The unique cis -triene structure of vitamin D and related metabolites makes it susceptible to oxidation, ultraviolet (UV) light-induced conformational changes, heat-induced conformational changes, and attacks by free radicals. Vitamin D 2 is much less bioactive than vitamin D 3 in humans. Metabolic activation and inactivation of vitamin D are well characterized and result in a plethora of metabolites, of which only 25-hydroxyvitamin D (25(OH)D) and 1,25-dihydroxyvitamin D (1,25(OH) 2 D) provide any clinically relevant information. 25(OH)D 2 and 25(OH)D 3 are commonly known as calcifediol and the 1,25(OH) 2 D metabolites as calcitriol. In this review the current state of the science on the clinical assessment of circulating 25(OH)D and 1,25(OH) 2 D is described.
Vitamin D is a 9,10-seco steroid, as shown by the numbering of its carbon skeleton. Vitamin D has 2 distinct forms: vitamin D 2 and vitamin D 3 . Vitamin D 2 is a 28-carbon molecule derived from the plant sterol ergosterol, whereas vitamin D 3 is a 27-carbon derivative of cholesterol. Vitamin D 2 differs from vitamin D 3 in that it contains an extra methyl group and a double bond between carbons 22 and 23.
The most important aspects of vitamin D chemistry center on its cis -triene structure. This unique structure makes vitamin D and related metabolites susceptible to oxidation, ultraviolet (UV) light-induced conformational changes, heat-induced conformational changes, and attacks by free radicals. Most of these transformation products have less biologic activity than does vitamin D. Research has now shown that vitamin D 2 is much less bioactive than vitamin D 3 in humans. The parent compounds vitamin D 2 and vitamin D 3 are sometimes referred to as calciferol.
Hydroxylation reactions at both carbon 25 of the side chain and, subsequently, carbon 1 of the A ring result in the metabolic activation of vitamin D. Metabolic inactivation of vitamin D takes place primarily through a series of oxidative reactions at carbons 23, 24, and 26 of the molecule’s side chain. Metabolic activation and inactivation are well characterized and result in a plethora of vitamin D metabolites. Of these metabolites, only 25-hydroxyvitamin D (25(OH)D) and 1,25-dihydroxyvitamin D (1,25(OH) 2 D) provide any clinically relevant information. 25(OH)D 2 and 25(OH)D 3 are commonly known as calcifediol and the 1,25(OH) 2 D metabolites as calcitriol.
In this review the current state of the science on the clinical assessment of circulating 25(OH)D and 1,25(OH) 2 D is described.
Methods of 25(OH)D quantitation
The assessment of circulating 25(OH)D started its journey approximately 4 decades ago with the advent of the competitive protein-binding assay (CPBA). From that early time to the present we have progressed to radioimmunoassay (RIA), high-performance liquid chromatography (HPLC), and liquid chromatography coupled with mass spectrometry (LC/MS). A brief description of each technique is given here.
Competitive Protein-Binding Assay
A major factor responsible for the explosion of information on vitamin D metabolism and its relation to clinical disease was the introduction of a CPBA for 25(OH)D. Haddad and Chyu introduced this CPBA almost 4 decades ago. The assay assessed circulating 25(OH)D concentrations using the vitamin D–binding protein (DBP) as a primary binding agent and 3 H-25(OH)D 3 as a reporter. Although this CPBA was valid, it was also relatively cumbersome. Technicians had to extract the sample with organic solvent, dry it under nitrogen, and purify it using column chromatography. This assay was suitable for the research laboratory but did not meet the requirements of a high-throughput clinical laboratory.
The major difficulty in measuring 25(OH)D is attributable to the molecule itself. 25(OH)D is probably the most hydrophobic compound measured by protein-binding assay (PBA), which constitutes either CPBA or radioimmunoassay (RIA). The fact that the molecule exists in 2 forms, 25(OH)D 2 and 25(OH)D 3 , compounds the difficulties with its quantitation by PBA. 25(OH)D’s lipophilic nature renders it especially vulnerable to the matrix effects of any PBA. Anything present in the sample assay vessel that is not present in the calibrator assay vessel can cause matrix effects. These matrix effect substances are usually lipid but in the newer direct assays, they could be anything contained in the serum or plasma sample. These matrix factors change the ability of the binding agent, antibody, or binding protein to associate with 25(OH)D in the sample or standard in an equal fashion. When this occurs, it markedly diminishes the assay’s validity. Experience has demonstrated that the DBP is more susceptible to these matrix effects than antibodies. The original Haddad procedure overcame the matrix problem by using chromatographic sample purification before CPBA.
