Vitamin D: marker, measurand & measurement

in Endocrine Connections
Authors:
Niek F DirksAtalmedial Diagnostics Centre, Spaarne Gasthuis, Haarlem, The Netherlands
Department of Clinical Chemistry, Hematology and Immunology, Noordwest Ziekenhuis, Alkmaar, The Netherlands

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Etienne CavalierDepartment of Clinical Chemistry, University of Liège, CHU de Liège, Liège, Belgium

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Annemieke C HeijboerAmsterdam UMC location Vrije Universiteit Amsterdam, Department of Clinical Chemistry, Endocrine Laboratory, Boelelaan, Amsterdam, The Netherlands
Amsterdam Gastroenterology, Endocrinology & Metabolism, Amsterdam, The Netherlands
Amsterdam UMC location University of Amsterdam, Department of Clinical Chemistry, Endocrine Laboratory, Amsterdam, The Netherlands
Amsterdam Reproduction & Development Research Institute, Amsterdam, The Netherlands

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https://orcid.org/0000-0002-6712-9955
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Correspondence should be addressed to A C Heijboer: a.heijboer@amsterdamumc.nl
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The measurement of vitamin D metabolites aids in assessing vitamin D status and in diagnosing disorders of calcium homeostasis. Most laboratories measure total 25-hydroxyvitamin D (25(OH)D), while others have taken the extra effort to measure 25(OH)D2 and 25(OH)D3 separately and additional metabolites such as 1,25-dihydroxyvitamin D and 24,25-dihydroxyvitamin D. The aim of this review is to provide an updated overview of the main markers of vitamin D metabolism, define the intended measurands, and discuss the advantages and disadvantages of the two most widely used assays, automated assays and liquid chromatography–tandem mass spectrometry (LC-MS/MS). Whether using the easy and fast automated assays or the more complex LC-MS/MS, one should know the pitfalls of the used technique in order to interpret the measurements. In conclusion, automated assays are unable to accurately measure 25(OH)D in all patient groups, including persons using D2. In these cases, an LC-MS/MS method, when appropriately developed and standardized, produces a more reliable measurement.

Abstract

The measurement of vitamin D metabolites aids in assessing vitamin D status and in diagnosing disorders of calcium homeostasis. Most laboratories measure total 25-hydroxyvitamin D (25(OH)D), while others have taken the extra effort to measure 25(OH)D2 and 25(OH)D3 separately and additional metabolites such as 1,25-dihydroxyvitamin D and 24,25-dihydroxyvitamin D. The aim of this review is to provide an updated overview of the main markers of vitamin D metabolism, define the intended measurands, and discuss the advantages and disadvantages of the two most widely used assays, automated assays and liquid chromatography–tandem mass spectrometry (LC-MS/MS). Whether using the easy and fast automated assays or the more complex LC-MS/MS, one should know the pitfalls of the used technique in order to interpret the measurements. In conclusion, automated assays are unable to accurately measure 25(OH)D in all patient groups, including persons using D2. In these cases, an LC-MS/MS method, when appropriately developed and standardized, produces a more reliable measurement.

Introduction

The percentage of people considered vitamin D deficient is ever-growing as a consequence of depletion of sufficient amounts of sunlight by our changing ways of life (1). Because of this, and as a consequence of the increasing variety of conditions known to be associated with vitamin D deficiency, vitamin D testing has skyrocketed. Nowadays, many laboratories, big and small, are running tests for the assessment of vitamin D status. While most run the well-known 25-hydroxyvitamin D (25(OH)D) metabolite, other vitamin D metabolites may offer vital information in diagnosing the more rare conditions. When measuring any of the vitamin D metabolites, it is essential to know what the actual measurand of the assay is, as it is not always the same for every method designed to measure vitamin D. Second, knowing the pitfalls of the used assay, most often an automated immunoassay or a liquid-chromatography coupled to tandem mass-spectrometry, is important for the interpretation of the results and adequate application in specific patient groups. Here, we review the different vitamin D markers that are in use today, articulate the intended measurand, and discuss the advantages and disadvantages of the two most used techniques for assessment of vitamin D status.

Metabolism

Vitamin D is not a single molecule that after production or ingestion rushes through our veins to exert its function on the target organs to maintain calcium homeostasis. In fact, it requires a whole cascade of metabolizing reactions that precede the formation of the active hormone (Fig. 1) (2).

Figure 1
Figure 1

Endogenous vitamin D3 metabolism. When our skin is penetrated by UVB light, 7-dehydrocholesterol is converted to pre-vitamin D. This rapidly isomerizes into vitamin D3, which then enters circulation and binds to vitamin D binding protein (VDBP). Liver enzyme 25α-hydroxylase then hydroxylates vitamin D3, which gains 25(OH)D3. 25(OH)D3 can be converted into the active hormone, 1,25(OH)2D3 by 1α-hydroxylase. Alternatively, it may be epimerized to epi-25(OH)D3 or to 24,25(OH)2D3, inactive metabolites with no or substantially lower affinity for 1α-hydroxylase.

