Clinical Chemistry 49:12
2050 –2055 (2003)
Endocrinology and
Metabolism
Inductively Coupled Plasma Mass Spectrometric
Analysis of Calcium Isotopes in Human Serum:
A Low-Sample-Volume Acid-Equilibration Method
Zhensheng Chen,1* Ian J. Griffin,1,2 Yana L. Kriseman,1 Lily K. Liang,1 and
Steven A. Abrams1,2
Background: Analytical methods for measuring the
calcium isotope distribution in enriched human serum
samples that use low blood volumes, simple preparation
methods, and rapid analysis are important in clinical
studies of calcium kinetics. Previously, sample preparation by oxalate precipitation typically required 500 ␮L
of serum. This method was time-consuming, and the
blood volume required was limiting in circumstances
when only a small amount of serum could be obtained.
Methods: Serum was collected from humans who were
administered 42Ca, and 20 ␮L of serum was mixed with
2 mL of 0.22– 0.67 mol/L HNO3 at room temperature for
between 1 min and 16 h. The 42Ca/43Ca ratio in the
supernatant was measured by a magnetic sector inductively coupled plasma mass spectrometer (ICP-MS).
Calcium isotope ratios from these equilibration solutions were compared with data from oxalate-precipitated serum samples to determine the optimum equilibrium time and the effect of acid concentration on
equilibrium.
Results: Various amounts of aggregated particles developed in different acid–serum mixtures. These affected
the time required for isotope equilibration in the mixture. The shortest equilibrium time needed for the
calcium isotopes varied from 1 to 6 h for samples
acidified with 0.22– 0.45 mol/L HNO3. Data obtained
from these solutions were consistent with data from
oxalate-precipitated calcium. The precision of 42Ca/43Ca
ratio measurements was better than 0.5%.
1
US Department of Agriculture/Agricultural Research Service Children’s
Nutrition Research Center, and 2 Section of Neonatology, Department of
Pediatrics, Baylor College of Medicine, Houston, TX 77030.
*Address correspondence to this author at: USDA/ARS Children’s Nutrition Research Center, 1100 Bates St., Houston TX 77030. Fax 713-798-7119;
e-mail [email protected].
Received August 7, 2003; accepted September 19, 2003.
DOI: 10.1373/clinchem.2003.025692
Conclusions: We have developed a simple, rapid sample preparation technique for ICP-MS analysis in which
20 ␮L of serum can be used for accurate measurement of
the calcium isotope distribution in a sample with good
precision and a rapid analysis time.
© 2003 American Association for Clinical Chemistry
Analysis of human serum samples for total calcium
concentration and isotope ratios has widespread application in biological sciences. This method has been used to
determine the rate of bone calcium deposition and resorption from diverse populations, including healthy premature infants, older adults, and even astronauts in space.
For example, a study of calcium kinetics was conducted
during the recent STS-107 space shuttle mission in which
all samples were lost during the shuttle break-up on
reentry. In planning those and other studies, a major
limitation has been the need for frequent venipuncture
and relatively large sample volumes. This is because
virtually all of these studies have used analytical approaches in which 500 ␮L or more of serum was required
for the oxalate precipitation or ion-exchange methods
used to prepare the samples for analysis. Most analyses
have involved thermal ionization mass spectrometry
(TIMS),3 using either quadrupole or magnetic sectorbased spectrometers (1, 2 ).
Application of this type of study to situations where
sample volume is limited would be very beneficial in
increasing the ability to perform calcium kinetic studies.
Additionally, the relatively large number of samples
needed for determination of kinetic parameters (typically
10 –15 blood samples/patient) requires improvement in
3
Nonstandard abbreviations: TIMS, thermal ionization mass spectrometry; ICP-MS, inductively coupled plasma mass spectrometry; and RSD, relative
SD.
2050
Clinical Chemistry 49, No. 12, 2003
the speed and simplicity of sample preparation and
analysis.
Stable calcium isotope tracers have been used extensively for ⬃30 years for metabolic studies of calcium
homeostasis in humans in a variety of clinical areas (1–7 ).
Stable isotopes are safe for use in humans of all ages and
do not have problems related to the disposal of contaminated waste, a major advantage over radioisotopes. Stable
isotope tracers, highly enriched in naturally low-abundance isotopes, are administered intravenously or orally.