Researchers had a strong desire to simplify this cumbersome CPBA for 25(OH)D, so Belsey and colleagues developed a streamlined CPBA in 1974. The goal of this second-generation CPBA was to eliminate chromatographic sample purification as well as individual sample recovery using 3 H-25(OH)D 3 . However, after several years of trying, researchers were unable to validate the Belsey assay due to matrix problems originating from ethanolic sample extraction.
The 25(OH)D CPBAs did have the advantage of being cospecific for 25(OH)D 2 and 25(OH)D 3 and thus provided a “total” 25(OH)D value if the assay was valid. The DBP’s binding cospecificity for 25(OH)D 2 and 25(OH)D 3 as well as its stability made it an attractive candidate for incorporation into automated direct chemiluminescent assays. In fact, Nichols Institute Diagnostics used this approach when its researchers developed the Advantage 25(OH)D Assay. The US Food and Drug Administration (FDA) approved this assay for clinical use, but Nichols ultimately withdrew it from the market place due to its propensity to overestimate total circulating 25(OH)D concentrations and its surprising inability to detect circulating 25(OH)D 2 . Although never described, these problems were probably linked to the DBP’s inability to resolve the matrix problems associated with direct sample assay. At present, the CPBA for 25(OH)D is rarely used. Also, one cannot accurately compare most CPBA results for circulating 25(OH)D concentrations from the past with values from current methods because many of the matrix interferences were not linear in the old CPBAs.
Radioimmunoassay
In the early 1980s, the author’s group decided that a nonchromatographic RIA for circulating 25(OH)D would be the best approach to measuring the substance. This group therefore designed an antigen that would generate an antibody that was cospecific for 25(OH)D 2 and 25(OH)D 3 . In addition, a simple extraction method was designed that allowed simple nonchromatographic quantification of circulating 25(OH)D. In 1985 Immunonuclear Corp., now known as DiaSorin, introduced this 3 H-based RIA as a kit on a commercial basis. This RIA was further modified in 1993 to incorporate a 125 I-labeled reporter and calibrators (standards) in a serum matrix. This modification finally made mass assessment of circulating 25(OH)D possible. In that same year this assay became the first FDA-approved device for the clinical diagnosis of nutritional vitamin D deficiency. Further, during the past 23 years these DiaSorin tests have been used in the vast majority of large clinical studies worldwide to define “normal” circulating 25(OH)D levels in a variety of disease states. This test still remains today the only RIA-based assay that provides a “total” 25(OH)D value.
Random-Access Automated Instrumentation
DiaSorin Corporation, Roche Diagnostics, and the now defunct Nichols Institute Diagnostics all introduced methods for the direct (no extraction) quantitative determination of 25(OH)D in serum or plasma using completive protein assay chemiluminescence technology. These assays appear quite similar on the surface but they are not.
In 2001, Nichols Diagnostics introduced the fully automated chemiluminescence Advantage 25(OH)D assay system. In this assay system, nonextracted serum or plasma was added directly into a mixture containing human DBP, acridinium-ester labeled anti-DBP, and 25(OH)D 3 -coated magnetic particles. Note that the primary binding agent was human DBP. Thus, this assay was a CPBA, much like the manual procedure introduced in 1974 by Belsey and colleagues. The major difference between these procedures was that Belsey depotenized the sample with ethanol before assaying it. The calibrators for the Belsey assay were in ethanol. In the Advantage assay, the calibrators were in a serum-based matrix, and its developers assumed that this matrix would replicate the serum or plasma sample introduced directly into the assay system. In the end, the 1974 Belsey assay never worked and neither did the Advantage 25(OH)D Assay. The company removed the assay from the market in 2006.
In 2004, the DiaSorin Corporation introduced the fully automated chemiluminescence Liaison 25(OH)D Assay System. This assay is very similar to the late Advantage assay, with one major difference: the Liaison assay uses an antibody as a primary binding agent as opposed to the human DBP in the Advantage system. Thus, the Liaison is a true RIA method. Details on this procedure are available elsewhere. The Liaison 25(OH)D assay is cospecific for 25(OH)D 2 and 25(OH)D 3 , so it reports a “total” 25(OH)D concentration. DiaSorin recently introduced a second-generation Liaison 25(OH)D assay. This new version has increased functional sensitivity and much improved assay precision. The Liaison 25(OH)D assay is the single most widely used 25(OH)D assay in the world for clinical diagnosis.
The most recent addition to the automated 25(OH)D assay platforms is from Roche Diagnostics. Their test is an RIA called vitamin D 3 (25-OH), which can be performed on their Elecsys and Cobas systems. Roche only released this assay in 2007, so very little information on it is available. However, the assay can only detect 25(OH)D 3 , so it will not be a viable product in countries in which vitamin D 2 is used clinically, including the United States.