Citation: Endocrine Connections 12, 4; 10.1530/EC-22-0269

The starting compound is 7-dehydrocholesterol, a final intermediate in the cholesterol biosynthesis. The enzyme 7-dehydrocholesterol reductase uses NADH to reduce this molecule to compose cholesterol. Alternatively, when UVB radiation penetrates the epidermal layers of our skin, part of the 7-dehydrocholesterol molecule can absorb light and break open. The resulting pre-vitamin D3 is unstable and immediately isomerizes into vitamin D3, which then enters circulation and binds to vitamin D binding protein (VDBP). At this point, vitamin D2, a very similar but vegetable form of vitamin D, which we obtain from certain foods or supplements may also enter this metabolic route. Both vitamin D3 and D2 are only present in small amounts in circulation. Liver cytochrome P450 CYP2R1 is the main 25-hydroxylase that catalyzes the 25-hydroxylation reaction to form, respectively, 25-hydroxyvitamin D3 (25(OH)D3) or 25-hydroxyvitamin D2 (25(OH)D2) (3). These vitamin D metabolites are most abundant in circulation but are still not bioactive. A second hydroxylation at the C1 position step by the kidney enzyme 1α-hydroxylase yields 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) or 1,25-dihydroxyvitamin D2 (1,25(OH)2D2), both are able to bind the nuclear vitamin D receptor (VDR). The renal 1α-hydroxylation is tightly regulated by parathyroid hormone (PTH), fibroblast growth factor 23 (FGF23), calcium, phosphate, and 1,25(OH)2D itself. PTH upregulates the expression of 1α-hydroxylation, while FGF23 and 1,25(OH)2D downregulate its expression. Apart from renal activation, many other cell types also harbor 1α-hydroxylase and are thus able to bioactivate 25(OH)D3 and 25(OH)D2 for autocrine or paracrine use (4). This amount of extra-renal bioactivation only contributes little to the circulating concentrations of total 1,25(OH)2D and is not under the regulation of PTH or FGF23. The 25-hydroxy and 1,25-dihydroxy forms of vitamin D may be inactivated and prepared for secretion by renal or extra-renal 24α-hydroxylase, which adds another hydroxyl group at the C24 position, resulting in, respectively, 24,25-dihydroxyvitamin D (24,25(OH)2D) or 1,24,25-trihydroxyvitamin D (1,24,25(OH)3D). This route is also under the regulation of PTH and FGF23, which, respectively, downregulate and upregulate the expression of the gene coding for 24,25(OH)2D.

Another enzyme, 3-epimerase, converts the orientation of the hydroxyl group at the C3 position of small amounts of 25(OH)D and 1,25(OH)2D resulting in 3-epi-25(OH)D and 3-epi-1,25(OH)2D. Only around 4% of circulating 25(OH)D is of the epimerized form, yet incidentally higher amounts up to 25% have been reported and maybe even higher in infants up to 1 year of age (5, 6, 7, 8). The function of the epimers is uncertain, yet seems of less clinical significance as they have a lower affinity for VDBP and 3-epi-1,25(OH)2D has a significantly lower affinity to the VDR (9).

Vitamin D metabolites as biomarkers for clinical use

25(OH)D

25(OH)D is the main circulating vitamin D metabolite and forms the pool from which 1,25(OH)2D can be formed by renal or extra-renal 1α-hydroxylation when required. Consequently, 25(OH)D is considered the best reflector of the body's vitamin D status as sufficient renal and local 1,25(OH)2D can only be generated if sufficient 25(OH)D is available. Measurement is recommended in individuals at increased risk of vitamin D deficiency and included obese individuals, pregnant and lactating women, older adults with a history of falls or fractures, individuals with darker skin pigmentation, patients with kidney disease, liver failure, bone disease, hyperparathyroidism, granuloma-forming disorders, lymphomas, and patients on certain medications (10). The debate on the exact target values for 25(OH)D sufficiency is still ongoing, but most studies set target values somewhere between 50 and 80 nmol/L (11, 12, 13).

1,25(OH)2D

Only a few rare conditions justify measuring 1,25(OH)2D, which have been reviewed elsewhere (10, 14). In short, disorders characterized by defective 1α-hydroxylase, such as vitamin D-dependent rickets type 1, result in an inability to produce 1,25(OH)2D and thus abnormally low concentrations (below 59 pmol/L) (15). Similarly, disorders in which FGF23 is increased, such as X-linked hypophosphatemia and tumor-induced osteomalacia, also result in very low 1,25(OH)2D combined with low phosphate levels. Defects in the VDR, impairing binding and subsequent hormonal activity by 1,25(OH)2D lead to vitamin D-dependent rickets type 2. Characteristically, very high 1,25(OH)2D levels are found in these individuals. Diseases displaying excessive amounts of extrarenal enzymatic formation of 1,25(OH)2D, such as sarcoidosis and tuberculosis are also associated with increased levels of 1,25(OH)2D (above 159 pmol/L) (15).

Vitamin D metabolite ratio

The ratio of 25(OH)D to 24,25(OH)2D is useful as a marker of 24α-hydroxylase activity (8). Its activity increases as a means to prevent overproduction of 1,25(OH)2D, for example upon supplementation of vitamin D. It has been revealed to be independent of VDBP and may serve as a better reflector of vitamin D status in patient groups with a larger variety of VDBP concentrations (16). In a recent study by Ginsberg et al., for example, it was shown to be more strongly associated with loss of BMD and fracture risk in a cohort of community-dwelling older adults compared to 25(OH)D. Some have suggested the ratio to be a good predictor of adequate vitamin D status after supplementation, but this has not been confirmed by recent publications (17). However, it is significantly increased in, and can therefore be used in the diagnosis of, idiopathic infantile hypercalcemia (IIH) (18, 19). IIH is caused by a mutation in the gene coding for 24α-hydroxylase which impairs the inactivation of 25(OH)D and 1,25(OH)2D and thus leads to overproduction of the active hormone, hypercalcemia and low PTH concentrations (20).

Free and bioavailable total 25-hydroxyvitamin D

Over 85% of 25(OH)D and 1,25(OH)2D is bound to VDBP, while most of the remaining are bound to albumin and only about 0.03% circulates free of any binding protein (21). Some have suggested that calculated bioavailable 25(OH)D (not bound to VDBP) or free 25(OH)D (not bound to VDBP or albumin) may be a more relevant biomarker of vitamin D status, especially in those with a different genotype of VDBP (22). However, a number of studies have since determined this not to be the case and showed that both free and bioavailable 25(OH)D only reflect total 25(OH)D and have no or limited added clinical utility (23, 24). This makes sense realizing that 25(OH)D is not the actual hormonally active compound but a prohormone and free 25(OH)D is as a consequence not regulated by feedback loops (14). As long as sufficient amounts of 25(OH)D can be oxidized to form 1,25(OH)2D, the exact pool of free or bioavailable 25(OH)D seems of little importance.