Isotope ratio data are collected from blood, urine, or fecal
samples to identify time-dependent tracer variation patterns. Such studies have a variety of applications ranging
from simple measurement of mineral absorption to measurement of mineral excretion into the gut and stableisotope-based multicompartmental modeling (8 ).
This report focuses on the development of a novel
serum sample preparation methodology for calcium isotope analysis that would allow the use of a smaller sample
size. It also describes a simple method of preparing
samples using low volumes and analysis by inductively
coupled plasma mass spectrometry (ICP-MS; ThermoFinnigan Element 2). We hypothesized that this approach would give results similar to those obtained with
conventional sample preparation methods and subsequent TIMS analysis. The accuracy of the calcium isotope
data that we obtain with TIMS and ICP-MS has been
documented previously (5, 9 ).
Materials and Methods
materials
Calcium salt enriched in 42Ca was purchased from Trace
Sciences Inc. and prepared for intravenous infusion as a
sterile, nonpyrogenic aqueous solution in saline. Certified
ACS Plus concentrated HNO3 (Fisher Scientific) and
deionized water (MilliQ Water System) were used to
prepare dilutions of the acid (0.22– 0.67 mol/L; pH 0.9 –
1.3) for human serum sample–acid equilibration experiments. Glassware was soaked overnight in a bath containing 2.25 mol/L HNO3, rinsed with deionized water, and
air-dried under cover before use.
methods
Study design. Samples were obtained from clinical studies
of calcium metabolism in children. Informed written
consent was obtained from the families of all children
who participated in these studies. These studies were
approved by the Institutional Review Board of Baylor
College of Medicine (Houston, TX).
Patients were admitted to the General Clinical Research Center of Texas Children’s Hospital at 0730 in the
morning and had a topical anaesthetic cream applied over
the vein of interest. One hour later, a heparin lock catheter
was inserted. Each child was subsequently infused with 5
mg of 42Ca over 1–2 min. Samples of whole blood (1 mL)
were obtained for calcium isotope ratio measurement at 6,
12, 20, 40, 120, 180, 240, and 480 min after the infusion in
2051
nonanticoagulant red-top tubes. The samples of whole
blood were then centrifuged, and the serum was separated from other blood components.
Sample preparation by acid–serum equilibration. Various concentrations of HNO3 were selected for the analytical
experiments to assess the effect of acid concentration on
calcium isotope ratios in sample solutions. We varied the
exchange time to determine the minimum time for reaching equilibrium and to ascertain the existence of any
time-dependent isotope fractionation during the sample
preparation process.
We mixed 20 ␮L of serum sample with 2 mL of HNO3
of a selected concentration at room temperature in a
conical tube. The sample was vigorously mixed three
times (15 s each time) in a Vortex-mix Genie (Fisher
Scientific), once at the initial mixing of the serum and acid,
once at the middle of the interaction period, and once
close to the end of the interaction experiment. The mixture
was then centrifuged at 530g for 12 min at room temperature in a Fisher Marathon 8K tabletop centrifuge. The
supernatant was extracted and transferred to an acidwashed glass tube for ICP-MS stable calcium isotope
analysis. If there were visible particulates present in the
solution, the supernatant was centrifuged a second time
to avoid blocking the uptake capillary of the ICP-MS inlet
system.
Sample preparation by calcium precipitation. Approximately
2 mL of saturated ammonium oxalate solution was added
to a 500-␮L serum sample, and the pH of the solution was
adjusted to ⬃10 for a 12-h period of calcium precipitation
(4, 10 ). A white calcium oxalate pellet was obtained after
centrifugation. The supernatant was decanted, and the
pellet was washed with an ammonium oxalate solution
(⬃10 g/L) and centrifuged again before being heated at
500 °C for 1 h. The ashed product was resuspended in
0.45– 0.67 mol/L highly purified HNO3 and diluted for
ICP-MS isotope ratio analysis.