Direct Physical Detection Methods
Direct detection methodologies for determining circulating 25(OH)D include both HPLC and LC/MS procedures. The HPLC methods separate and quantitate circulating 25(OH)D 2 and 25(OH)D 3 individually. HPLC followed by UV detection is highly repeatable and, in general, most people consider it the gold standard method. However, these methods are cumbersome and require a relatively large sample as well as an internal standard. Sample throughout is slow and is not suited to a high-demand clinical laboratory processing up to 10,000 25(OH)D assays per day.
Researchers have recently revitalized LC/MS as a viable method to assess circulating 25(OH)D. As with HPLC, LC/MS quantitates 25(OH)D 2 and 25(OH)D 3 separately. When performed properly, LC/MS is a very accurate testing method. However, the equipment is very expensive and its overall sample throughput cannot, when performed properly, match that of the automated instrumentation format. As a methodology, LC/MS can compare favorably with RIA techniques. One unique problem with LC/MS is its relative inability to discriminate between 25(OH)D 3 and its inactive isomer 3-epi-25(OH)D 3 . This problem has been especially noticeable in the circulation of newborn infants. Next to the DiaSorin assays, LC/MS is the next most used procedure for the clinical assessment of circulating 25(OH)D.
Determining analytical recovery of 25(OH)D 2 and 25(OH)D 3 in human serum or plasma
Questions constantly arise regarding the various 25(OH)D assay procedures’ ability to accurately measure total 25(OH)D (25(OH)D 2 + 25 (OH)D 3 ) levels in human samples. 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 with the gold standard HPLC/UV quantitation of 25(OH)D 2 and 25(OH)D 3 . Baseline serum samples that contained only 25(OH)D 3 were obtained from 9 volunteers. All subjects then consumed 50,000 IU/d vitamin D 2 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)D 2 or 25(OH)D 3 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)D 2 and 25(OH)D 3 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)D 2 attempting to equal the amount of endogenous 25(OH)D 3 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 ( x ¯ ± SD) 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 D 3 form, and only 2 of 9 subjects had detectable (>1.0 ng/mL) 25(OH)D 2 . Following 14 days of oral vitamin D 2 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)D 2 ranged from 25 to 88 ng/mL. In these postsupplementation samples, HPLC-UV analysis also revealed 25(OH)D 3 to be 43.5% of the total while the remaining 56.5% was 25(OH)D 2 . The regression relationship of pre and post samples between HPLC-UV and Liaison was Liaison Total-D = 1.04 (HPLC-UV) − 5.27, r 2 = 0.95 ( Fig. 1 ). The recovery of exogenously added 25(OH)D 2 or 25(OH)D 3 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.
NIST Level 1 concentrations measured by the Liaison compared favorably with HPLC results. However, NIST Level 2 was 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 reveal an important artifact that could lead to false conclusions about the ability of direct competitive antibody-based chemiluminescence assays to quantitatively detect 25(OH)D 2 and/or 25(OH)D 3 in patient samples. It has proven difficult to produce an antibody that is cospecific for the detection of 25(OH)D 2 and 25(OH)D 3 in human serum. In fact, only one such antibody has been reported, being the antibody used in the DiaSorin 25(OH)D assays.
In the United States it is imperative that any 25(OH)D assay used for clinical diagnosis has the ability to detect total 25(OH)D, a sum of 25(OH)D 2 and 25(OH)D 3 . With a single exception, all competitive protein-binding assays introduced commercially have discriminated against 25(OH)D 2 including the now defunct Nichols Advantage 25(OH)D assay system. It is also a fact that approximately 99% of the United States population has undetectable 25(OH)D 2 in their circulation, because vitamin D 2 is rarely used as a supplement nowadays and patients only receive it when being treated for vitamin D deficiency by a physician. Because blood samples in the general population rarely contain significant amounts of 25(OH)D 2 , 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 1970s that when one adds exogenous 25(OH)D to a blood sample it rapidly binds to its carrier protein, the DBP, with little interaction to other blood components. Up to this point in vitamin D assay technology, exogenous addition of 25(OH)D 2 or 25(OH)D 3 has served clinicians well in the testing of quantitative analytical recoveries of these compounds. 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 one employed in the Liaison assay.
