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1 Departments of Pediatrics, Biochemistry and Molecular Biology, Medical University of South Carolina, 171 Ashley Ave., Charleston, SC 29425. Fax 843-792-1844; e-mail Hollisb{at}musc.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Methods: We used two commercially available, Food and Drug Administration-approved, radioiodine (125I)-based RIA kits for the detection of 25(OH)D (DiaSorin, Stillwater, MN and IDS Ltd, Tyne and Wear, United Kingdom). These methods were tested for general assay performance, including antibody specificity. Results were compared with those of an HPLC-based direct ultraviolet detection method.
Results: Within- and between-run CVs were 
10%. Both methods
quantitatively recovered 25(OH)D3 added to serum, but only
the DiaSorin kit quantitatively recovered 25(OH)D2. The
primary antibody in the IDS kit had unequal reactivities with pure
25(OH)D2 and 25(OH)D3, whereas the DiaSorin
primary antibody reacted with them equally. In 50 patient samples
assayed by HPLC, the IDS method, but not the DiaSorin method,
underestimated total circulating 25(OH)D when significant circulating
25(OH)D2 was present in patient samples.
Conclusions: Some immunoassays may underestimate total 25(OH)D when 25(OH)D2 constitutes an appreciable part of the total.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Many assays have been developed to assess circulating 25(OH)D. Various competitive protein-binding assays for 25(OH)D dominated the literature until 1978 (5), when the first valid direct ultraviolet (UV) quantitative HPLC assay was introduced (6). HPLC detection provided the advantage of being able to individually quantify 25-hydroxylated ergocalciferol [25(OH)D2] and cholecalciferol [25(OH)D3]. However, the disadvantages of HPLC quantification methods include their requirements for expensive equipment, large sample volumes, and technical expertise to perform this type of analysis.
As the clinical demand for circulating 25(OH)D analysis increased, it was clear that simpler, rapid but valid assay methods would be required. Thus, in 1985, the first valid RIA to assess circulating 25(OH)D was introduced (7). This RIA eliminated the need for sample prepurification before assay and had no requirement for organic solvent evaporation. However, the method was still based on the use of 3H-25(OH)D3 as a tracer. This final shortcoming was solved in 1993 when an 125I-labeled tracer was developed and incorporated into the RIA for 25(OH)D (8). This assay has become the method of choice for assessing 25(OH)D status and was the first test for vitamin D approved for clinical diagnosis by the Food and Drug Administration (FDA) and available through DiaSorin Corporation (Stillwater, MN). Recently, another manufacturer (IDS Ltd, Tyne and Wear, United Kingdom) received FDA approval for a similar device. This report compares the performance of these two methods.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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clinical samples
Serum or plasma samples from 50 apparently healthy subjects from
previous studies in our laboratory were used for this comparative
study. These subjects ranged in age from 4 months to 70 years.
Some subjects received daily supplements of vitamin
D2 or vitamin D3 (400–1200
IU/day) for various periods of time. Other subjects received no known
supplemental vitamin D.
methods
Spectroscopy.
Concentrations of 25(OH)D2
and 25(OH)D3 were determined by UV spectroscopy
with molar absorptivity (
264) values of
19 400 and 18 300 mol-1
cm-1, respectively.
RIA.
25(OH)D was measured as directed by the manufacturers’
product inserts. To perform the reactivity studies, pure
25(OH)D2 and 25(OH)D3 were
dissolved in acetonitrile to concentrations of 0, 2, 4, 10, 20, 40,
100, 200, and 400 pg in 40 µL for the IDS RIA and the same
concentrations in 23 µL for the DiaSorin RIA. The respective RIAs
were performed by placing these volumes of each concentration in
12 x 75 mm borosilicate culture tubes. From this point, the
methods were performed as directed by the manufacturers’ product
inserts. Each concentration was measured in triplicate. Radioactivity
was determined with a gamma well-counting system.