Measurand

As both 25(OH)D3 and 25(OH)D2 may be hydroxylated to an active hormone, measurement of vitamin D status should encompass total 25(OH)D, meaning both the isomers 25(OH)D3 and 25(OH)D2. 25(OH)D2 has an additional double bond compared to 25(OH)D3 and as a result differs in mass. As mentioned earlier, we are able to produce 25(OH)D3 with the help of UVB radiation. Usually, most if not all of the total 25(OH)D is therefore 25(OH)D3. However, supplementation may be either of the two forms. In the United States, for instance, it is customary to prescribe the D2 form, while in Europe, most formulations contain the D3 form. This means that the D2/D3 ratio found in clinical practice differs per country. To that end, methods should either distinguish the two components and may sum their concentrations or, if their distinguishing is not possible or wanted, quantify their concentrations together in an equimolar manner. Furthermore, epi-25(OH)D is not part of total 25(OH)D and should therefore ideally not be included in the calculation of total 25(OH)D. Likewise, total 1,25(OH)2D includes isomers 1,25(OH)2D3 and 1,25(OH)2D2 and total 24,25(OH)2D includes isomers 24,25(OH)2D2 and 24,25(OH)2D3. The D2 and D3 isomers differ in their molecular weight. When using a method that does not distinguish between both components, conversion of molar concentrations to weight (e.g. ‘ng/mL’) is not justifiable as it cannot take the different molecular weights into account (25). Measurement results are therefore to be reported in ‘nmol/L’ in the case of 25(OH)D and 24,25(OH)2D or ‘pmol/L’ for 1,25(OH)2D.

Measurement preanalysis

For 25(OH)D, both automated assays and liquid chromatography–tandem mass spectrometry (LC-MS/MS) methods mostly do not require a specific sample tube as serum, EDTA or heparin plasma may all be used, but should be checked in the manual before use in case of automated assay and validated in case of LC-MS/MS (26). Samples have been proven very stable for days to months at different storage conditions (−80° to room temperature) and the effects of repeated freeze-thaw cycles are reported to be insignificant (27, 28, 29, 30). For the other two metabolites, 1,25(OH)2D and 24,25(OH)2D, preanalysis has been less extensively studied unfortunately (14).

Automated assays

Many laboratories rely on automated immunoassays or protein binding assays for the determination of total 25(OH)D. The number of tests they process requires a method that is easy in operation and fast. Unlike LC-MS/MS, the automated immunoassay platforms offer such. However, by choosing to focus on easy operation and high throughput, they sacrifice on accuracy. As discussed earlier, the method used should either distinguish 25(OH)D3 and 25(OH)D2 and may sum their concentrations or quantify their concentrations together in an equimolar manner. Automated immunoassays are, by virtue of their use of mostly polyclonal antibodies directed toward 25(OH)D with variable affinities that differ for 25(OH)D3 and 25(OH)D2, unable to truly quantify both components in an equimolar manner (31, 32, 33). The antibodies used show various cross-reactivity for 25(OH)D2 (34). This is especially problematic in countries where D2 is frequently described. While cross-reactivity with epi-25(OH)D is not observed in immunoassays, variable cross-reactivity is observed with other more hydroxylated vitamin D metabolites, such as 24,25(OH)D (35). Another pitfall of using an automated immunoassay platform for the measurement of 25(OH)D is their lack of accuracy in certain patient groups. Due to varying concentrations of VDBP, and the difficulty the automated assays experience removing vitamin D from its binding proteins, they have difficulty measuring accurately in pregnant women, women on oral contraceptives, patients admitted to the intensive care unit and patients with liver failure (36). Additionally, automated assays struggle with hemodialysis and osteoporotic patients (37, 38). Interestingly, while long-term stability of samples from similar patients measured with LC-MS/MS does not affect the results, a number of the automated immunoassays did show variation in results over time (29).

Similarly, for measurement of 1,25(OH)2D and the vitamin D metabolite ratio, automated immunoassays lack the ability to distinguish the two isomers (D3 and D2) and experience cross-reactivity with other metabolites, making these assays less reliable (39).

On the other side, sample work-up is often diminished completely and tubes, either serum or plasma, may be directly placed on the instrument and no further action is required.

Comparing the costs of running an automated assay and an LC-MS/MS method for vitamin D testing is not easily done as these costs rely on many conditions. In general, machinery and maintenance fee is relatively low in automated assays as the machines are often already in use in clinical laboratories and additional testing will not increase these costs substantially. On the other hand, automated assays need relatively expensive reagents that need to be bought from the manufacturers of the specific machines.

LC-MS/MS

During recent years, more labs have turned to LC-MS/MS for their measurement of vitamin D metabolites as the technique offers superior specificity compared to automated immunoassays. Importantly, LC-MS/MS does not suffer from cross-reactivity with most analogous vitamin D metabolites that differ little from the desired measurand. Differences in mass can lead to easy separation by mass spectrometry, or alternatively, these metabolites may be separated by liquid chromatography. Only epi-25(OH)D, with the same molecular mass as 25(OH)D and rather difficult to separate on the LC, is co-measured in many LC-MS/MS methods. Luckily, for most adults, concentrations of epi-25(OH)D are low compared to 25(OH)D and do not often significantly alter total 25(OH)D quantification (40). In infants, greater epi-25(OH)D concentrations may falsely increase total 25(OH)D results when using an LC-MS/MS method not able to separate the epimers (6, 41). The current mass spectrometer methods are sensitive enough to accurately quantify the lower concentrations of 24,25(OH)2D and 1,25(OH)2D. Although for 1,25(OH)2D measurements an immunoprecipitation may be helpful to remove interferences and increase sensitivity (15). Another advantage of LC-MS/MS measurements is the fact that this technique allows quantification of the multiple metabolites at once, thereby supporting studying the relationship between the different vitamin D metabolites, such as those expressed in the Vitamin D metabolite ratio. The technique, however, requires sufficiently trained technicians capable of developing, validating, and running the applications. Today, this may be the biggest hurdle to overcome for LC-MS/MS as a technique to be overall superior to the automated immunoassay, as the quality of the used LC-MS/MS methods seems to differ substantially among laboratories (42). Just as with the automated immunoassays, standardization is still lacking and may improve the overall quality. This should be feasible, as certified reference material is available for the aforementioned measurands (43). Much effort has been put into the assessment of the commutability and stability of these reference materials (44, 45). It again shows the superiority of LC-MS/MS compared to the automated assays. Currently, fully automated LC-MS/MS machines enter the market which are designed to measure 25(OH)D3 and 25(OH)D2 separately. This makes it easier for laboratories without the expertise to run LC-MS/MS methods to measure vitamin D. However, these machines are not yet capable of measuring other metabolites in the same run, and up till now studies do not show outstanding method comparisons (46). This might be a result of the compromises a fully automated LC-MS/MS machine has to make on accuracy for easy and fast operation.

