three-milliliter
quantities of water are added to each flask
3 times. Following each addition of water,
digestion is continued until the appearance
of white fumes.
Digestion of ribonucleic acid (RNA) or
protein. Samples of RNA or protein weighing 15 to 50 mg. are transferred to 100-ml.
Kjeldahl flasks. Five milliliters of nitric
acid and a few Pyrex beads are added to
each flask, and the contents of the flask are
digested to a volume of 1 to 2 ml. The flask
is cooled and 1 ml. of sulfuric acid is added.
The digestion is continued as described for
samples of urine.
Blank and standard samples containing
0, 1, 2, 3, 4, and 5 Mg- of nickel are digested
by the same procedure that is used for the
unknown samples.
Extraction of nickel. Each digestion flask
is cooled to room temperature in a water
bath. Five milliliters of citrate buffer are
added and the contents of the flasks are
again cooled to room temperature. One drop
of phenolphthalein indicator is added, and
the contents of each flask are titrated with
ammonium hydroxide until the appearance
of a pink color. After the solution has cooled,
3 ml. of dimethylglyoxime solution are
added and the contents of the flask are
mixed. Three milliliters of chloroform are
added; the flask is stoppered with a Teflon
plug, and the contents of the flask are
shaken for 1 min. The flask is allowed to
stand for approximately 10 min., in order to
complete the separation of the aqueous and
chloroform phases. Using a long Pasteur
pipet, the chloroform phase is removed
quantitatively and transferred to a 12-ml.
centrifuge tube. Extraction of the aqueous
phase is repeated with a second 3-ml. volume
of chloroform. In analyses of urine, a white
precipitate of inorganic salts remains in the
aqueous phase. One milliliter of 0.5 N hydrochloric acid is added to the centrifuge tube
which contains the combined chloroform
extracts. The tube is stoppered, shaken
vigorously for 1 min., and centrifuged at
2500 r.p.m. for 3 min. The chloroform phase
is removed by aspiration and discarded.
184.
Vol. 44
SUNDERMAN
Adjustment of the atomic absorption apparatus. The power supply to the nickel
hollow cathode lamp is adjusted to 30 ma.
The beam of light from the hollow cathode
lamp is focused upon the monochromator
aperture. Monochromator entrance and.
exit slits of 25 n are placed in the slit-holders.
The monochromator is adjusted to measure
the absorption at 2320.0 A. The oxygen
regulator valve is opened first, followed by
the acetylene regulator valve, and the
burners are ignited. The gas regulator
valves are adjusted to provide an oxygen
pressure of 15 p.s.i. and an acetylene pressure of 9.5 p.s.i.
An opaque card is placed in front of the
monochromator entrance slit and the
recorder is adjusted to 100 per cent absorption. The card is removed and the recorder
is adjusted to 0 per cent absorption while
water is aspirated through the burners.
Fluctuations in the baseline are minimized
by use of the damping adjustment. The
stability of the burners, optical system, and
electronic system is verified by monitoring
the base line while water is aspirated through
the burners.
Measurements of atomic absorption. The
polyethylene tubes connected to the burners
are inserted into a test tube which contains
1 of the standards or samples. The absorption reading does not become stable until
the sample has been aspirated into the
flames for 2 to 3 sec. The atomic absorption
of the sample is then recorded for 5 sec.
Immediately following the aspiration of each
sample, water is aspirated through the
burners until the absorption reading has
returned to the base line.
EXPERIMENTAL STUDIES AND RESULTS
Instrumental Parameters
The sensitivity of detection of nickel with
the atomic absorption spectrometer was
determined for the nickel emission lines at
2310.9 A, 2320.0 A, 2345.5 A, 3002.5 A,
3050.8 A, 3414.8 A, 3461.7 A, and 3524.5 A.
These measurements confirmed Allan's
report1 that greatest sensitivity was achieved
with the emission line at 2320.0 A. Under
the operating conditions described for the
procedure, the sensitivity (1 % A) of detection of nickel in aqueous solution without
preliminary extractions was 0.10 p.p.m.
Comparisons were made of the measurements of nickel with fuel mixtures of acetylene and oxygen, acetylene and air, propane
and oxygen, and propane and air. The
sensitivities which were obtained with
acetylene-oxygen flames were greater than
those obtained with the other fuel mixtures.
A systematic evaluation was made of measurements of nickel with oxygen pressures
ranging from 7 to 18 p.s.i., and with acetylene pressures ranging from 5 to 12 p.s.i.
