Mass Spectrometry Assays of Vitamin D Metabolites

C H A P T E R

50 Mass Spectrometry Assays of Vitamin D Metabolites Martin Kaufmann1, Lusia Sepiashvili2, Ravinder J. Singh2 1Queen’s

University, Kingston, ON, Canada; 2Mayo Clinic, Rochester, MN, United States

O U T L I N E Sample Separation Mass Spectrometry

Introduction909 Approaches and Utility of Vitamin D Metabolite Measurements in the Clinical Laboratory Setting Overview of Methodology for Analysis of Circulating Vitamin D Metabolites by LC-MS/MS Sample Extraction Sample Derivatization

910 912 913 913

INTRODUCTION Vitamin D is acquired from the diet or from photolysis of 7-dehydrocholesterol in the skin [1–5]. Total vitamin D is the sum of vitamins D2 and D3 which are derived from plant and animal sources, respectively. Vitamin D is metabolized to 25-hydroxyvitamin D (25(OH)D) by CYP2R1 and CYP27A1 in the liver [6–10], as described in detail in Chapter 8. The metabolite 25(OH)D is the most abundant circulating form of vitamin D, and measurement of this metabolite is the accepted index of vitamin D nutritional status [11–14]. The physiological function of vitamin D is carried out by the active form, 1,25-dihydroxyvitamin D (1,25 (OH)2D), formed from 25(OH)D in the proximal tubule of the kidney by CYP27B1 [15–18]. 1,25(OH)2D acts via a vitamin D receptor (VDR)-mediated genomic mechanism in the maintenance of calcium and phosphate homeostasis as well as broad range of extraskeletal functions [19,20] (see Chapter 9). CYP27B1 is therefore tightly regulated by calcium/phosphate status via parathyroid hormone and fibroblast growth factor 23; as well as a negative feedback loop where 1,25 (OH)2D3 itself acts to suppress CYP27B1 [21,22]

Vitamin D, Volume 1: Biochemistry, Physiology and Diagnostics, Fourth Edition http://dx.doi.org/10.1016/B978-0-12-809965-0.00050-1

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Application of LC-MS/MS to the Study of Vitamin D Metabolism in Animal Models

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Future Directions

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(see Chapter 7) and induce its own catabolism by CYP24A1 via a five-step process commencing with C24-hydroxylation to 1,24,25-trihydroxyvitamin D (1,24,25(OH)3D3) and culminating with calcitroic acid [23] which is excreted in the bile (Chapter 5). The metabolite 25(OH)D is also catabolized by CYP24A1 via 24,25-dihydroxyvitamin D (24,25(OH)2D) which can be detected in circulation. The identification of extraskeletal functions of vitamin D [20], as well as extrarenal production of 1,25(OH)2D [24–26], has emphasized the importance of adequate vitamin D intakes and has spawned an increase in demand in number, as well as quality of clinical 25(OH) D measurements. Furthermore, the complexity of vitamin D metabolism described previously has driven the continuous development of sophisticated methodologies to interrogate associated biochemical processes in the clinical research setting by measurement not only of 25(OH)D, but lower abundance metabolites including 24,25(OH)2D and 1,25(OH)2D as well. Liquid chromatography tandem mass spectrometry (LCMS/MS) has emerged as the gold standard technique with the capability for reliable, accurate, and high-throughput quantification of various circulating vitamin D metabolites. The first

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50.  MASS SPECTROMETRY ASSAYS OF VITAMIN D METABOLITES

aim of this chapter is to discuss in detail the variations of this approach, providing considerations in assay development and methods for improving assay performance. The second, and equally important aim, is to describe the applications of this technology for the investigation of animal models and human disease. Assay utilization in research and clinical laboratory settings will also be described.

APPROACHES AND UTILITY OF VITAMIN D METABOLITE MEASUREMENTS IN THE CLINICAL LABORATORY SETTING Historically, various methods have been used for measurement of vitamin D metabolites, including radioimmunoassay (RIA), vitamin D-binding protein (DBP) assays, high performance liquid chromatography coupled with ultra-violet spectrophotometry (HPLC-UV), and LC-MS/MS methods [27]. It is desirable that antibodies used in immunoassays have equal cross-reactivity to both 25(OH)D2 and 25(OH)D3. However in practice, most of the immunoassays have been reported to underestimate 25(OH)D2 because of either lack of crossreactivity or incomplete release from the DBP [28–30]. The recoveries are also inconsistent resulting in higher coefficient of variations (CVs) for the tests [31]. LC-MS/MS methods published by the National Institute of Standards and Technology (NIST) and Centers for Disease Control and Prevention are considered candidate reference methods for the quantification of circulating 25(OH)D and 3-epi-25(OH)D, but it is impractical for every laboratory to perform the reference method. NIST has also developed quality control materials (human serum, standard reference material SRM 972) that contain the metabolite 3-epi-25(OH)D at four different concentrations as characterized by LC-MS/MS [32,33] (Fig. 50.1). Although