Contrary to the automated assays, the costs of running an LC-MS/MS method for vitamin D testing are primarily made up of instrument, maintenance, and labor costs, as the machines are expensive and well-trained technicians are required yet chemicals are available at relatively low costs. The costs for personnel totally depend on the degree of automation of processing the samples before putting them on the LC-MS/MS machine.

Conclusion

25(OH)D remains the best indicator of vitamin D status, while only specific conditions may require measurement of the other described metabolites. Whether using an automated immuno- or protein binding assay platform or LC-MS/MS for measurement of vitamin D metabolites, be aware of the intended measurand and the inherent pitfalls of the technique. The automated assays are fast and easily operated but lack the accuracy to produce accurate total 25(OH)D results. LC-MS/MS has proven excellent at the determination of total 25(OH)D and other vitamin D metabolites and further standardization efforts will improve the overall quality of LC-MS/MS methods worldwide. While automated assays are still widely used and may be adequate for a largely healthy population not using D2, LC-MS/MS allows for vitamin D metabolite profiling, enabling us to study vitamin D metabolism in detail and aids us in more complex cases such as samples containing D2 or samples from specific patient groups (47, 48). For a concise summary of the current applicability, advantages, and disadvantages of the two techniques, see Table 1.

Table 1

Availability of reference material and reference measurement procedures (RMP) and advantages and disadvantages of immunoassays and LC-MS/MS.

25(OH)D 1,25(OH)2D 24,25(OH)2D
NIST standard(s) SRM 972a, SRM 2969, SRM 2970, SRM 2972a, SRM 2973 None SRM 972a, SRM 2972a, SRM 2973
RMP Yes (LC-MS/MS based) No Yes (LC-MS/MS based)
Advantages Disadvantages
Immunoassay Fast, easy operation, no cross-reactivity with epi-forms Reduced accuracy in certain patient groups, no distinction between D2 and D3 forms, variable cross-reactivity with other related metabolites
LC-MS/MS Specificity, possibility of metabolite profiling Complexity, difficulty to separate isomers

Declaration of interest

The authors declare there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Funding

This study did not receive any specific grant from any funding agency in the public, commercial or not-for-profit sector.

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    van der Vorm LN, Le Goff C, Peeters S, Makris K, Cavalier E, Heijboer AC. 25-OH vitamin D concentrations measured by LC-MS/MS are equivalent in serum and EDTA plasma. Steroids 2022 187 109096. (https://doi.org/10.1016/j.steroids.2022.109096)

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  • 27

    Wielders JPM, Wijnberg FA. Preanalytical stability of 25(OH)-vitamin D3 in human blood or serum at room temperature: solid as a rock. Clinical Chemistry 2009 55 15841585. (https://doi.org/10.1373/clinchem.2008.117366)

    • Search Google Scholar
    • Export Citation
  • 28

    Colak A, Toprak B, Dogan N, Ustuner F. Effect of sample type, centrifugation and storage conditions on vitamin D concentration. Biochemia Medica 2013 23 321325. (https://doi.org/10.11613/bm.2013.039)

    • Search Google Scholar
    • Export Citation
  • 29

    Cavalier E Long-term stability of 25-hydroxyvitamin D: importance of the analytical method and of the patient matrix. Clinical Chemistry and Laboratory Medicine 2021 59 e389e391. (https://doi.org/10.1515/cclm-2021-0382)

    • Search Google Scholar
    • Export Citation
  • 30

    Borai A, Khalil H, Alghamdi B, Alhamdi R, Ali N, Bahijri S, Ferns G. The pre-analytical stability of 25-hydroxyvitamin D: storage and mixing effects. Journal of Clinical Laboratory Analysis 2020 34 e23037. (https://doi.org/10.1002/jcla.23037)

    • Search Google Scholar
    • Export Citation
  • 31

    Wyness SP, Straseski JA. Performance characteristics of six automated 25-hydroxyvitamin D assays: mind your 3s and 2s. Clinical Biochemistry 2015 48 10891096. (https://doi.org/10.1016/j.clinbiochem.2015.08.005)

    • Search Google Scholar
    • Export Citation
  • 32

    Nguyen VTQ, Li X, Castellanos KJ, Fantuzzi G, Mazzone T, Braunschweig CA. The accuracy of vitamin D assays of circulating 25-hydroxyvitamin D values: influence of 25-hydroxylated ergocalciferol concentration. Journal of AOAC International 2014 97 10481055. (https://doi.org/10.5740/jaoacint.13-305)

    • Search Google Scholar
    • Export Citation
  • 33

    Wise SA, Camara JE, Burdette CQ, Hahm G, Nalin F, Kuszak AJ, Merkel J, Durazo-Arvizu RA, Williams EL & Popp C, et al.Interlaboratory comparison of 25-hydroxyvitamin D assays: vitamin D standardization program (VDSP) intercomparison study 2 – part 2 immunoassays – impact of 25 hydroxyvitamin D2 and 24R,25-dihydroxyvitamin D3 on assay performance. Analytical and Bioanalytical Chemistry 2022 414 351–366. (https://doi.org/10.1007/s00216-021-03577-0)

    • Search Google Scholar
    • Export Citation
  • 34

    Shu I, Pina-Oviedo S, Quiroga-Garza G, Meng QH, Wang P. Influence of vitamin D2 percentage on accuracy of 4 commercial total 25-hydroxyvitamin D assays. Clinical Chemistry 2013 59 12731275. (https://doi.org/10.1373/clinchem.2013.206128)

    • Search Google Scholar
    • Export Citation
  • 35

    Carter GD, Jones JC, Shannon J, Williams EL, Jones G, Kaufmann M, Sempos C. 25-Hydroxyvitamin D assays: potential interference from other circulating vitamin D metabolites. Journal of Steroid Biochemistry and Molecular Biology 2016 164 134138. (https://doi.org/10.1016/j.jsbmb.2015.12.018)

