CLIN.
24/2, 303-308(1978)
CHEM.
Determination
of Chromium
Activation Analysis
and Cobalt in Human Serum
Jacques Versieck,1 Julien Hoste,2 Fabrice Barbier,’ Herman
Michels1
Confusion
exists
about
the
chromium
and
cobalt
con-
centrations
in the serum of healthy individuals.
We determined these elements
by neutron
activation
analysis.
The
samples were irradiated during 12 days at a flux of ,_.,1014
neutrons#{149}cm2s1.
Chromium
was selectively
separated
by distillation
after the irradiation.
We obtained the following values (mean ± standard deviation): 0.160 ± 0.083
pig/liter
for chromium,
and 0.108 ± 0.060 ag/liter
for
cobalt.
Addftlonal Keyphrases:
normal values
trace elements
contamination
In view of the
of trace elements,
analyses
for serum
and biological
analytical
.
increasing
reliable
are needed.
chromium
Previously
mean
this
study
systematic
described
previously
the
errors
due
to contaminations
same
in this
journal
precautions
as
(5, 6).
Materials and Methods
Materials
and Preliminary
Procedures
Subjects.
We examined
20 apparently
healthy
individuals.
Serum
from
17 could
be analyzed
in duplicate.
However,
three chromium
determinations
were rejected
as being outlying
(vide infra: statistical
methods);
thus
only
14 duplicate
metric
mean
value
for
the
Samples
taken,
trocar
purity
determinations
of the
two
was
The
considered
geoas the
individual.
and standards.
after
an
(Intranule
quartz
‘Department
remained.
results
Venous
overnight
fast,
110 16; Vygon)
tubes
of Internal
(Spectrosil;
Medicine,
Rijksuniversiteit
with
and
blood
samples
were
a plastic
collected
cannula
in high-
Thermal
Quarz-
Division
of Gastroenterology,
Gent,
De Pintelaan
the
serum
cannot
samples
irradiation
room.
the
be irradiated
of ,,4014 neutrons.cm2.s.
Indeed,
radiation
develops
so large a pressure
in the sealed quartz
ampoules
that
the risk of explosion
is too great.
Therefore,
we decided
to ash the serum
before
the ir-
values
we applied
lyophilized
water.
Before
in a dust-free
damage
justified.
During
handled
at a flux
and cobalt in normal
subjects
vary
over extremely
wide ranges,
namely
from 0.73 sg/liter
(1) up to 150 tg/liter
(2) for chromium
and from 0.03
zg/liter
(3) up to 72 pg/liter
(4) for cobalt.
Because
of
the prevailing
confusion,
further
investigations
seemed
to avoid
distilled-in-quartz
samples
were
Large
in the metabolism
for quantitative
reported
Steyaert,3 Julien De Rudder,2 and Hilde
Schmelze;
outside
diameter
16 mm, wall thickness
1.1-1.5 mm, length
approximately
120 mm),
previously
cleaned
with twice-distilled
water
(Bi-Destillier-Apparat.
Vitreosil.
Thermal
Quarz-Schmelze),
boiled for
two successive
periods
of 2 h in a mixture
of equal volumes
of nitric
and sulfuric
acid (Suprapur;
Merck),
rinsed
again,
and finally
steam-cleaned
for 3 h with
variation
interest
techniques
by Neutron
Academisch
Ziekenhuis,
135,
B-9000
Gent,
Belgium.
2 Institute
for Nuclear
Sciences,
Laboratory
for Analytical
Chemistry,
Rijksuniversiteit
Gent,
Proeftuinstraat
86, B-9000
Gent, Belgium.
‘ Division
of Mathematical
Statistics,
Rijksuniversiteit
Gent,
Krijgslaan
273, B-9000
Gent,
Belgium.
Received
Aug. 9, 1977; accepted
Dec. 1, 1977.
radiation.
Furthermore,
this
procedure
allows
to fivefold
increase
in sample
size. Thus,
approximately,
100 mg of lyophilized
weighed
into high-purity
quartz
ampoules
a four-
samples
of
serum
were
(Spectrosil;
diameter
8-9 mm, wall thickness
1.0-1.2
mm,
length approximately
45 mm). The serum was dried and
ashed in a Simon-Muller
oven during
24 h at 100, 200,
350, and 450 #{176}C
successively.
The ampoules
were then
sealed by fusion. Tracer experiments
with 51Cr and 60Co
showed there were no losses during this process.