the biological significance of the 3-epi-25(OH)D remains to be elucidated, the preparation of this SRM is important for assays for which the cross-reactivity with these metabolites is not well defined [34,35], as well as for quantitatively assessing resolution of 3-epi-25(OH)D from 25(OH)D in LC-MS/ MS–based assays. NIST has also recently prepared a reference material for 24R,25-dihydroxyvitamin D3 using the reference method (reference values: level 1 at 2.66 ± 0.10 ng/mL; level 2 at 1.4 ± 0.05 ng/mL; level 3 at 1.62 ± 0.06 ng/mL; level 4 at 2.64 ± 0.09 ng/mL) [36]. Clinical laboratories should be aware of the advantages and disadvantages of various methods. We at Mayo Clinic have been performing 25(OH)D testing since 1981. In the beginning, we used to perform 25-hydroxyvitamin D testing by HPLC-UV methodology when our testing volume used to be low, for example, 50 samples/day. HPLC method is accurate and specific compared to immunoassays but is laborious and has lower throughput. As the demand was increasing in 2000, our laboratories switched to RIA and were able to perform more than 150 samples/day. Later in 2004, we implemented LC-MS/MS into the Endocrine Lab and were able to perform 3000–3500 samples a day to determine the level of 25-hydroxyvitamin D in the blood [37]. The amount of specimen required has also decreased significantly over time. The method comparison analysis of LC-MS/MS assay with the automated immunoassays proved that immunoassays not only lack accuracy but also had inconsistent recovery of 25(OH)D from binding proteins. Clinical laboratories monitor the quality of 25(OH) D testing by participating in proficiency testing offered by the College of American Pathologists (CAP) and proficiency testing offered by Vitamin D External Quality Assessment Scheme (DEQAS). These proficiency testing schemes are precision based and compare the performance of a given assay in an individual laboratory with method- or platform-specific

FIGURE 50.1  LC-MS/MS multiple reaction monitoring chromatograms for level 4 of SRM 972. Spectra for 25(OH)D3 and 3-epi-25(OH)D3 (A) and

25(OH)D2 (B) are shown. Further experimental details are provided in the article [33]. LC-MS/MS, liquid chromatography tandem mass spectrometry; SRM, standard reference material. Reprinted courtesy of the National Institute of Standards and Technology, U.S. Department of Commerce. Not copyrightable in the United States.

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Approaches and Utility of Vitamin D Metabolite Measurements in the Clinical Laboratory Setting

FIGURE 50.2  Relative percent difference among various clinical 25(OH)D assays available in the market in 2013. Similar biases among various methods have been published numerous times and demand for standardization of 25(OH)D assays for improved patient care. LC-MS/MS, liquid chromatography tandem mass spectrometry. EIA, enzyme immunoassay; HPLC, high performance liquid chromatography; IDS, immunodiagnostic systems; RIA, radioimmunoasay.

peer group means at different analyte concentrations. Under optimal conditions, patient 25(OH)D results from LC-MS/ MS, immunoassays, and receptor-binding method should not differ from each other. But unfortunately, the observed differences are large and can impact individual patient care especially those being treated for vitamin D deficiency with supplementation. Fig. 50.2 highlights the differences that can be observed in various methods against the mean of all the methods. Although there is excellent agreement between LC-MS/MS and the all-method mean—reemphasizing the criterion-standard nature of LC-MS/MS—the results of individual immunoassays can easily deviate by 20% or more from those of LC-MS/MS. In particular, some immunoassays that are reported to have inconsistent recoveries of 25(OH)D thus may show large differences from LC-MS/MS and immunoassay methods with good recovery for patients taking vitamin therapy. The discordance between 25(OH)D values may be due to differences in standardization of each assay relative to HPLC. Therefore it is recommended that serial testing be performed with the same assay whenever possible and that LC-MS/MS be used if vitamin D2 is being given. If vitamin D2 is used as form of supplementation for treatment of deficiency, it is imperative to use an assay for which the total 25(OH)D result will be reflective of both vitamin D2 and D3. In some countries, including the United States, vitamin D2 is the only form of supplementation, whereas others prescribe vitamin D3 [38]. In one data set from the United States, vitamin D2 was measurable in 23% of specimens (n = 7614) highlighting the frequent necessity to detect this analyte in this population [39]. An advantage of LC-MS/MS-based measurements of total 25(OH)D is that 25(OH)D3 and 25(OH)2D2 are measured individually, which enables assessment of the proportion of total 25(OH)D arising from supplementation with vitamin D2 (Fig. 50.1). 25(OH)D3 concentrations seem to be stable at room temperature and under the common preanalytical conditions experienced in medical laboratories. There appears to be no