    • Search Google Scholar
    • Export Citation
  • 36

    Heijboer AC, Blankenstein MA, Kema IP, Buijs MM. Accuracy of 6 routine 25-hydroxyvitamin D assays: influence of vitamin D binding protein concentration. Clinical Chemistry 2012 58 543548. (https://doi.org/10.1373/clinchem.2011.176545)

    • Search Google Scholar
    • Export Citation
  • 37

    Depreter B, Heijboer AC, Langlois MR. Accuracy of three automated 25-hydroxyvitamin D assays in hemodialysis patients. Clinica Chimica Acta; International Journal of Clinical Chemistry 2013 415 255260. (https://doi.org/10.1016/j.cca.2012.10.056)

    • Search Google Scholar
    • Export Citation
  • 38

    Cavalier E, Lukas P, Bekaert AC, Peeters S, Le Goff C, Yayo E, Delanaye P, & Souberbielle JC. Analytical and clinical evaluation of the new Fujirebio Lumipulse®G non-competitive assay for 25(OH)-vitamin D and three immunoassays for 25(OH)D in healthy subjects, osteoporotic patients, third trimester pregnant women, healthy African subjects, hemodialyzed and intensive care patients. Clinical Chemistry and Laboratory Medicine: CCLM / FESCC 2016 54 13471355. (https://doi.org/10.1515/cclm-2015-0923)

    • Search Google Scholar
    • Export Citation
  • 39

    Hawkes CP, Schnellbacher S, Singh RJ, Levine MA. 25-Hydroxyvitamin D Can interfere with a common assay for 1,25-dihydroxyvitamin D in vitamin D intoxication. Journal of Clinical Endocrinology and Metabolism 2015 100 28832889. (https://doi.org/10.1210/jc.2015-2206)

    • Search Google Scholar
    • Export Citation
  • 40

    Wright MJP, Halsall DJ, Keevil BG. Removal of 3-epi-25-hydroxyvitamin D(3) interference by liquid chromatography-tandem mass spectrometry is not required for the measurement of 25-hydroxyvitamin D(3) in patients older than 2 years. Clinical Chemistry 2012 58 17191720. (https://doi.org/10.1373/clinchem.2012.191460)

    • Search Google Scholar
    • Export Citation
  • 41

    Stepman HCM, Vanderroost A, Stöckl D, Thienpont LM. Full-scan mass spectral evidence for 3-epi-25-hydroxyvitamin D in serum of infants and adults. Clinical Chemistry and Laboratory Medicine 2011 49 253256. (https://doi.org/10.1515/CCLM.2011.050)

    • Search Google Scholar
    • Export Citation
  • 42

    Dirks NF, Ackermans MT, Martens F, Cobbaert CM, Jonge de R, Heijboer AC. We need to talk about the analytical performance of our laboratory developed clinical LC-MS/MS tests, and start separating the wheat from the chaff. Clinica Chimica Acta; International Journal of Clinical Chemistry 2021 514 8083. (https://doi.org/10.1016/j.cca.2020.12.020)

    • Search Google Scholar
    • Export Citation
  • 43

    Phinney KW, Tai SS, Bedner M, Camara JE, Chia RRC, Sander LC, Sharpless KE, Wise SA, Yen JH & Schleicher RL et al.Development of an improved Standard reference material for vitamin D metabolites in human serum. Analytical Chemistry 2017 89 49074913. (https://doi.org/10.1021/acs.analchem.6b05168)

    • Search Google Scholar
    • Export Citation
  • 44

    Camara JE, Wise SA, Hoofnagle AN, Williams EL, Carter GD, Jones J, Burdette CQ, Hahm G, Nalin F & Kuszak AJ et al.Assessment of serum total 25-hydroxyvitamin D assay commutability of Standard Reference Materials and College of American Pathologists Accuracy-Based Vitamin D (ABVD) Scheme and vitamin D External Quality Assessment Scheme (DEQAS) materials: Vitamin D Standardization Program (VDSP) Commutability Study 2. Analytical and Bioanalytical Chemistry 2021 413 50675084. (https://doi.org/10.1007/s00216-021-03470-w)

    • Search Google Scholar
    • Export Citation
  • 45

    Sempos CT, Williams EL, Carter GD, Jones J, Camara JE, Burdette CQ, Hahm G, Nalin F, Duewer DL & Kuszak AJ et al.Assessment of serum total 25-hydroxyvitamin D assays for vitamin D External Quality Assessment Scheme (DEQAS) materials distributed at ambient and frozen conditions. Analytical and Bioanalytical Chemistry 2022 414 10151028. (https://doi.org/10.1007/s00216-021-03742-5)

    • Search Google Scholar
    • Export Citation
  • 46

    Benton SC, Tetteh GK, Needham SJ, Mücke J, Sheppard L, Alderson S, Ruppen C, Curti M, Redondo M, Milan AM. Evaluation of the 25-hydroxy vitamin D assay on a fully automated liquid chromatography mass spectrometry system, the Thermo Scientific Cascadion SM Clinical Analyzer with the Cascadion 25-hydroxy vitamin D assay in a routine clinical laboratory. Clinical Chemistry and Laboratory Medicine 2020 58 10101017. (https://doi.org/10.1515/cclm-2019-0834)

    • Search Google Scholar
    • Export Citation
  • 47

    Jones G, Kaufmann M. Diagnostic aspects of vitamin D: clinical utility of vitamin D metabolite profiling. JBMR Plus 2021 5 e10581. (https://doi.org/10.1002/jbm4.10581)

    • Search Google Scholar
    • Export Citation
  • 48

    Kaufmann M, Schlingmann KP, Berezin L, Molin A, Sheftel J, Vig M, Gallagher JC, Nagata A, Masoud SS & Sakamoto R et al. Differential diagnosis of vitamin D-related hypercalcemia using serum vitamin D metabolite profiling. Journal of Bone and Mineral Research 2021 36 13401350. (https://doi.org/10.1002/jbmr.4306)

    • Search Google Scholar
    • Export Citation

 

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    Figure 1

    Endogenous vitamin D3 metabolism. When our skin is penetrated by UVB light, 7-dehydrocholesterol is converted to pre-vitamin D. This rapidly isomerizes into vitamin D3, which then enters circulation and binds to vitamin D binding protein (VDBP). Liver enzyme 25α-hydroxylase then hydroxylates vitamin D3, which gains 25(OH)D3. 25(OH)D3 can be converted into the active hormone, 1,25(OH)2D3 by 1α-hydroxylase. Alternatively, it may be epimerized to epi-25(OH)D3 or to 24,25(OH)2D3, inactive metabolites with no or substantially lower affinity for 1α-hydroxylase.