Results
preliminary signal test
Preliminary tests focused on the minimum amount of
serum required for preparation of a sample solution with
a calcium concentration and solution volume adequate for
our ICP-MS analysis. The minimum calcium concentration and solution volume can vary depending on the
particular ICP-MS sample-introducing setup and operating conditions. For our ICP-MS analysis, a calcium concentration of ⬃ 1 mg/L was sufficient to generate isotope
ratio data with a precision better than 0.5% relative SD
(RSD) (9 ), and a 2 mL solution was adequate for ⬃14 min
of analysis. Initially, 50 ␮L of 42Ca-enriched serum was
mixed with 100 ␮L of concentrated HNO3 in a clean
12-mL disposable glass test tube, and a clear solution was
obtained. Deionized water was then added to the mixture
to dilute the acid to ⬃0.90 mol/L (⬃ 1.8 mL total volume)
2052
Chen et al.: ICP-MS Analysis of Calcium Isotopes
to make the solution suitable for ICP-MS analysis. Lightyellow particulates developed in the solution after the
addition of deionized water. Heating the mixture slightly
to ⬃70 °C for 10 min did not help to redissolve the
particulates. The solution was therefore centrifuged for 10
min at 420g, and the supernatant was extracted. Test
results showed that this solution had a calcium concentration of ⬃2.5 mg/L.
determination of equilibrium time
A 42Ca-enriched serum sample collected 20 min after
dosing was selected for a series of 10 acid–serum equilibrium tests with different mixing times: 1, 15, 30, 45, 60, 75,
90, 120, 240, and 960 min. We mixed 2 mL of 0.67 mol/L
HNO3 with 25 ␮L of serum sample in each of the 10 test
tubes. The mixtures were vortex-mixed several times
during the interaction period, except for the sample with
a 1-min interaction time, before being centrifuged to
separate the aggregated materials from the solution. After
centrifugation, the supernatant from the equilibration
solution was collected and transferred to a clean glass
tube for ICP-MS calcium isotope ratio (42Ca/43Ca) analysis (Table 1). The isotope data collected from the equilibration solutions were similar for samples from the 1- to
16-h interaction times (Fig. 1). Apparently the solutions
achieved a maximum ⌬% excess [⌬% Excess ⫽ 100 ⫻
(ratio in sample ⫺ ratio in natural abundance)/(ratio in
natural abundance); 37% excess for this sample] when the
interaction time was ⱖ1 h. If the maximum ⌬% excess
from the equilibration solution at a 240-min (6-h) interaction time was assumed to be in calcium isotope equilibrium and its degree of equilibration was 100%, the degrees of equilibration for other solutions at different
interaction times could be calculated as shown in Table 1.
The degree of equilibration reached 90% at an interaction
time of 1 min and increased to 95% at 30 min and 99.5%
at 1h. This showed that interaction time played an important role in the acid–serum calcium isotope equilibration
process and suggested that calcium isotope equilibrium
might be established in a serum-diluted HNO3 mixture in
a period of as short as 1 h.
Table 1. Results of equilibration tests using 0.67 mol/L
nitric acid–serum interaction solution.
Reaction
time, min
1
15
30
45
60
75
90
120
240
960
42
Ca/43Ca
6.417
6.428
6.511
6.515
6.577
6.581
6.564
6.553
6.584
6.583
⌬% excess
Degree of
equilibration, %
33.88
34.12
35.84
35.92
37.21
37.30
36.96
36.71
37.37
37.35
90.66
91.29
95.90
96.11
99.56
99.82
98.88
98.24
100.00
99.94
Fig. 1. Results of time-dependent equilibration tests for calcium
isotopes in a mixture of HNO3 and a 42Ca-enriched serum sample.
determination of data accuracy
A complete series of eight serum samples from a study
participant (6, 12, 20, 40, 120, 180, 240, and 480 min after
intravenous infusion of the 42Ca isotope) was selected to
prepare calcium isotope ICP-MS analytical samples by
both the conventional precipitation methodology and the
new acid–serum equilibration methodology presented
here. Because 0.22– 0.67 mol/L HNO3 is commonly used
as sample solvent for ICP-MS analysis, 0.45 mol/L HNO3
was selected for this acid–serum interaction experiment.
We added 2 mL of 0.45 mol/L HNO3 to 20 ␮L of each
serum sample in a test tube and vortex-mixed the mixture
for 15 s. The mixture was centrifuged immediately after
vortex-mixing, and the supernatant was extracted for
ICP-MS calcium isotope analysis.