The
whole procedure
was particularly
tested
with human
serum
albumin-51Cr
(-.0.03
Ci per gram of albumin,
>97% organic
chromium)
as well as with serum
from two
patients
who received
intravenously
20 tCi of 51CrCl3
(specific activity
>50 Ci/g of chromium),
and from rats
which were fed 60 JLCi of 51CJ13 (same specific activity)
or 100 1zCi of Na251CrO4
(specific
activity
50-200
Ci/g
of chromium).
We obtained
the following
analytical
recoveries:
97.5-99.8%
for human
serum albumin-51Cr,
98.3-99.7%
for the serum from patients,
98.4-101.9%
for
outside
the
serum
from
5tCrCl3-treated
rats,
and
94.8-110.3%
for the serum from Na251CrO4-treated
rats.
Chromium
metal (2.0-4.0
mg) and cobalt/aluminum
wire (containing
2% cobalt)
(3.0-4.0
mg) were used
as
standards.
Other
Methods
Irradiation.
to long-lived
can see from
Reactor
chromium
the nuclear
irradiation
of serum
gives rise
and cobalt
radioisotopes,
as one
data summarized
in Table 1 (7,
CLINICAL CHEMISTRY,
Vol. 24, No. 2, 1978
303
Table 1. Nuclear Data
IsotopIc
abundance
Nuclear
reactIons
50Cr(n,y)
Cross
sections.
th.rrnaI
or fast
(barns)
of th
target
nudild.
(%)
4.31
51Cr
S&ected
photopeaks,
key
(IntensIty,
Half-lIfe
(T1,2)
16
%)
27.8 days
320.0
(9.0)
59Co(n,y) 60Co
54Fe(n,a)
8). The
samples
were
_.,1014 neutrons#{149}cm2s1
Belgium).
Two samples
irradiated
in
could
for 12 days
the BR
be fitted
fast
at a flux of
container.
neutron
cross
(0.00074
barns)
(Table
when
serum
samples
mg/liter
0.00074
2 reactor
(Mol,
into a graphite
The fast neutron
flux of the nuclear
reactor
gives
to the threshold
reaction
MFe (n,a)51Cr.
However,
are
irradiated
section
being
only
74.10
rise
the
m2
1), the interference
is negligible
with an iron content
of -.1.5
in an
adequately
thermalized
reactor
neutron
flux.
Post-irradiation
procedure.
After a decay of about
30 days, the quartz
ampoules
were immersed
for 10 mm
in a 2/1 (by vol) mixture
of HF (50%) and HNO3
(14
mol/liter)
at room
temperature,
to remove
outside
contaminations.
After
scouring
with silicon
carbide
paper
and rinsing
with tap water,
the ampoules
were
immersed
in liquid nitrogen
and the top was removed
with a diamond
saw. As it is impossible
to remove
the
samples
quantitatively
from the ampoules
as a consequence
of radiation
damage,
each was placed in a 25-ml
spherical
flask equipped
with an efficient
reflux
condenser,
3 ml of an equivolume
mixture
of HC1O4 (70%)
and HNO3 (14 mol/liter)
was added,
and the flask was
heated
on a hot plate with magnetic
stirring
until the
sample
had completely
dissolved.
After
the irradiation,
direct
measurement
of the
1173.1 or 1332.4 keV gamma
peak of 60Co is possible.
However,
the measurement
of the 320.0 keV gamma
peak
of 51Cr
is strongly
hindered
by the
32
Bremsstrahlung.
Therefore,
a selective
separation
of the
element
after the irradiation
was performed
according
to a modification
of an earlier-described
technique
(9).
After
dissolution
the content
is transferred
into the
distillation
apparatus
shown
in Figure
1, and 5 mg of
chromium
carrier
(as Cr03) and 10 ml of perchloric
acid
(70%) are added.
The HNO3 is removed
by distillation
until the temperature
reaches
135 #{176}C.
Chromium
is
distilled
as CrO2Cl2
by increasing
the temperature
to
210 #{176}C
and slowly introducing
dry HC1 gas. The distillation is repeated
twice, each time after addition of 5 mg
of chromium
carrier.
The procedure
ensures
a quantitative
recovery
of chromium,
whereas
cobalt
remains
in the residue,
as was ascertained
from tracer
experiments with 51Cr and 60Co. The distillate
and the residue
were transferred
into their respective
vials and the radioactivity
was counted.
304
CLINICAL
CHEMISTRY,
Vol.