TABLE 50.1 Reference Intervals for Vitamin D Metabolitesa Analyte

Reference Interval

Units

25(OH)Db

<10 (severe deficiency)

ng/mL

10–19 (mild–mod deficiency) 20–50 (optimum levels) 51–80 (risk of hypercalciuria) >80 (toxicity possible) 1,25(OH)2D

Children: <16 years: 24–86

pg/mL

Males: ≥16 years: 18–64 Females: ≥16 years: 18–78 24,25(OH)2D

1–4

ng/mL

25(OH)D/24,25(OH)2D

5–35

Ratio

25(OH)D, 25-hydroxyvitamin D. aIn all cases, D and D metabolites can be quantified separately. 2 3 bIn patients younger than 1 year, 3-epi-25(OH)D is subtracted.

need for serum to be frozen for transport, and whole blood might even be the specimen of choice for transport for up to 3 days. Storage conditions of serum at 4°C for at least 7 days and up to four freeze-thawing cycles are permissible. To monitor an index of nutritional vitamin D, status measurement of 25(OH)D is sufficient (reference interval shown in Table 50.1). It has been established that testing for additional vitamin D metabolites is of no added value for confirming vitamin D deficiency. A number of studies have shown that vitamin D deficiency is very prevalent in developed and developing countries. This has resulted in high demand of 25(OH)D testing, and the volume of samples coming to the hospital laboratories has increased exponentially. The laboratories were not prepared for this situation, and only one manual RIA assay was available. To meet the demand, tens of new methods have been developed

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50.  MASS SPECTROMETRY ASSAYS OF VITAMIN D METABOLITES

to measure 25(OH)D for human serum or plasma. These detection methods are based on competitive immunoassays, chromatography using either UV or mass spectrometry principals. Advantages and disadvantages of various methodologies have been studied very well and published. Because the therapeutic levels and toxicity window of 25(OH)D vitamin D are very wide, most of these assays do reasonably well at a population basis. For individual patient care, an accurate and specific method is critical for optimal patient outcome. Because of their accuracy and specificity, LC-MS/MS methods are considered the gold standard methods. Yet, the vast majority of laboratories perform 25(OH)D analyses using immunoassays because of minimal requirement for preanalytic specimen workup, complete automation, and historic familiarity [40]. LC-MS/MS is becoming the technique of choice for various reference laboratories. Laboratories that use in-house LC-MS/ MS have responsibility for many steps of the assay. The LC-MS/ MS technology for testing of human samples is not approved by the FDA, and manufacturers of LC-MS/MS instrumentation are not responsible for troubleshooting the assays. Laboratories performing 25(OH)D testing by LC-MS/MS technology have differences in their standard operating procedures, and thus interlaboratory CVs are in the range of 20%. The preparation of the reagents required for in-house LC-MS/MS assays is conducted by individual laboratories under their institutionally regulated standard procedures. The complexity of the LC-MS/ MS technology in its present form demands a robust, fully automated platform that can meet the need for throughput, precision, and accurate testing of vitamin D and metabolites. Multiplexed immunoassays may have the potential of achieving accuracy and precision for multiple vitamin D metabolites. For better patient care, the goal should be to have not only an accurate 25(OH)D value but also precision for 25(OH)D testing, with a CV <1%. Challenges with 1,25(OH)2D testing in clinical laboratories will not be discussed in detail here. The analytical issues are further magnified because the concentration of 1,25(OH)2D is 1000-fold lower (pg/mL or pmol/L) than 25(OH) D (ng/mL or nmol/L) (Table 50.1) [41]. Recently, there has been renewed interest in measuring 24,25(OH)2D owing to the discovery of inactivating mutations to CYP24A1 as a cause of idiopathic infantile hypercalcemia (IIH) [42–52]. A number of groups have now concluded that patients with biallelic (homozygous or compound heterozygous) inactivating mutations in CYP24A1 have inappropriately low 24,25(OH)2D as a result of reduced 24-hydroxylase activity. In normal individuals, there is a strong correlation between 25(OH)D3 and 24,25(OH)2D [53,54]; 24,25(OH)2D typically circulates in the 1–4 ng/mL range, at approximately 5%–25% of 25(OH)D concentration thus the 25(OH)D3:24,25(OH)2D ratio remains within a relatively narrow range of approximately 5–35 (Table 50.1). Conversely, patients with IIH-CYP24A1 exhibit a strikingly elevated ratio of >80–100. Thus determination of 25(OH)D3:24,25(OH)2D ratio based on simultaneous measurement of these two metabolites by LC-MS/MS serves as an important cost-effective tool to rationalize expensive genetic testing to confirm the presence of CYP24A1 mutations. Vitamin D-deficient subjects [53] as well as patients