The results clearly demonstrate that exogenously added 25(OH)D 2 or 25(OH)D 3 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, suggested by the clear linear relationship observed from in vivo human samples containing elevated amounts of 25(OH)D 2 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)D 2 or 25(OH)D 3 to the same samples when the assay methods are compared ( Table 1 ). This study describes an in vitro anomaly that really has no physiologic 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. Extreme caution is warranted when preparing samples for such comparisons, as is being done by the Vitamin D External Quality Assessment Scheme (DEQAS) and NIST.
| Sample ID | DiaSorin Liaison | HPLC |
|---|---|---|
| Total 25(OH)D (ng/mL) | ||
| Baseline | 43.7 ± 16.8 | 48.3 ± 19.0 |
| Vitamin D 2 | 81.1 ± 21.9 | 80.0 ± 23.5 |
| Baseline + 25(OH)D 2 a | 51.0 ± 16.8 (22.8%) | 79.7 ± 19.0 (98.3%) |
| Baseline + 25(OH)D 3 a | 63.7 ± 20.4 (62.7%) | 80.0 ± 18.5 (99.0%) |
| Horse serum | 12.7 ± 1.0 | 4.7 ± 0.2 |
| NIST Level 1 [22–24] b | 24.4 ± 0.8 (106%) | 26.0 ± 1.1 (113%) |
| NIST Level 2 [12–14] b | 19.8 ± 0.5 (152%) | 15.9 ± 0.7 (122%) |
| NIST Level 3 [42–46] b | 27.2 ± 1.0 (61.8%) | 48.1 ± 3.0 (109%) |
a 32 ng/mL was added to each of 9 samples. Values in parentheses represent amount of 25(OH)D recovered as a percentage of mean values.
b Values in brackets are expected values provided by NIST. Values in parentheses represent amount of 25(OH)D recovered as a percentage of mean values.
Determining analytical recovery of 25(OH)D 2 and 25(OH)D 3 in human serum or plasma
Questions constantly arise regarding the various 25(OH)D assay procedures’ ability to accurately measure total 25(OH)D (25(OH)D 2 + 25 (OH)D 3 ) levels in human samples. 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 with the gold standard HPLC/UV quantitation of 25(OH)D 2 and 25(OH)D 3 . Baseline serum samples that contained only 25(OH)D 3 were obtained from 9 volunteers. All subjects then consumed 50,000 IU/d vitamin D 2 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)D 2 or 25(OH)D 3 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)D 2 and 25(OH)D 3 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)D 2 attempting to equal the amount of endogenous 25(OH)D 3 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 ( x ¯ ± SD) 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 D 3 form, and only 2 of 9 subjects had detectable (>1.0 ng/mL) 25(OH)D 2 . Following 14 days of oral vitamin D 2 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)D 2 ranged from 25 to 88 ng/mL. In these postsupplementation samples, HPLC-UV analysis also revealed 25(OH)D 3 to be 43.5% of the total while the remaining 56.5% was 25(OH)D 2 . The regression relationship of pre and post samples between HPLC-UV and Liaison was Liaison Total-D = 1.04 (HPLC-UV) − 5.27, r 2 = 0.95 ( Fig. 1 ). The recovery of exogenously added 25(OH)D 2 or 25(OH)D 3 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.
NIST Level 1 concentrations measured by the Liaison compared favorably with HPLC results. However, NIST Level 2 was 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 reveal an important artifact that could lead to false conclusions about the ability of direct competitive antibody-based chemiluminescence assays to quantitatively detect 25(OH)D 2 and/or 25(OH)D 3 in patient samples. It has proven difficult to produce an antibody that is cospecific for the detection of 25(OH)D 2 and 25(OH)D 3 in human serum. In fact, only one such antibody has been reported, being the antibody used in the DiaSorin 25(OH)D assays.
In the United States it is imperative that any 25(OH)D assay used for clinical diagnosis has the ability to detect total 25(OH)D, a sum of 25(OH)D 2 and 25(OH)D 3 . With a single exception, all competitive protein-binding assays introduced commercially have discriminated against 25(OH)D 2 including the now defunct Nichols Advantage 25(OH)D assay system. It is also a fact that approximately 99% of the United States population has undetectable 25(OH)D 2 in their circulation, because vitamin D 2 is rarely used as a supplement nowadays and patients only receive it when being treated for vitamin D deficiency by a physician. Because blood samples in the general population rarely contain significant amounts of 25(OH)D 2 , 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 1970s that when one adds exogenous 25(OH)D to a blood sample it rapidly binds to its carrier protein, the DBP, with little interaction to other blood components. Up to this point in vitamin D assay technology, exogenous addition of 25(OH)D 2 or 25(OH)D 3 has served clinicians well in the testing of quantitative analytical recoveries of these compounds. 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 one employed in the Liaison assay.
The results clearly demonstrate that exogenously added 25(OH)D 2 or 25(OH)D 3 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, suggested by the clear linear relationship observed from in vivo human samples containing elevated amounts of 25(OH)D 2 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)D 2 or 25(OH)D 3 to the same samples when the assay methods are compared ( Table 1 ). This study describes an in vitro anomaly that really has no physiologic 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. Extreme caution is warranted when preparing samples for such comparisons, as is being done by the Vitamin D External Quality Assessment Scheme (DEQAS) and NIST.
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