24, No. 2, 1978
1173.1
5.25 years
(99.9)
5.84
51Cr
irradiation
37
100
27.8 days
320.0
Instrumentation.
The gamma
spectra
were measured
with a Ge(Li)
detector
and associated
equipment:
#{149}
coaxial
Ge(Li)
detector
(Philips)
and preamplifier:
size 70 cm3, resolution
1.9 keV, relative
detection
efficiency
15.6%.
#{149}
amplifier
(Canberra)
with integration
and differentiation
constant
at 4 his.
#{149}
4000-channel
analyzer
(Didac,
Intertechnique).
The data were handled
by a PDP-9
computer
(Digital
Equipment
Corp.,
Marlboro,
Mass. 01752).
The analytical
results
were obtained
by comparing
the photopeak areas of the sample
and standard
by means
of a
program
developed
by Op de Beeck and Hoste
(10). It
makes
use of the square-wave
convoluted
spectrum
obtained
by convoluting
the original
data with a symmetric
one-period
square
wave
of 8 channels
width,
and
varying
between
the values
-1 and + 1.
The Ge(Li)
detector
is placed
in a lead castle with
10 cm 6#{176}Co-freeold steel plate inside.
In an ordinary
lead castle placed on a steel frame, the 60Co background
DRY
HCI
200
C
GAS
THERMOMETER
C,
CARRIER
AIR
lee
00
THERMOMETER
DISTIL
LATE
COLLECTION
HEATING
MANTLE
COOL INC
(ICE)
Fig. 1. Apparatus
for CrO2CI2
distillation
a
ENERGY
Fig.
2. Gamma
Irradiation
time
(t1)
spectrum
=
12 dayS.
of the distillate
time
Decay
(td)
of a serum
= 31 days.
Counting
sample
time
irradiated
is relatively
high because
of contamination
of modern
steel with that isotope.
Blanks.
Four
blank determinations
were done in the
absence
of serum.
A mean
value
of 47.8 pg (range
26.2-70.4
pg) was obtained
for chromium
and of 26.7 pg
(range, 16.7-33.8
pg) for cobalt. These blank means were
subtracted
from each sample
value.
Statistical
methods.
Important
contaminations
cause
outlying
values
and discrepancies
in duplicate
determinations.
The usual methods
for detecting
the former
could not be used because
the total number
of the results is small relative
to the number
of possible
outlying
observations.
We used the likelihood-ratio
test (11) to answer
the
following
questions:
are the observations
samples
from
two populations
(one non-contaminated,
another
contaminated),
and are the populations
normally
or lognormally
distributed?
Applied
to the total number
of
observations
by this
(n
=
37) we obtained
the
following
results
test:
#{149}
For chromium
we got three outlying
values,
whereas
for cobalt
we found none.
#{149}
In both cases a log-normal
distribution
was seen. This
is probably
due to experimental
errors, namely
minor
contaminations.
Therefore,
in the case of duplicate
determinations,
the value of the subject
should
be
given by the geometric
mean:
C
=
These
geometric
means
could then be considered
as
samples
from a normal
distribution.
Having
14 duplicate
chromium
and 17 duplicate
cobalt determinations,
it was possible
to estimate
the reproducibility(s)
of the analytical
technique
by the usual
formula
(12):
--
/ d2
5 =
V
at 1014 neutrons#{149}cm2#{149}s
(4,) = 48 h
where
d is the
duplicate
determinations
difference
between
the two results
in a
and N the number
of duplicate
determination
performed.
The mean, standard
deviation
(SD), and coefficient
of variation
(CV) of the concentrations
in the sera of the
20 subjects
were calculated
from the results of the single
determinations
(six for chromium
and three for cobalt)
and the geometric
means
of the duplicate
determinations (14 for chromium
and 17 for cobalt)
by standard
statistical
techniques
(13,
14).
Recalculations.
For comparison
of our values
those of other investigators
we had to recalculate
of their values
so as to express
them in SI units
liter).
Concentrations
in ppb (parts
per billion)
multiplied
by 1.026 (serum
specific
gravity).
with
some
(zg/
were
Results
Accuracy
and precision
of the assays.
We analyzed
six times the multi-element
serum standard
of Cornelis
et al. (15) (concentration
of chromium:
19.32 mg/kg;
concentration
of cobalt:
6.46 mg/kg).
The following
means
± SD were obtained:
19.51 ± 1.38 mg/kg
for
chromium
and 6.17 ± 0.23 mg/kg for cobalt.