  • 1

    Amrein K, Scherkl M, Hoffmann M, Neuwersch-Sommeregger S, Köstenberger M, Tmava Berisha A, Martucci G, Pilz S, Malle O. Vitamin D deficiency 2.0: an update on the current status worldwide. European Journal of Clinical Nutrition 2020 74 14981513. (https://doi.org/10.1038/s41430-020-0558-y)

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  • 2

    Christakos S, Li S, De La Cruz J, Bikle DD. New developments in our understanding of vitamin metabolism, action and treatment. Metabolism: Clinical and Experimental 2019 98 112120. (https://doi.org/10.1016/j.metabol.2019.06.010)

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  • 3

    Zhu JG, Ochalek JT, Kaufmann M, Jones G, & Deluca HF. CYP2R1 is a major, but not exclusive, contributor to 25-hydroxyvitamin D production in vivo. PNAS 2013 110 1565015655. (https://doi.org/10.1073/pnas.1315006110)

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    Shi M, Grabner A, Wolf M. Importance of extra-renal CYP24A1 expression for maintaining mineral homeostasis. Journal of the Endocrine Society 2021 5 A234A234. (https://doi.org/10.1210/jendso/bvab048.476)

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    Lensmeyer G, Poquette M, Wiebe D, Binkley N. The C-3 epimer of 25-hydroxyvitamin D(3) is present in adult serum. Journal of Clinical Endocrinology and Metabolism 2012 97 163168. (https://doi.org/10.1210/jc.2011-0584)

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  • 6

    van den Ouweland JMW, Beijers AM, & van Daal H. Overestimation of 25-hydroxyvitamin D3 by increased ionization efficiency of 3-epi-25-hydroxyvitamin D3. In LC-MS/MS methods not separating both metabolites as determined by an LC-MS/MS method for separate quantification of 25-hydroxyvitamin D3, 3-epi-25-hydroxyvitamin D3 and 25-hydroxyvitamin D2 in human serum. Journal of Chromatography. Part B 2014 967 195202. (https://doi.org/10.1016/j.jchromb.2014.07.021)

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    Singh RJ, Taylor RL, Reddy GS, Grebe SKG. C-3 epimers can account for a significant proportion of total circulating 25-hydroxyvitamin D in infants, complicating accurate measurement and interpretation of vitamin D status. Journal of Clinical Endocrinology and Metabolism 2006 91 30553061. (https://doi.org/10.1210/jc.2006-0710)

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    Bailey D, Veljkovic K, Yazdanpanah M, Adeli K. Analytical measurement and clinical relevance of vitamin D(3) C3-epimer. Clinical Biochemistry 2013 46 190196. (https://doi.org/10.1016/j.clinbiochem.2012.10.037)

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    Alonso N, Zelzer S, Eibinger G, Herrmann M. Vitamin D metabolites: analytical challenges and clinical relevance. Calcified Tissue International 2023 112 158177. (https://doi.org/10.1007/s00223-022-00961-5)

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    Dirks NF, Ackermans MT, Lips P, Jongh de RT, Vervloet MG, Jonge de R, Heijboer AC. The when, what & how of measuring vitamin D metabolism in clinical medicine. Nutrients 2018 10 482. (https://doi.org/10.3390/nu10040482)

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  • 11

    Holick MF Vitamin D status: measurement, interpretation, and clinical application. Annals of Epidemiology 2009 19 7378. (https://doi.org/10.1016/j.annepidem.2007.12.001)

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    Holick MF, Binkley NC, Bischoff-Ferrari HA, Gordon CM, Hanley DA, Heaney RP, Murad MH, Weaver CM & Endocrine Society. Evaluation, treatment, and prevention of vitamin D deficiency: an Endocrine Society clinical practice guideline. Journal of Clinical Endocrinology and Metabolism 2011 96 19111930. (https://doi.org/10.1210/jc.2011-0385)

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    Ross AC. The 2011 report on dietary reference intakes for calcium and vitamin D. Public Health Nutrition 2011 14 938939. (https://doi.org/10.1017/S1368980011000565)

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  • 14

    Makris K, Bhattoa HP, Cavalier E, Phinney K, Sempos CT, Ulmer CZ, Vasikaran SD, Vesper H, Heijboer AC. Recommendations on the measurement and the clinical use of vitamin D metabolites and vitamin D binding protein - A position paper from the IFCC Committee on bone metabolism. Clinica Chimica Acta; International Journal of Clinical Chemistry 2021 517 171197. (https://doi.org/10.1016/j.cca.2021.03.002)

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    Dirks NF, Martens F, Vanderschueren D, Billen J, Pauwels S, Ackermans MT, Endert E, Heijer den MD, Blankenstein MA, Heijboer AC. Determination of human reference values for serum total 1,25-dihydroxyvitamin D using an extensively validated 2D ID-UPLC-MS/MS method. Journal of Steroid Biochemistry and Molecular Biology 2016 164 127133. (https://doi.org/10.1016/j.jsbmb.2015.12.003)

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  • 16

    Ginsberg C, Hoofnagle AN, Katz R, Becker JO, Kritchevsky SB, Shlipak MG, Sarnak MJ, Ix JH. The vitamin D metabolite ratio is independent of vitamin D binding protein concentration. Clinical Chemistry 2021 67 385393. (https://doi.org/10.1093/clinchem/hvaa238)

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  • 17

    Francic V, Ursem SR, Dirks NF, Keppel MH, Theiler-Schwetz V, Trummer C, Pandis M, Borzan V, Grübler MR & Verheyen ND et al.The effect of vitamin D supplementation on its metabolism and the vitamin D metabolite ratio. Nutrients 2019 11 2539. (https://doi.org/10.3390/nu11102539)