Direct UV detection of 25(OH)D2 and
25(OH)D3 in plasma or serum after HPLC.
25(OH)D2 and 25(OH)D3 were
determined in samples by direct quantification by UV absorbance after
HPLC purification as described previously (9).
Analytical recovery.
Twenty-five microliters of acetonitrile
containing 0, 50, 100, or 200 ng of pure 25(OH)D2
or 25(OH)D3 was added to a 5-mL pooled serum
sample from five individuals. This provided addition concentrations of
0, 10, 20, and 40 µg/L for each metabolite. The samples, which were
in 16 x 100 mm borosilicate glass culture tubes, were
vortex-mixed and incubated for 30 min at room temperature to
equilibrate. The samples were then assayed as directed by the
manufacturers’ product inserts.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. The antibody from the DiaSorin method reacted equally with
25(OH)D2 and 25(OH)D3. The
detection limits of the DiaSorin method were 0.30 and 0.31 pg/tube for
25(OH)D2 and 25(OH)D3,
respectively. The median effective doses (ED50s)
in this assay were 14.6 and 15.5 pg/tube for
25(OH)D2 and 25(OH)D3,
respectively. Conversely, the antibody from the IDS method did not
react equally with 25(OH)D2 and
25(OH)D3. The detection limits for the IDS method
were 5.0 and 1.0 pg/tube for 25(OH)D2 and
25(OH)D3, respectively. The
ED50s also were markedly different: 23.6 pg/tube
for 25(OH)D3 and 50.3 pg/tube for
25(OH)D2.
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detection limit, analytical recovery, and precision
The detection limit, defined as 3 SD from the mean for the zero
calibrator, was 1 µg/L for both the DiaSorin and the IDS RIA. The
ED50s for 25(OH)D3 were
24.0 and 15.3 µg/L for the DiaSorin and IDS methods, respectively.
The analytical recovery data for both methods, using human serum, are
shown in Table 1
. Recovery for the DiaSorin method was 91–100% for both
25(OH)D2 and 25(OH)D3. In
comparison, the IDS method recovered 92–95% of
25(OH)D3 added to serum samples. However,
recovery of 25(OH)D2 was only 21–29% and was
uniform at all concentrations of 25(OH)D2 tested
(Table 1
).
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Imprecision (CV), both within and between assays, was determined with serum samples at various points on the calibration curves. The within- and between-assay CVs were 2.2–8.6% for the DiaSorin method and 2.1–10% for the IDS method. These values are similar to those reported in the respective product inserts.
comparison of RIAs with each other and with an
independent assay method
Separate quantification of 25(OH)D2 and
25(OH)D3 by direct UV detection after HPLC
allowed the human serum samples be divided into two groups. The first
group had no detectable circulating 25(OH)D2 (<2
µg/L), but all had detectable 25(OH)D3. This
group will be referred to as having "minimal circulating
25(OH)D2". In the second group, both
25(OH)D2 and 25(OH)D3 were
detectable, 18.3 ± 5.3 and 9.0 ± 5.2 (mean ± SD)
µg/L, respectively. This group will be referred to as having
"significant circulating 25(OH)D2". For the
patient population with minimal circulating
25(OH)D2, linear regression analysis comparing
the two RIAs displayed excellent results (Fig. 2
A). The mean circulating 25(OH)D concentrations for this group
were 20.3 ± 9.8 and 20.3 ± 8.2 µg/L as measured by the
DiaSorin and IDS methods, respectively. However, in the group with
significant circulating 25(OH)D2, the
relationship between the two RIA methods was poor (Fig. 2B
). The mean
circulating 25(OH)D concentrations in this group were 26.3 ± 4.8
and 18.6 ± 4.0 µg/L as measured by the DiaSorin and IDS
methods, respectively.