Furthermore, we compared
the results
of two chromium
determinations
in human
liver by our technique
with the
results
obtained
by the separation
scheme
of Lievens
et al. (16). The following
values were obtained:
5.66 and
6.84 pg/kg wet weight vs. 5.72 and 7.00 zg/kg wet weight.
Finally,
the estimate
of the reproducibility
calculated
from the results
of the duplicate
determinations
as
outlined
above
gave the following
s = 0.084 gig/liter,
and for cobalt,
values:
for chromium,
s = 0.051 tg/liter.
Figure
2 shows the gamma
spectrum
of a 48-h measurement
of the distillate
of a sample.
It appears
that
the chromium
320.0 keV photopeak
is large enough
to
ensure
good counting
statistics.
We accepted
2.5% as
upper limit for the standard
deviation
of the total peak
CLINICAL
CHEMISTRY,
Vol. 24, No. 2, 1978
305
ENERGY
Fig. 3. Gamma
t,
spectrum
of the residue
of a serum
sample
irradiated
area. The spectrum
also shows the 391.4 keV photopeak
of 3mIn,
daughter
isotope of ‘13Sn (which probably
will
permit
the determination
of serum
tin, work in progress), as well as traces of 75Se activity
and a number
of
background
peaks. The mCo peaks are also mainly
due
to background
activity.
Figure
3 shows
the gamma
spectrum
of a 2-h measurement
of the residue.
Besides
the cobalt
1173.1
and 1332.4
keV photopeaks,
again a
number
of other ‘y energies
are seen, including
those of
ilOmAg.
Three chromium
values were outlying-621,
642, and
665 ng/liter-and
were excluded
from further
calculations. The paired
values are, respectively,
252,91.8,
and
259 ng/liter.
Chromium.
The mean serum
chromium
concentration ± 1 SD is 0.160 ± 0.083 pg/liter
(CV = 5 1.9%). The
median
of the frequency
distribution
is 0.158 gig/liter.
Cobalt.
The mean serum cobalt concentration
± 1 SD
is 0.108 ± 0.060 gig/liter
(CV = 55.6%).
The median
of
the frequency
distribution
is 0.093 fig/liter.
Discussion
It appears
from the data of Table
2 that previously
reported
mean plasma
or serum chromium
concentrations in normal
subjects
vary widely,
namely
from 0.73
zg/liter
(1) to 150 fig/liter
(2, 17-25).
A similar situation
was found
for manganese
some years
ago (26). The
values
found
fig/liter.
Whether
during
our
study
are
considerably
lower
than reported
by other
investigators.
However,
they
approximate
the lower limit
recently
mentioned
by
Grafflage
et al. (1) and Pekarek
et al. (18). Furthermore,
our findings
agree perfectly
with
the observations
of
Seeling et al. (17), who used flameless
atomic
absorption
spectroscopy
and concluded
that the serum
chromium
concentration
in normal subjects
must be lower than 0.5
CLINICAL CHEMISTRY,
Vol. 24, No. 2, 1978
any
chromium
is lost
during
dry
ashing
of biological
material
is a matter
of debate.
A
group of investigators
has suggested
that naturally
occurring
chromium
compounds
may volatilize
(27-29),
but in a later publication
some of them attribute
the
variations
in analytical
results
to a number
of processes
(30).
Indeed,
brewer’s
yeast and bovine
liver samples
showed
even higher values after muffle-furnace
ashing
at 450-600
#{176}C
than by low-temperature
ashing
and direct analysis.
Moreover,
Jones
et al. (31)
did not find
significant
losses from brewer’s
yeast grown in a medium containing
SlCr3+ with oven drying at temperatures
up to 800 #{176}C.
These observations
agree with the results
of Gorsuch
(32),
Koirtyohann
and Hopkins
(33), and
Kumpulainen
(34). The first found no important
losses
of radio-chromium
(as chromate)
heated
at 600 #{176}C
for
16 h in presence
of inorganic
chlorides.
The second
saw
Table 2. Summary of Previously Reported Plasma
or Serum Chromium
Concentrations in Normal
Subjects
Reference
no.