Optimal sensitivity was achieved with an
oxygen pressure of 15 p.s.i. and an acetylene
pressure of 9.5 p.s.i.
In order to prevent disturbance of the
flames by drafts of air, as well as to reduce
the auditory and visual irritation produced
by the flames, a flame-shield was constructed
of %6-in. asbestos sheeting. The flameshield surrounded the burner and optical
systems of the atomic absorption apparatus.
Use of the flame-shield improved the constancy of the recorder baseline, and thereby
resulted in a slight increase in the sensitivity
of the apparatus for the detection of nickel.
At the conclusion of the analyses, water To avoid inhalation of nickel, the atomic
is aspirated through the burners for 5 min. absorption spectrometer was operated within
The acetylene regulator valve is closed and a fume hood.
the burners are dried in a stream of oxygen
Additions of octyl alcohol, caprylic alfor 1 to 2 min.
cohol, and Sterox* to standards and urine
Calculations. Measurements of per cent samples did not improve the performance
absorption (% A) are converted into ab- of the burners or increase the sensitivity of
the method. Additions of a variety of orsorbance (O.D.) units by the equation:
ganic
solvents, including isopropyl alcohol,
Absorbance(O.D-) = 2-logi 0 (100 - % A).
A calibration curve is used in computing
* Sterox is a non-ionic detergent, produced by the
the concentrations of nickel in the unknown Monsanto Chemical Corporation, St. Louis, Missamples.
souri.
Aug. 1965
N I C K E L IN BIOLOGICAL
propyl alcohol, and acetone, were likewise
without significant effect.
Digestion and Extraction Procedures
The sensitivity of the atomic absorption
spectrometer was insufficient to permit
direct measurements of nickel in normal
urine; therefore, it was necessary to employ
a chemical procedure to concentrate nickel,
prior to atomic absorption spectrometry.
Destruction of organic materials is an essential preliminary step in such procedures.
The wet digestion technic which is described
in the present method was found to be more
rapid and convenient than the digestion
procedure previously developed in our
laboratory, which used a mixture of perchloric, sulfuric, and nitric acids.8,16
Several methods were attempted for
extraction and concentration of nickel from
the digestion mixture. In 1 approach, the
digestion mixture was adjusted to pH 8.5,
and sodium diethyldithiocarbamate was
added. Nickel diethyldithiocarbamate was
extracted into isoamyl alcohol, and the
isoamyl alcohol extract was aspirated
directly into the burners of the atomic
absorption apparatus. In a second approach,
the digestion mixture was adjusted to
alkaline pH and dimethylglyoxime was
added. Nickel dimethylglyoxime was extracted into butanol, and the butanol extract was aspirated directly into the burners.
Dissatisfaction with the results obtained
with both of these approaches led to the
development of the extraction technic which
is described here. Under the conditions
outlined for the procedure, extraction of
nickel dimethylglyoxime with chloroform
was found to be preferable to the use of
carbon tetrachloride, methylene chloride,
ethylene dichloride, or a mixture of equal
parts of chloroform and carbon tetrachloride.
Extraction of the chloroform phase with
1 ml. of 0.5 N hydrochloric acid, as described
above, resulted in a mean recovery of 80.3
per cent (S.D. = ±3.4) of the nickel which
was originally present in the digestion
mixture. The recovery of nickel could be
increased to 96 per cent by a second extraction of the chloroform phase with 1 ml.
MATERIALS
185
of hydrochloric acid. Since the concentration
of nickel in the combined hydrochloric
acid extracts was only 60 per cent of that
in the first hydrochloric acid extract, greatest sensitivity of analysis was accomplished
by a single extraction with hydrochloric acid.
Tests for Interference
To a series of standard samples containing
1 ng. of nickel were added soluble salts containing 100 ng. of metals which might possibly constitute sources of interference. The
metals that were tested included sodium,
magnesium, aluminum, potassium, calcium,
vanadium, chromium, manganese, iron,
cobalt, copper, zinc, arsenic, strontium,
molybdenum, silver, cadmium, gold, mercury, lead, bismuth, and uranium. Cobalt
and chromium salts were the only apparent
sources of interference. Measurements with
salts of cobalt and chromium from several
commercial sources indicated that the apparent interferences were probably attributable to contamination of the reagents with
traces of nickel.
To a series of standard samples containing
1 Mg- of nickel were added sodium salts
containing 100 ng. of anions which might
possibly constitute sources of interference.