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  • 18

    Kaufmann M, Morse N, Molloy BJ, Cooper DP, Schlingmann KP, Molin A, Kottler ML, Gallagher JC, Armas L, Jones G. Improved screening test for idiopathic infantile hypercalcemia confirms residual levels of serum 24,25-(OH)2 D3 in Affected Patients. In Journal of Bone and Mineral Research 2017 32 15891596. (https://doi.org/10.1002/jbmr.3135)

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  • 19

    Cavalier E, Huyghebaert L, Rousselle O, Bekaert AC, Kovacs S, Vranken L, Peeters S, Le Goff C, Ladang A. Simultaneous measurement of 25(OH)-vitamin D and 24,25(OH)2-vitamin D to define cut-offs for CYP24A1 mutation and vitamin D deficiency in a population of 1200 young subjects. Clinical Chemistry and Laboratory Medicine 2020 58 197201. (https://doi.org/10.1515/cclm-2019-0996)

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  • 20

    Schlingmann KP, Kaufmann M, Weber S, Irwin A, Goos C, John U, Misselwitz J, Klaus G, Kuwertz-Bröking E & Fehrenbach H et al. Mutations in CYP24A1 and idiopathic infantile hypercalcemia. New England Journal of Medicine 2011 365 410421. (https://doi.org/10.1056/NEJMoa1103864)

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  • 21

    Bikle DD, Gee E, Halloran B, Kowalski MA, Ryzen E, Haddad JG. Assessment of the free fraction of 25-hydroxyvitamin D in serum and its regulation by albumin and the vitamin D-binding protein. Journal of Clinical Endocrinology and Metabolism 1986 63 954959. (https://doi.org/10.1210/jcem-63-4-954)

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  • 22

    Chun RF, Peercy BE, Orwoll ES, Nielson CM, Adams JS, Hewison M. Vitamin D and DBP: the free hormone hypothesis revisited. Journal of Steroid Biochemistry and Molecular Biology 2014 144 132137. (https://doi.org/10.1016/j.jsbmb.2013.09.012)

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  • 23

    Nielson CM, Jones KS, Chun RF, Jacobs JM, Wang Y, Hewison M, Adams JS, Swanson CM, Lee CG & Vanderschueren D et al. Free 25-hydroxyvitamin D: impact of vitamin D binding protein assays on racial-genotypic associations. Journal of Clinical Endocrinology and Metabolism 2016 101 22262234. (https://doi.org/10.1210/jc.2016-1104)

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  • 25

    Thienpont LM, Stepman HCM, Vesper HW. Standardization of measurements of 25-hydroxyvitamin D3 and D2. Scandinavian Journal of Clinical and Laboratory Investigation. Supplementum 2012 243 4149. (https://doi.org/10.3109/00365513.2012.681950)

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    • Export Citation
  • 26

    van der Vorm LN, Le Goff C, Peeters S, Makris K, Cavalier E, Heijboer AC. 25-OH vitamin D concentrations measured by LC-MS/MS are equivalent in serum and EDTA plasma. Steroids 2022 187 109096. (https://doi.org/10.1016/j.steroids.2022.109096)

    • Search Google Scholar
    • Export Citation
  • 27

    Wielders JPM, Wijnberg FA. Preanalytical stability of 25(OH)-vitamin D3 in human blood or serum at room temperature: solid as a rock. Clinical Chemistry 2009 55 15841585. (https://doi.org/10.1373/clinchem.2008.117366)

    • Search Google Scholar
    • Export Citation
  • 28

    Colak A, Toprak B, Dogan N, Ustuner F. Effect of sample type, centrifugation and storage conditions on vitamin D concentration. Biochemia Medica 2013 23 321325. (https://doi.org/10.11613/bm.2013.039)

    • Search Google Scholar
    • Export Citation
  • 29

    Cavalier E Long-term stability of 25-hydroxyvitamin D: importance of the analytical method and of the patient matrix. Clinical Chemistry and Laboratory Medicine 2021 59 e389e391. (https://doi.org/10.1515/cclm-2021-0382)

    • Search Google Scholar
    • Export Citation
  • 30

    Borai A, Khalil H, Alghamdi B, Alhamdi R, Ali N, Bahijri S, Ferns G. The pre-analytical stability of 25-hydroxyvitamin D: storage and mixing effects. Journal of Clinical Laboratory Analysis 2020 34 e23037. (https://doi.org/10.1002/jcla.23037)

    • Search Google Scholar
    • Export Citation
  • 31

    Wyness SP, Straseski JA. Performance characteristics of six automated 25-hydroxyvitamin D assays: mind your 3s and 2s. Clinical Biochemistry 2015 48 10891096. (https://doi.org/10.1016/j.clinbiochem.2015.08.005)

    • Search Google Scholar
    • Export Citation
  • 32

    Nguyen VTQ, Li X, Castellanos KJ, Fantuzzi G, Mazzone T, Braunschweig CA. The accuracy of vitamin D assays of circulating 25-hydroxyvitamin D values: influence of 25-hydroxylated ergocalciferol concentration. Journal of AOAC International 2014 97 10481055. (https://doi.org/10.5740/jaoacint.13-305)

    • Search Google Scholar
    • Export Citation
  • 33

    Wise SA, Camara JE, Burdette CQ, Hahm G, Nalin F, Kuszak AJ, Merkel J, Durazo-Arvizu RA, Williams EL & Popp C, et al.Interlaboratory comparison of 25-hydroxyvitamin D assays: vitamin D standardization program (VDSP) intercomparison study 2 – part 2 immunoassays – impact of 25 hydroxyvitamin D2 and 24R,25-dihydroxyvitamin D3 on assay performance. Analytical and Bioanalytical Chemistry 2022 414 351–366. (https://doi.org/10.1007/s00216-021-03577-0)

    • Search Google Scholar
    • Export Citation
  • 34

    Shu I, Pina-Oviedo S, Quiroga-Garza G, Meng QH, Wang P. Influence of vitamin D2 percentage on accuracy of 4 commercial total 25-hydroxyvitamin D assays. Clinical Chemistry 2013 59 12731275. (https://doi.org/10.1373/clinchem.2013.206128)