Chromium
306
at 1014 neutrons#{149}cm2s
l2days,4,=6months.t02h
This study
SD
g/IIter
Mean
0.160
0.73
0.083
(18)
1.62
0.31
(19)
5.1
(1)
(17)
(20)
9.3
(21)
10.3
(22)
28
28
28
(23)
(24)
(25)
(2)
45
150
Range
0.0382-0.351
0.23-1.90
<0.50
0.2O
3.1-7.2
5.6
6.2
23-34
9-56
48
14-77
41-251
no volatility
porated
losses
from
blood
radiochromium
The
last
51Cr
from
samples
recovered
at 550
from
brewer’s
over
vation
offers
errors
our
As we also
is much
techniques.
irradiation
checked
the
are
great
ashing
found
the
we
Indeed,
contaminations
This study
(3)
2 are
actithe
with
other
the
(37)
of consequence.
(38)
(39)
for
cobalt
3, previously
concentrations
subjects
also vary
to 72 fig/liter
reported
in plasma
widely,
mean
or serum
namely
from
(4, 20, 21, 35-41).
Our
values
(40)
of normal
0.03 fig/liter
results
agree
with
the
values
of Lins and Pehrsson
(3) and Thiers
et al.
(35).
However,
both reported
only a very small number
of determinations,
three and two, respectively.
We also
checked
our technique
for possible
cobalt
losses. Our
experiments
confirm
the results
of van Raaphorst
et al.
(42),
who
did
not
find
significant
volatilization
of cobalt
the fact
agreement
exists
between
results
of the Pratt
Trace
lottesville,
Va.) published
fig/liter)
pears
(35)
between
(see
our
whereas
an excellent
our cobalt
values
and the
Analysis
Laboratory
(Charin 1955 (range,
0.064-0.085
Table
3), a striking
chromium
values
discrepancy
and
of contaminated
the
ap-
results
of
samples.
It must be noticed
that the dispersion
of the serum
chromium
and cobalt
values
in our subjects
is greater
than those
for several
other
trace elements.
The CV
amounts
to 51.9%
for chromium
and 55.6% for cobalt,
but to only 22.8% for manganese,
22.4% for copper,
13.8% for zinc (5, 6, 45), 15.4% for selenium,
23.5% for
rubidium,
and 27.0% for cesium,
as published
recently
in this journal
(46). Apart
from the risk of contamination, two other
major
difficulties
are encountered
in
determining
serum
chromium
or cobalt:
the relatively
low sensitivity
of the method
(mainly
for chromium)
and the important
blank values. In order to obtain more
precise
chromium
and cobalt values it will be of utmost
importance
to have
rials available
the samples.
the analytical
The
existing
for
Only
from
still
much
collecting,
then will
more
handling,
it be possible
the biological
discrepancies
nearly
shown
0.11
0.29
0.46
0.52
0.16
0.08-0.60
0.2-1.3
0.43
1.32
1.2-36
1.9
5.6-9.8
72
(4)
create doubt concerning
previous
statements
about the
plasma
or serum
chromium
and cobalt
response
after
a glucose load (21,22,47,48,49),
or about the variations
in health
and disease
(49, 50).
References
that laboratory
published
in 1956 (mean,
185 fig/liter;
range, 82-308
fig/liter)
(44) or in 1960 (mean,
28 fig/liter;
range,
9-56 fig/liter)
(23) (see Table
2). Based
on our
experience
concerning
contaminations
during collection
and handling
of biological
material,
we hypothesize
that
the questioned
chromium
values
are the result
of
analyses
0.23
We thank
Prof. Dr. Marc
Bogaert
for advice
on the animal
experiments,
Dr. Albert
Speecke
for stimulating
discussions,
and Dr. Patrick
Lievens
for valuable
suggestions.
We appreciate
the excellent
technical
assistance
and secretarial
aid of Miss
Lidia
Vanballenberghe,
Mrs.
Yvette
Odent,
and
Miss
Marie.Rose
Luyckx.
This
work
was
supported
by the Nationaal
Fonds
voor Wetenschappelijk
Onderzoek
and the Interuniversitair
Instituut
voor Kernwetenschappen.
(43).
that,
0.0394-0.271
during
ashing at temperatures
up to 1000 #{176}C.
Previous
studies
in our laboratory
proved
that the risk of obtaining
falsely
high values
due to contaminations
is also important
in the case
We wish to stress
Range
62
(41)
(3)
0.060
1.85
6.6
7.7
(20)
in Table
--
0.02-0.06
0.064-0.085
(36)
Cobalt
As shown
SD
0.108
(35)
(21)
risk
after
Mean
Mg/liter
no losses,
that
in comparison
Reference
no.
exhaus-
in Table
Neutron
advantage
Table 3. Summary
of Previously
Reported
Plasma
or Serum Cobalt Concentrations
in Normal
Subjects
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incor-
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