TABLE 50.2  Various Serum Biochemical Profiles in the Workup of Hypercalcemia Cause of Hypercalcemia

Calcium

Hypervitaminosis D associated

↑

PTH associated

↑

Granulomatous disease/lymphoma

1,25(OH)2D PTH

25(OH)D/ 24,25(OH)2D

↓

N

↑

↑

N

↑

↑

↓

N

CYP24A1 enzyme dysfunction

↑

↑

↓/N

↑

SLC34A1 associated

↑

↑

↓

N

25(OH)D, 25-hydroxyvitamin D; PTH, parathyroid hormone; N, within reference value concentration; ↓, below lower reference interval value concentration; ↑, above upper reference interval value concentration [56].

with chronic kidney disease can also exhibit elevated 25(OH) D:24,25(OH)2D ratios [55]. Table 50.2 lists some of the different causes of hypercalcemia where a simultaneous 25(OH)D and 24,25(OH)2D assay could play an important role in differential diagnosis, in combination with assays for 1,25(OH)2D as well as other physiological markers. Because the prevalence of CYP24A1-associated hypercalcemia is rare, estimated to be in the order of 1/40,000, the demand for 24,25(OH)2D testing is expected to remain a fraction of the total 25(OH)D testing in the clinical setting. Although 25(OH) D and 24,25(OH)2D can be assayed simultaneously, the lower serum concentration of 24,25(OH)2D often demands more timeconsuming derivatization steps to improve assay sensitivity. Therefore high-volume reference laboratories may choose to assay these low abundance metabolites on separate platforms in an effort to maintain throughout for 25(OH)D-only requisitions, which currently outweighs the demand for 24,25(OH)2D in large measure. However, multimetabolite assays are of tremendous utility in the research setting especially in cases when specimen volume is limited such as in animal studies and when analytical run times are not constrained by clinical demands. In such cases, multiplexing beyond the aforementioned two analytes could be of additional value and enable comprehensive quantitative examination of vitamin D metabolism.

OVERVIEW OF METHODOLOGY FOR ANALYSIS OF CIRCULATING VITAMIN D METABOLITES BY LC-MS/MS LC-MS/MS has become a method of choice for the quantitative analysis of vitamin D metabolites in biological fluids. Among the technical challenges faced by the vitamin D analyst include the lipophilic nature of the vitamins D and strong binding affinity to DBP which can cause substantial matrix contamination during the extraction process. Vitamin Ds lack easily ionizable functional groups which can pose assay sensitivity challenges in the analysis of low-abundance metabolites

VI.  DIAGNOSIS AND MANAGEMENT

Overview of Methodology for Analysis of Circulating Vitamin D Metabolites by LC-MS/MS

Extraction

Derivatization

Separation

LLE SPE/SLE IE

(none) PTAD DMEQ-TAD

HPLC UHPLC SFC

913

Mass spectrometry ESI/APCI QqQ/QqLIT

FIGURE 50.3  Steps involved in LC-MS/MS methodology for quantification of vitamin D metabolites. General overview of the basic steps involved in LC-MS/MS–based assays, including some of the more common specific procedures presented in the literature. APCI, atmospheric pressure chemical ionization; ESI, electrospray ionization; LC-MS/MS, liquid chromatography tandem mass spectrometry; LLE, liquid–liquid extraction; QqLIT, quadrupole linear ion trap; QqQ, triple quadrupole; SFC, supercritical fluid chromatography; SLE, supported liquid extraction; SPE, solid-phase extraction; UHPLC, ultra-high performance liquid chromatography.