    • Search Google Scholar
    • Export Citation
  • 35

    Carter GD, Jones JC, Shannon J, Williams EL, Jones G, Kaufmann M, Sempos C. 25-Hydroxyvitamin D assays: potential interference from other circulating vitamin D metabolites. Journal of Steroid Biochemistry and Molecular Biology 2016 164 134138. (https://doi.org/10.1016/j.jsbmb.2015.12.018)

    • Search Google Scholar
    • Export Citation
  • 36

    Heijboer AC, Blankenstein MA, Kema IP, Buijs MM. Accuracy of 6 routine 25-hydroxyvitamin D assays: influence of vitamin D binding protein concentration. Clinical Chemistry 2012 58 543548. (https://doi.org/10.1373/clinchem.2011.176545)

    • Search Google Scholar
    • Export Citation
  • 37

    Depreter B, Heijboer AC, Langlois MR. Accuracy of three automated 25-hydroxyvitamin D assays in hemodialysis patients. Clinica Chimica Acta; International Journal of Clinical Chemistry 2013 415 255260. (https://doi.org/10.1016/j.cca.2012.10.056)

    • Search Google Scholar
    • Export Citation
  • 38

    Cavalier E, Lukas P, Bekaert AC, Peeters S, Le Goff C, Yayo E, Delanaye P, & Souberbielle JC. Analytical and clinical evaluation of the new Fujirebio Lumipulse®G non-competitive assay for 25(OH)-vitamin D and three immunoassays for 25(OH)D in healthy subjects, osteoporotic patients, third trimester pregnant women, healthy African subjects, hemodialyzed and intensive care patients. Clinical Chemistry and Laboratory Medicine: CCLM / FESCC 2016 54 13471355. (https://doi.org/10.1515/cclm-2015-0923)

    • Search Google Scholar
    • Export Citation
  • 39

    Hawkes CP, Schnellbacher S, Singh RJ, Levine MA. 25-Hydroxyvitamin D Can interfere with a common assay for 1,25-dihydroxyvitamin D in vitamin D intoxication. Journal of Clinical Endocrinology and Metabolism 2015 100 28832889. (https://doi.org/10.1210/jc.2015-2206)

    • Search Google Scholar
    • Export Citation
  • 40

    Wright MJP, Halsall DJ, Keevil BG. Removal of 3-epi-25-hydroxyvitamin D(3) interference by liquid chromatography-tandem mass spectrometry is not required for the measurement of 25-hydroxyvitamin D(3) in patients older than 2 years. Clinical Chemistry 2012 58 17191720. (https://doi.org/10.1373/clinchem.2012.191460)

    • Search Google Scholar
    • Export Citation
  • 41

    Stepman HCM, Vanderroost A, Stöckl D, Thienpont LM. Full-scan mass spectral evidence for 3-epi-25-hydroxyvitamin D in serum of infants and adults. Clinical Chemistry and Laboratory Medicine 2011 49 253256. (https://doi.org/10.1515/CCLM.2011.050)

    • Search Google Scholar
    • Export Citation
  • 42

    Dirks NF, Ackermans MT, Martens F, Cobbaert CM, Jonge de R, Heijboer AC. We need to talk about the analytical performance of our laboratory developed clinical LC-MS/MS tests, and start separating the wheat from the chaff. Clinica Chimica Acta; International Journal of Clinical Chemistry 2021 514 8083. (https://doi.org/10.1016/j.cca.2020.12.020)

    • Search Google Scholar
    • Export Citation
  • 43

    Phinney KW, Tai SS, Bedner M, Camara JE, Chia RRC, Sander LC, Sharpless KE, Wise SA, Yen JH & Schleicher RL et al.Development of an improved Standard reference material for vitamin D metabolites in human serum. Analytical Chemistry 2017 89 49074913. (https://doi.org/10.1021/acs.analchem.6b05168)

    • Search Google Scholar
    • Export Citation
  • 44

    Camara JE, Wise SA, Hoofnagle AN, Williams EL, Carter GD, Jones J, Burdette CQ, Hahm G, Nalin F & Kuszak AJ et al.Assessment of serum total 25-hydroxyvitamin D assay commutability of Standard Reference Materials and College of American Pathologists Accuracy-Based Vitamin D (ABVD) Scheme and vitamin D External Quality Assessment Scheme (DEQAS) materials: Vitamin D Standardization Program (VDSP) Commutability Study 2. Analytical and Bioanalytical Chemistry 2021 413 50675084. (https://doi.org/10.1007/s00216-021-03470-w)

    • Search Google Scholar
    • Export Citation
  • 45

    Sempos CT, Williams EL, Carter GD, Jones J, Camara JE, Burdette CQ, Hahm G, Nalin F, Duewer DL & Kuszak AJ et al.Assessment of serum total 25-hydroxyvitamin D assays for vitamin D External Quality Assessment Scheme (DEQAS) materials distributed at ambient and frozen conditions. Analytical and Bioanalytical Chemistry 2022 414 10151028. (https://doi.org/10.1007/s00216-021-03742-5)

    • Search Google Scholar
    • Export Citation
  • 46

    Benton SC, Tetteh GK, Needham SJ, Mücke J, Sheppard L, Alderson S, Ruppen C, Curti M, Redondo M, Milan AM. Evaluation of the 25-hydroxy vitamin D assay on a fully automated liquid chromatography mass spectrometry system, the Thermo Scientific Cascadion SM Clinical Analyzer with the Cascadion 25-hydroxy vitamin D assay in a routine clinical laboratory. Clinical Chemistry and Laboratory Medicine 2020 58 10101017. (https://doi.org/10.1515/cclm-2019-0834)

    • Search Google Scholar
    • Export Citation
  • 47

    Jones G, Kaufmann M. Diagnostic aspects of vitamin D: clinical utility of vitamin D metabolite profiling. JBMR Plus 2021 5 e10581. (https://doi.org/10.1002/jbm4.10581)

    • Search Google Scholar
    • Export Citation
  • 48

    Kaufmann M, Schlingmann KP, Berezin L, Molin A, Sheftel J, Vig M, Gallagher JC, Nagata A, Masoud SS & Sakamoto R et al. Differential diagnosis of vitamin D-related hypercalcemia using serum vitamin D metabolite profiling. Journal of Bone and Mineral Research 2021 36 13401350. (https://doi.org/10.1002/jbmr.4306)

    • Search Google Scholar
    • Export Citation