Questions constantly arise regarding the various 25(OH)D assay procedure’s ability to accurately measure total 25(OH)D [25(OH)D2
+ 25 (OH)D3
] levels in human samples [8
]. A brief study recently has described the ability of the DiaSorin Liaison Total-D 25(OH)D Assay System to perform this task as compared to the “gold standard” hplc/UV quantitation of 25(OH)D2
]. Baseline scrum samples were obtained from nine volunteers which contained only 25(OH)D3
. All subjects then consumed 50,000 IU/d vitamin D2
for a period of 14 days. Seven days following the final dose serum samples were again obtained. For exogenous in vitro
recovery experiments 32 ng/ml of either 25(OH)D2
were added, in a small volume of ethanol, to each baseline serum sample. All samples were then subjected to direct hplc/UV quantitation to determine individual levels of 25(OH)D2
] or the DiaSorin Liaison Total-D Assay.
25(OH)D calibrators from The National Institute of Standards and Technology (NIST) were also tested. NIST describes the samples as Level 1; “normal” human serum; Level 2 “normal” human serum diluted 1/1 with horse serum; and Level 3 “normal” human serum “spiked” with 25(OH)D2 attempting to equal the amount of endogenous 25(OH)D3 contained in the sample. Horse serum from Sigma Chemical Company was also accessed.
In the group of volunteers the baseline total 25(OH)D was 48.3 ± 19.0 and 43.7 ± 16.8 ng/ml (
) by hplc-UV and Liaison, respectively. In these baseline samples hplc-UV analysis demonstrated 99% of the circulating 25(OH)D to be of the D3
form, only 2 of 9 subjects had detectable (>1.0 ng/ml) 25(OH)D2
. Following 14 days of oral vitamin D2
supplementation, total 25(OH)D levels were determined to be 81.1 ± 21.9 and 80.0 ± 25.5 ng/ml by hplc-UV and Liaison, respectively. By hplc analysis the elevations in 25(OH)D2
ranged from 25-88 ng/ml. In these post-supplementation samples, hplc-UV analysis also revealed 25(OH)D3
to be 43.5% of the total while the remaining 56.5 was 25(OH)D2
. The regression relationship of pre and post samples between hplc-UV and Liaison was Liaison Total-D = 1.04 (hplc-UV) – 5.27, r2
= 0.95 (). The recovery of exogenously added 25(OH)D2
to baseline samples was 98.3 ± 5.7 and 99.0 ± 6.7%, respectively by hplc-UV analysis, and 22.8 ± 19.7 and 62.7 ± 24.8%, respectively by Liaison analysis [18
Fig. 1 Elevations in plasma total 25(OH)D in volunteers following supplementation with vitamin D2 as measured by the DiaSorin Liaison Total method versus HPLC. Volunteers were given vitamin D2. Presupplementation concentrations are represented by the closed (more ...)
NIST Level 1 concentrations measured by the Liaison compared favorably with HPLC results. However, NIST Level 2 as higher (Liaison vs. HPLC) and Level 3 was lower Liaison vs. HPLC. The higher concentration in the NIST Level 2 can be attributed to the impact of the horse serum matrix and lower levels in NIST Level 3 can be attributed to the lack of recovery of exogenous material by the Liaison system.
The data reveals an important artifact that could lead to false conclusions about the ability of direct competitive antibody-based chemiluminescence assays to quantitatively detect 25(OH)D2
in patient samples. It has proven difficult to produce an antibody that is co-specific for the detection of 25(OH)D2
in human serum. In fact, only one such antibody has been reported and that is the antibody utilized in the DiaSorin 25(OH)D assays [9
In the U.S. it is imperative that any 25(OH)D assay used for clinical diagnosis have the ability to detect total 25(OH)D, a sum of 25(OH)D2 and 25(OH)D3. With a single exception, all competitive protein binding assays introduced commercially have discriminated against 25(OH)D2 including the now defunct Nichols Advantage 25(OH)D assay system. It is also a fact that approximately 99% of the U.S. population has undetectable 25(OH)D2 in their circulation. This is because vitamin D2 is rarely used as a supplement anymore and patients only receive it when being treated for vitamin D deficiency by a physician. Since blood samples in the general population rarely contain significant amounts of 25(OH)D2, and because the compound is usually discriminated against by most antibody-based assays, it is the compound most often added exogenously to human serum to assess cross-reactivity and determine analytical recovery.
We have assumed since the early 1970’s that when one adds exogenous 25(OH)D to a blood sample it rapidly binds to its carrier protein, the vitamin D-binding protein (DBP) with little interaction to other blood components [19
]. Up to this point in vitamin D assay technology, exogenous addition of 25(OH)D2
has served us well in our testing of quantitative analytical recoveries of these compounds [9
]. Problems were never encountered because extraction procedures were based on organic solvents of one kind or another and they all destroyed the DBP and liberated the 25(OH)D into solution. The direct serum or plasma assays emerging today do not destroy the carrier proteins. Instead they rely on pH changes and/or blocking agents that liberate the 25(OH)D from its carrier protein but do not affect the ability of the steroid to bind to a specific antibody. This later disruption method is the method employed in the Liaison assay [11
The results clearly demonstrate that exogenously added 25(OH)D2
do not distribute themselves on the DBP as occurs when assembled in vivo
. The other possibility is that exogenously added 25(OH)D distribute to moieties other than the DBP. This is suggested by the clear linear relationship observed from in vivo
human samples containing elevated amounts of 25(OH)D2
when assayed by the Liaison method versus HPLC-UV. On the other hand the failure of quantitative recovery is apparent from exogenously added 25(OH)D2
to the same samples and the assay methods compared (). This study describes an in vitro
anomaly that really has no physiological relevance but could result in erroneous conclusions about 25(OH)D assay performance when comparing sample destruction methods such as HPLC-UV versus the newer sample disruption method such as the Liaison assay [18
]. Extreme caution is warranted when preparing samples for such comparisons as is being done by vitamin D External Quality Assessment Scheme (DEQAS) and NIST.
Comparison of 25(OH)D concentrations measured by the DiaSorin Liaison and HPLC as a result of various exogenous and endogenous treatments.