such as 1,25(OH)2D3 and 24,25(OH)2D3, and the presence of matrix and isobaric interferences must be excluded to achieve truly selective measurements. Unlike UV265-based measurements, mass spectrometry–based ion counts are not absolute. Furthermore, ionization efficiency (as well as recovery) can vary from sample to sample and can be suppressed by phospholipid as well as other factors, requiring mass spectrometry–based measurements to be normalized against a specific isotope-labeled internal standard for each metabolite under investigation, added at the beginning of the extraction process. The detector response can then be interpolated against a calibration line comprising blank matrix, supplemented with known amounts of the metabolite under investigation. As LC-MS/MS continues to become increasingly adopted for measurement of vitamin D metabolites, so has the availability of deuterated internal standards, standard reference material, as well as the use of external quality assessment schemes mentioned previously, including DEQAS and CAP. The flowchart in Fig. 50.3 outlines the key basic steps involved in LC-MS/ MS–based analysis of serum vitamin D metabolites, from sample preparation, LC-separation through to MS analysis and detection. The specific methods presented in Table 50.3 focus on more recent examples of assays mostly involving lower abundance vitamin D metabolites measured individually, or simultaneously with 25(OH)D, and are intended to provide an overview of the different combinations of tools available to the vitamin D LC-MS/MS analyst.

Sample Extraction Considerations for assay sensitivity include selective isolation metabolites from the lipid fraction. Phospholipids are abundant in serum and plasma and are known to cause significant ion suppression if coeluted with vitamin D metabolites of interest, particularly in electrospray applications [66,67]. Most extraction protocols used in vitamin D analytical studies involve an initial protein precipitation step, combined with solid phase extraction (SPE), supported liquid extraction (SLE), or liquid–liquid extraction (LLE) optimized for recovery of vitamin D metabolites while maximizing exclusion of interfering substances such as phospholipids (Table 50.3). Strategies for assessing effective removal of phospholipid include monitoring formation of m/z 184, the trimethylammonium ethyl phosphate moiety of glycerophosphocholines, the most abundant phospholipid [66]. Furthermore, postcolumn infusion studies

can be used to assess regions of the elution profile affected by suppression by phospholipid and other factors, which may identify the need to further optimize chromatographic conditions so that analytes of interest are sufficiently resolved from matrix interferences [66,67]. Selective extraction and removal of interferences that cause ion suppression are the most critical for quantitating the least abundant circulating metabolites of clinical relevance, 1,25(OH)D which circulate in the 15–60 pg/mL range. Jenkinson and colleagues have shown that SLE can be used for quantification of 1,25(OH)2D3 in clinical samples down to 20 pg/mL, without the need for derivatization; however, the most common approach appears to be immunoextraction using an anti-1,25 antibody immobilized on a solid support, available from at least two commercial sources, with the extraction procedure involving incubation, washing, and elution steps [62,68]. Although immunoextraction may prove to be a more specific extraction procedure compared with LLE and SPE/SLE, matrix effects can still occur and should be evaluated. Liquid–liquid extraction after protein precipitation is simple, effective, and relatively inexpensive; however, SLE and SPE approaches are more easily scalable for high-throughout studies as the solid phases are available in 96-well plate formats, and collection plates can be directly introduced into the LC autosampler.

Sample Derivatization Relatively poor ionizability of vitamin D metabolites can be circumvented by derivatization with Cookson-type reagents, which can increase the detector signal by 10- to 100-fold, an approach that has been studied extensively by Higashi and colleagues [69]. The simplest of these dienophiles is 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) (Fig. 50.4), which quantitatively reacts with conjugated dienes to give a stable Diels–Alder product. PTAD adducts of vitamin D metabolites increase the number of proton-affinitive nitrogen atoms, which enhances assay sensitivity in positive-ion mode, while increasing the m/z ratio of the parent compound into a range where there is less noise in electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) applications, effectively increasing the signal-to-noise ratio by using two different strategies. The Diels–Alder product comprises characteristic 6S and 6R isomers, which can be chromatographically resolved on many systems [69]. Although separation of the 6S and 6R isomers sacrifices some sensitivity because the total signal is divided between two peaks, the presence

VI.  DIAGNOSIS AND MANAGEMENT

TABLE 50.3 Comparison of Selected LC-MS/MS Methods Used for Quantification of Vitamin D Metabolites

Study Satoh [57]

Year 2016

Volume (μL)a Extract. 20

SPE

Derivatization LC DAP-TAD

HPLC

Columnb C18

Run Time (min)

Ionization

5.5

ESI+

QqQ

MSc

Target/Parent

Transitiond (m/z)

Metabolite

LOQ (ng/mL)

QqQ

[M + H]+

635 → 341

24,25(OH)2D3

0.024

[M + H – H2

399 → 105

1,25(OH)2D3

0.20

Jenkinson [58]

2016

220

SLE

None

UHPLC

Cell.