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The relationships between direct UV quantification of 25(OH)D and the
two RIA methods are displayed on Figs. 3
and 4. The methods, as compared by linear regression analysis, were
in good agreement for subjects with minimal circulating
25(OH)D2 (Fig. 3
). The mean concentrations of
circulating 25(OH)D in this comparison were 20.3 ± 10.5,
20.3 ± 8.2, and 20.3 ± 9.8 µg/L for HPLC, IDS, and
DiaSorin, respectively. In subjects with significant circulating
25(OH)D2, the DiaSorin RIA and HPLC method
exhibited excellent agreement (Fig. 4A
), whereas the IDS RIA and HPLC
did not (Fig. 4B
). The mean circulating 25(OH)D concentrations, as
determined by each method, in this comparison were 27.3 ± 4.4,
26.2 ± 4.8, and 18.6 ± 4.0 µg/L for HPLC, DiaSorin, and
IDS, respectively.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the determination of nutritional vitamin D status, it is imperative
that the method of choice measure circulating
25(OH)D2 and 25(OH)D3
equally to provide total circulating 25(OH)D. Both of the methods
evaluated in this study claim to measure total 25(OH)D. The
reactivities of both RIAs were first investigated by the use of pure
25(OH)D2 and 25(OH)D3 to
generate competition curves with the primary antibody from each RIA.
These data are displayed in Fig. 1
, which demonstrates that the primary
antibody in the DiaSorin RIA recognizes both
25(OH)D2 and 25(OH)D3
equally, whereas the antibody used in the IDS method does not. Data
from the calibration curves suggest that the IDS method will
underestimate total 25(OH)D when significant
25(OH)D2 is present in the circulation.
Underestimation of circulating 25(OH)D by the IDS RIA was confirmed
when the analytical recoveries of 25(OH)D2 and
25(OH)D3 were assessed. Table 1
shows that both
the IDS and DiaSorin methods quantitatively recovered
25(OH)D3 from human serum, whereas only the
DiaSorin method quantitatively recovered
25(OH)D2. In fact, the recovery of
25(OH)D2 from human samples was poor at all
concentrations tested when the IDS method was used (Table 1
). This is because the calibration curves for the IDS method, like the
DiaSorin method, are constructed using
25(OH)D3. The IDS method does appear to be valid
when 25(OH)D3 is the only circulating 25(OH)D
species. Figs. 2
, 3
, and 4
show some disparity among the DiaSorin, IDS,
and HPLC methods for samples containing primarily
25(OH)D3. These differences are most likely
attributable to the different calibrators used in the DiaSorin and IDS
methods and exemplify the point that every laboratory should establish
its own range of values for whatever method is chosen.
The in vitro data suggest that the DiaSorin method will accurately
estimate total 25(OH)D in the presence of significant circulating
25(OH)D2, whereas the IDS method will
underestimate total 25(OH)D under the same conditions. Figs. 2
, 3
, and 4
exemplify this point. When human samples containing minimal
circulating 25(OH)D2 were analyzed by the IDS and
DiaSorin methods, the results were in good agreement (Fig. 2A
).
However, in samples containing significant circulating
25(OH)D2, the IDS method underestimated total
circulating 25(OH)D by an average of 30% (Fig. 2B
). These data are
confirmed in Figs. 3
and 4
, which show that the DiaSorin method agreed
with UV-HPLC analysis for samples with significant or minimal
circulating 25(OH)D2, whereas the IDS method
underestimated 25(OH)D, on average, by 
30% in samples containing
significant circulating 25(OH)D2 (Fig. 4B
).
Vitamins D2 and D3 are both widely utilized in the food supply and are interchangeably supplemented in the milk supply in the United States (10)(11)(12). Furthermore, vitamin D2 is widely used in pharmaceutical preparations worldwide, including the United States, Europe, and Japan. Hence, it is very important to select an analytical method that will accurately estimate total circulating 25(OH)D independent of the circulating concentrations of 25(OH)D2 and 25(OH)D3. From the data generated in this study, it is clear that the DiaSorin and HPLC-based methods fill this purpose, whereas the IDS method does not.
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Acknowledgments |
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Footnotes |
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References |
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