The anions that were tested included borate,
citrate, oxalate, nitrite, nitrate, fluoride,
metasilicate, phosphate, sulfite, sulfate,
thiosulfate, persulfate, chloride, and bromide. None of these anions interfered in the
measurement of nickel.
Calibration Curve
As illustrated in Figure 1, the calibration
curve obtained with the atomic absorption
procedure adhered to the Beer-Lambert
law with quantities of nickel in the range of
1 to 5 jug. With quantities of nickel greater
than 5 fig., the calibration curve deviated
from linearity. Since the slope of the calibration curve was influenced by small
fluctuations in gas pressures and in monochromator adjustments, it was necessary to
prepare a calibration curve with each set of
analyses. It may be noted from Figure 1
that the atomic absorption technic was
approximately 20 per cent less sensitive
than the laborious ultraviolet spectrophoto-
186
SUNDERMAN
0.6
0.5
ULTRA-VIOLET
SPECTROPHOTOMETRY
PROCEDURE
(I cm. cuvels, 3200 A)
0.4.
1
1«
ATOMIC ABSORPTION
SPECTROPHOTOMETRY
(2320.0A)
0.2
0.1
Vol. 44
marized in Table 1. The subjects were
physicians and laboratory technologists
who had no occupational exposure to nickel.
The mean concentration of nickel in urine
was 1.8 tig. per 100 ml. (S.D. = ± 0.8),
with a range from 0.4 to 3.1 Mg- per 100 ml.
The mean excretion of nickel in urine was
19.8 Mg- per 24 hr. (S.D. = ±10.0), with a
range from 7.2 to 37.6 Mg- per 24 hr. As
indicated in Table 1, the observed concentrations of nickel in urine are comparable to the concentrations reported by
previous investigators. 7, 8- 12- 13
Measurements of Nickel in Ribonucleic Acids
1
2
NICKEL ( M g
3
4
/sample)
F I G . 1. Comparison of nickel calibration curves
by spectrophotometry and atomic absorption.
metric method which was previously reported.10
Measurements of Precision and Recovery
Statistical analysis of differences between duplicate measurements of nickel in
urine samples from 17 normal persons indicated that the S.D. of the method was
±0.12 Mg- of Ni per 100 ml., (coefficient of
variation = 6.3 per cent). Ten replicate
determinations of nickel were performed
upon a single specimen of human ^-globulin.*
The samples of /3-globuliu taken for analysis
ranged in weight from 33 to 38 mg. and
contained an average of 1.61 Mg- of nickel.
The mean concentration of nickel was 45.9
jug. of Ni per Gm. of protein (S.D. ±2.7),
with a range of values from 41.3 to 50.2.
The coefficient of variation of replicate
analyses was 5. 9 per cent.
The recovery of 2.5 Mg- of nickel added
to 4 samples of urine averaged 96 per cent,
with a range from 94 to 97. The recovery of
1 Mg- of nickel added to 4 samples of human
0-globulin averaged 101 per cent, with a
range from 96 to 104.
Measurements of Nickel in Urine
Measurements of the urinary excretion
of nickel by 17 normal persons are sum* Pentex Biochemicals, Inc., Kankakee, Illinois.
Measurements of nickel were made upon
specimens of ribonucleic acids isolated from
the ultracentrifugal (109,000 X g) supernatants of homogenates of lungs and livers
of normal rats, by methods previously developed in our laboratory.16- "• l9 The mean
concentration of nickel in 5 preparations of
supernatant RNA from rat lung was 48
Mg. of Ni per Gm. of RNA with a range
from 34 to 64. The mean concentration of
nickel in 5 preparations of supernatant RNA
from rat liver was 29 Mg- of Ni per Gm. of
RNA, with a range from 21 to 39. In studies
which are currently in progress, the concentrations of nickel in preparations of
RNA from normal rats are being contrasted
with the concentrations of nickel in RNA
from rats which have been chronically
exposed to the inhalation of nickel carbonyl to carcinogenic levels.
Measurements of Nickel in Serum Proteins
The mean concentrations of nickel in
purified preparations of human serum proteins are listed in Table 2. Electrophoretic
fractions of serum proteins from a normal
subject were prepared in our laboratory by
continuous-flow electrophoresis, lyophilization, lipid extraction, and dialysis, as previously described.20 Purified serum proteins, prepared by cold-ethanol fractionation, were obtained from 2 commercial
sources.* As indicated in Table 2, the
* Nutritional Biochemicals Corporation, Cleveland, Ohio, and Pentex Biochemicals, Inc., K a n k a kee, Illinois.