A resuspended solution was also prepared from the
product of the precipitation method. The solution was
diluted with 0.45 mol/L HNO3 to make its calcium
concentration nearly identical to the counterpart solution
from the acid–serum equilibration. Both sets of sample
solutions were then analyzed side by side by ICP-MS to
determine the 42Ca/43Ca ratios.
Measured calcium isotope ratios from the two sets of
solutions were similar (Table 2). This was surprising
because the interaction time for the equilibration solution
was only a matter of ⬃1 min before centrifugation. Both
Table 2. Comparison of calcium isotope ratios from a
complete set of serum samples prepared with two different
sample preparation techniques.
Equilibration
Sample
s1
s2
s3
s4
s5
s6
s7
s8
42
43
Ca/ Ca
15.658
7.949
7.885
7.269
6.045
5.669
5.466
5.264
⌬%
Excess
226.68
65.84
64.50
51.65
26.11
18.27
14.03
9.83
Precipitation
42
43
Ca/ Ca
16.129
8.053
7.914
7.336
6.113
5.714
5.505
5.278
⌬%
Excess
236.51
68.01
65.11
53.05
27.53
19.20
14.85
10.12
Difference Degree of
in ⌬%
equilibration,
excess
%
9.83
2.17
0.61
1.41
1.42
0.93
0.82
0.29
95.84
96.81
99.07
97.35
94.84
95.13
94.49
97.15
2053
Clinical Chemistry 49, No. 12, 2003
Fig. 2. Comparison of calcium isotope data obtained with two different
sample preparation techniques.
sets of data had the same trend in variability (Fig. 2). The
difference between the two sets of analytical data, however, was consistent. Calcium isotope ratios in the equilibration solutions were always slightly lower than the
ratios in the precipitated calcium. The difference between
the two sets of data apparently increased with increasing
isotope enrichment in the sample. The difference could be
up to 10% for a serum sample (Table 2, sample s1) with
⬎200% excess (not shown in Fig. 2), the very first sample
collected from the studied individual after isotope infusion. This indicated that data from the equilibration
solutions were not accurate enough to be considered
representative in this case. If the isotope excess of the
precipitation calcium was used as the representative data,
a degree of equilibration (as a percentage) for each equilibration solution could be calculated on the basis of the
difference in isotope excess between the two sets of
sample solutions (Table 2). Our experiments suggested
that these acid–serum interaction solutions could achieve
95–99% equilibration after a very short period of interaction.
Tests were further conducted on the serum sample (s1)
that had the largest (⬃ 10%) difference between the two
sets of 42Ca/43Ca ratios (Table 2) to better define the
equilibrium time required for samples with extraordinarily enriched 42Ca. Equilibration solutions were prepared
using 0.45 and 0.67 mol/L HNO3, with interaction times
varying from 1 to 6 h. The supernatants were collected
and analyzed for 42Ca/43Ca ratios, and the results are
shown in Table 3. We observed that with increasing
interaction time, the degree of equilibration increased.
The differences in isotope data obtained from the 0.45 and
0.67 mol/L HNO3 equilibration solutions were consistent
(Table 3, sample s1). The degrees of equilibration for the
0.67 and 0.45 mol/L HNO3 solutions at an interaction
time of 1 h were 96% and 99%, respectively. At an
interaction time of 6 h, they were 98% and 99.5%, respectively. These results suggest that 0.45 mol/L HNO3 was
more effective than 0.67 mol/L HNO3 in the equilibration
tests. The equilibration solution from the 6-h, 0.45 mol/L
acid–serum mixture gave an isotope ratio virtually identical to the ratio from the precipitation solution (within
the instrumental error of 0.5% RSD), with a degree of
equilibration of 99.5%. It therefore appears that when 0.45
mol/L acid is used for acid–serum equilibration, the
solution will achieve equilibrium in 6 h for samples with
highly enriched calcium isotopes.
Further tests were conducted using both 0.22 mol/L
HNO3 and deionized water solutions. Either 0.22 mol/L
HNO3 or deionized water was mixed with a serum
sample and allowed to equilibrate for 1 h. The results
were compared with the results obtained with 0.67 mol/L
acid for the same sample (Table 3, samples f1 and f2).
Table 3. Effects of interaction time and acid concentration on human serum–acid calcium isotope equilibrium.