Aug.
1965
I\ICKEL IN BIOLOGICAL MATERIALS
•
•
187
TABLE 1
R E S U M ^ OF NORMAL, V A L U E S FOR U R I N E
Urine Nickel
No. of
Subjects
Year
NICKEL
Mean
S.D.
Range
lig./100 ml.
Kinkuid and associates 8
Perry and Perry 13
Morgan 12
Imbrus and associates'
Present, study
1956
1959
1960
1963
1965
154
17
1.1
2.0
4.0
1.0
1.8
Present study
1965
17
19.8*
69
24
±0.9
±2.6
±2.0
±0.7
±0.8
0-3.0
1.0-7.0
0-8.0
0.1-8.1
0.4-3.1
±10.0*
7.2-37.6*
* These results are given in micrograms per 24 hr.
TABLE 2
CONCENTRATIONS OF N I C K E L IN
SERUM
Serum Protein Fractions
PURIFIED
PROTEINS
ContinuousFlow
Electrophoresis
ColdEthanol
Fractionation*
ColdEthanol
Fractionation!
M. Ni/Gm. pre tein
Albumin
a-Globulins
Fraction IV-1
Fraction FV-4
/3-Globulin
7-Globulin
9
22
51
19
5
7
<1
7
93
3
<1
10
46
<1
* Nutritional Biocliemicals Corp., Cleveland,
Ohio.
j Pentex
Biochemicals,
Inc.,
Kankakee,
Illinois.
highest concentrations of nickel were present in preparations of serum /3-globulin.
Attempts to isolate and to characterize a
nickel-binding constituent of serum /3globulin are currently being undertaken in
our laboratory.
DISCUSSION
The sensitivity of the atomic absorption
procedure for the determination of nickel in
biological materials is greater than the sensitivity of the colorimetric procedures8, 12
and less than the sensitivities of the ultraviolet spectrophotometric16 and emission
spectrographic technics.7' 13 The time required for measurements of nickel by the
atomic absorption procedure is approxi-
mately one half of that required for the ultraviolet spectrophotometric procedure. The
cost of an atomic absorption spectrometer is
less than that of a suitable emission spectrograph, and the atomic absorption spectrometer is more adaptable to use in clinical
laboratories. For these reasons, the atomic
absorption procedure should be especially
useful for routine determinations of urinary
nickel, in the detection of industrial exposures to the inhalation of nickel carbony l.8- 12
SUMMARY
An atomic absorption procedure has been
developed which facilitates quantitative
measurements of nickel in biological materials, including urine, ribonucleic acids
(RNA), and serum proteins.
The sensitivity of detection of nickel by
the atomic absorption spectrometer (0.10
p.p.m.) was insufficient to permit direct
measurements of nickel in normal urine.
Therefore, it was necessary to employ a
dimethylglyoxime extraction procedure to
concentrate the nickel, prior to atomic
absorption spectrometry.
The coefficients of variation of measurements of nickel in urine and human /3globulins were 6.3 and 5.9 per cent, respectively. The recovery of nickel added to
urine averaged 96 per cent, with a range
from 94 to 97, and the recovery of nickel
added to human /3-globulin averaged 101
per cent, with a range from 96 to 104.
The mean concentration of nickel in 24-hr.
188
SUNDERMAN
collections of urine from 17 normal subjects
was 1.8 jug. per 100 ml. (S.D. = ±0.8), with
a range from 0.4 to 3.1. The mean urinary
excretion of nickel was 19.8 jug. per 24 hr.
(S.D. = ±10.0), with a range from 7.2
to 37.6.
The mean concentration of nickel in 5
preparations of ribonucleic acids from ultracentrifugal supernatants of homogenates of
rat lung was 48 ng. of Ni per Gm. of RNA,
with a range from 34 to 64. The mean concentration of nickel in 5 preparations of
RNA from ultracentrifugal supernatants of
homogenates of rat liver was 29 jug. of Ni
per Gm. of RNA, with a range from 21 to 39.
Measurements of nickel were performed
upon fractions of human serum proteins,
prepared by continuous-flow electrophoresis,
and by cold-ethanol precipitation. The highest concentrations of nickel were found in
preparations of serum j3-globulins.
Acknowledgments.
F . William
Sundermiin,
M . D . , gave advice and encouragement in this
research, and William R. A. Boben, M . D . , a Student Research Trainee of the American Cancer
Society, assisted with this work.
1.
2.
3.
4.
5.
6.
7.
8.
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