Sample
Method
s1
Precipitation
Equilibration
Reagent used
0.45 mol/L HNO3
0.67 mol/L HNO3
f1
f2
Precipitation
Equilibration
Precipitation
Equilibration
Time, h
6
1
⬍0.1
6
1
Deionized water
0.22 mol/L HNO3
0.67 mol/L HNO3
1
1
1
Deionized water
0.22 mol/L HNO3
0.67 mol/L HNO3
1
1
1
42
Ca/43Ca
16.129
16.073
16.002
15.658
15.852
15.654
6.900
6.786
6.887
6.791
6.910
6.946
6.903
6.733
⌬% excess
236.51
235.34
233.86
226.68
230.73
226.60
44.00
41.57
43.68
41.68
44.17
44.92
44.03
40.46
Degree of equilibration, %
99.50
98.88
95.84
97.56
95.81
94.48
99.28
94.72
101.69
99.68
91.61
2054
Chen et al.: ICP-MS Analysis of Calcium Isotopes
Deionized water in our laboratory is slightly acidic (pH
5.9 – 6.0), probably because of dissolved CO2 and the
extensive use of acid in this working environment. Analytical results from the deionized water–serum solutions
fluctuated because of unstable signals during ICP-MS
analysis, with a precision of ⬎1.5% RSD for calcium
isotope ratios. As a result, data from the deionized water
solutions were not precise enough to be used for our data
comparison and evaluation purposes. This suggests that
water is not the best sample matrix and that a weak acid
is preferable for introduction of equilibration solutions
into an ICP-MS inlet system.
Similar to the results from the comparison of 0.45 and
0.67 mol/L acid–sample solutions, there were systematic
differences in isotope data from the 0.22 and 0.67 mol/L
acid solutions (Table 3). At an interaction time of 1 h, the
degree of equilibration was ⬍94.7% for the 0.67 mol/L
acid and was ⬎99.3% for the 0.22 mol/L acid. Apparently,
0.22 mol/L HNO3 induced acid–serum calcium isotope
equilibration faster than 0.45 mol/L HNO3, and 0.45
mol/L HNO3 induced equilibration faster than 0.67
mol/L HNO3. This was based on the observation that at
1 h of interaction time, the degree of equilibration for the
0.67 mol/L HNO3 solution was lower than that for the
0.45 mol/L HNO3 solution and the degree of equilibration
for the 0.45 mol/L HNO3 solution was lower than that for
the 0.22 mol/L HNO3 solution. A 6-h interaction solution
using 0.45 mol/L acid might barely reach a degree of
equilibration equivalent to one achieved in 1 h by a 0.22
mol/L acid solution. This result confirmed our previous
test results that a more diluted acid reagent might act
more efficiently in calcium isotope equilibrium tests.
Discussion
Calcium is particularly well suited for stable-isotope
studies because there are six naturally occurring calcium
isotopes, four of which are of very low abundance (natural abundance ⬍1% by mass). Selection of the isotopes
and dosing concentrations used in pediatric and clinical
studies depends greatly on the study design, the anticipated degree of calcium absorption, and the capability of
analytical instruments (11 ). To study calcium kinetics,
highly enriched calcium tracers are given intravenously
and multiple blood and urine samples are collected.
Isotope ratios are measured in these samples and used to
estimate kinetic values, including calcium absorption,
endogenous fecal calcium excretion, and bone calcium
deposition rate using a compartmental modeling
(5, 6, 12, 13 ). The calcium compartmental model illustrates the distribution and turnover of calcium after
absorption and can provide insight into how these processes vary with growth and hormonal changes. This
information can be used to establish dietary mineral
requirements for populations in various life stages, such
as infants, children, and pregnant or lactating women
(14 ).
Before this study, an oxalate precipitation method was
usually applied to extract calcium from 500 ␮L of a serum
sample for TIMS analysis; total calcium recovery was
usually ⬎93% (4, 15 ). Measured calcium isotope ratios
from the precipitation method agreed well with the ratio
expected from calculations, with a difference of 0.2–3.7%
and a mean difference of 1.3%, which was not significant
(4 ). Isotope ratios from the precipitation calcium are taken
as representative data in this report.
Because of the relatively large number of blood samples collected per individual, a fast, simple method for the
analysis of these samples would be highly advantageous.
In this study, we developed a sample preparation method
that uses 20 ␮L of human serum. Calcium isotope ratios
obtained from the equilibrated solutions are consistent
with those from the precipitation calcium. In fact, 10 ␮L of
serum should be adequate to conduct a similar ICP-MS
calcium isotope analysis if a microflow inlet system is
used. The time required for sample preparation with this
new method is only several hours, which is substantially
shorter than time required for the oxalate precipitation
method.
The various amounts of aggregated materials formed
at different concentrations of acid solutions might be
affected by pH and protein net charges, which are pH
dependent (16 ). Studies have indicated that at pH ⬎5
there is a high percentage of calcium bound to serum
albumin and globulin mainly because of electrostatic
attraction (16, 17 ). Studies have also shown that when
human serum albumin is exposed to an increasing pH,
albumin ionization increases, causing repulsion between
molecules, which leads to decreased formation of aggregates (18 ). This corresponds to our observations that
although aggregates formed in the testing tubes for 0.45
mol/L acid–serum solutions and that even more aggregates formed in the 0.67 mol/L acid-serum equilibration
mixture, very few aggregates formed in the 0.22 mol/L
acid–serum equilibration mixture.
Total calcium loss was expected to be proportional to
the amount of aggregates that formed in the mixing
solution. The loss of calcium in the aggregates, however,
may not have noticeable effects on isotope ratios in the
equilibrated solution. Very limited calcium isotope fractionation was reported in geological and biological systems, typically 0.1– 0.2% in biological samples (19, 20 ). In
our experiments, small 42Ca and 43Ca isotope fractionations were observed only in the short-interaction-time,
nonequilibrated solutions (Tables 1–3). The magnitude of
fractionation was, again, directly related to the amount of
aggregates formed in the solutions and the interaction
time. This small degree of isotope fractionation may be
attributable to the “isotope effect” and the difference in
isotope concentrations in the solution. More 40Ca2⫹ and
42
Ca2⫹ (lighter, more abundant) were expected to incorporate into the aggregates than 43Ca2⫹ (heavier, less
abundant) at the initial stage of mixing. Hence, 42Ca/43Ca
ratios obtained from the early mixing solutions were
lower than 42Ca/43Ca ratios from the equilibrated solu-
Clinical Chemistry 49, No. 12, 2003
tion, and small isotope fractionations observed (mostly
⬍2%, but up to 10% in extreme cases; see Table 2).
The problem of calcium isotope fractionation with this
new sample preparation technique for ICP-MS may be
overcome by giving the mixture enough time to equilibrate, as demonstrated in Tables 2 and 3. The number of
calcium ions bound to aggregates was expected to be
exchangeable. Our experiments suggested that 0.22
mol/L HNO3 might reach calcium isotope equilibrium
with serum in 1 h, faster than either 0.45 or 0.67 mol/L
HNO3. The time length required to achieve acid–serum
calcium isotope equilibrium is probably related to the
amount of aggregate formed in the mixing solutions. A
more reliable 6-h equilibration was recommended for
unknown samples with exceptionally high ⌬% excess of
calcium isotope. Vigorous periodic mixing may promote
the equilibration process. On the other hand, although the
0.22 mol/L acid solutions required less time to reach
isotope equilibrium, supernatants from the 0.45 mol/L
solutions were cleaner than their 0.22 mol/L counterparts
because more aggregated materials were separated from
the testing solutions. Separation of more aggregated materials from the mixing solution might benefit the ICP-MS
sample inlet and ion extraction systems in the long term.
This work is a publication of the US Department of
Agriculture (USDA)/Agricultural Research Service (ARS)
Children’s Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine and Texas Children’s
Hospital, Houston, TX. This project has been financed in
part with federal funds from the USDA/ARS under
Cooperative Agreement 58-6250-6-001. Contents of this
publication do not necessarily reflect the views or policies
of the USDA, nor does mention of trade names, commercial products, or organizations imply endorsement by the
US Government. We thank Keli Hawthorne and Cynthia
Edwards for help in recruiting participants. We also thank
Holly Olvey; Melissa Knox; Michelle Precourt; Courtney
Edwards; Rachel Wolfson; Michelle Lopez; Dalia Galicia,
MD; and Lora Plumlee for whole-blood sample collection
and preparation.
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