8

ESI+

O]+

Jumaah [59]

2016

2000

LLE

None

SFC

AA

≤20

APCI+

QqTOF

[M + H]+

417

24,25(OH)2D3

1.2

Geib [60]

2015

50

SLE

None

UHPLC

PFP

ESI+

QqTOF

[M + H]+

401 → 383

25(OH)2D

<9.6

Ketha [56]

2015

500

SPE

PTAD

UHPLC

C8

7.2

ESI+

QqQ

[M + H – H2O]+

574 → 298

24,25(OH)2D

0.1–0.5

Tai [36]

2015

2000

LLE

None

HPLC

C18

35

APCI+

QqQ

[M + H]+

417 → 381

24,25(OH)2D3

0.2

Hedman [61]

2014

200

SPE

Amplifex

HPLC

20

ESI+

QqLIT

[M + H]+

748 → 689

1,25(OH)2D3

0.002

Kaufmann [53]

2014

100

LLE

DMEQ-TAD

UHPLC

Phenyl

6

ESI+

QqQ

[M + H]+

762 → 468

24,25(OH)2D3

0.1–0.3

Strathman [62]

2011

400

IE

PTAD

UHPLC

C18

4.6

ESI+

QqQ

[M + H – H2O]+

574 → 298

1,25(OH)2D3

0.007

Ding [63]

2010

50/200

SPE/LLE

PTAD

UHPLC

C18

5

ESI+

QqLIT

[M + H – H2O]+c

574 → 314

1,25(OH)2D3

0.01–0.02

Casetta [64]

2010

200

LLE

None

HPLC

C18

18

ESI+

QqLIT

[M + Li]+

423 → 367

1,25(OH)2D3

0.015

Aaronov [65]

2008

500

LLE/SPE

PTAD

UHPLC

C18

8

ESI+

QqQ

[M + H – H2O]+

558 → 298

1,25(OH)2D3

0.025

25(OH)D, 25-hydroxyvitamin D; AA, aminoanthracene; APCI, atmospheric pressure chemical ionization; Cell, cellulose; DMEQ-TAD, 4-[2-(6,7-dimethoxy-4-methyl-3-oxo-3,4-dihydroquinoxalyl)ethyl]-1,2,4triazoline-3,5-dione; ESI, electrospray ionization; IE, immunoextraction; HPLC, high performance liquid chromatography; LC, columns; LC-MS/MS, liquid chromatography tandem mass spectrometry; LLE, liquid–liquid extraction; LOQ, lower of quantification; PFP, pentafluorophenyl; PTAD, phenyl-TAD; SFC, supercritical fluid chromatography; SLE, supported liquid extraction; SPE, solid phase extraction; QqLIT, quadrupole linear ion trap; QqQ, triple quadrupole; QqTOF, quadrupole time of flight; UHPLC, ultra-high performance liquid chromatography. aVolume in μL, Tai et al. gravimetrically aliquoted 2 g of serum, corresponding to approximately 2 mL. bCasetta et al. used a two-dimensional system comprising a poly(styrene-divinylbenzene) and a monolithic C18 column [1]. cDing et al. also used methylamine adduct as a target ion [M + CH NH ]+, m/z 623 → 314. 3 3 dRepresentative transitions from the lowest abundant metabolite detected in the assay.

Overview of Methodology for Analysis of Circulating Vitamin D Metabolites by LC-MS/MS

of Cookson-type reagents have been synthesized, comprising a range of substituents added to the 4 position of TAD moiety, and used in assays of low-abundance vitamin D metabolites. Because many of these reagents were synthesized in-house and not commercially available, PTAD appears to remain the most frequently used TAD, although 4-[2-(6,7-dimethoxy-4-methyl3-oxo-3,4-dihydroquinoxalyl)ethyl]-1,2,4-triazoline-3,5-dione (DMEQ-TAD) and precursors of DMAP-TAD and MBO-TAD are commercially available [69]. Recently, novel Cookson-type reagents have been developed that contain positively charged functional groups, well suited to detecting adducts with vitamin D metabolites in positive-ion mode applications, including Amplifex Diene and SecoSet. Amplifex Dienes contains a positively charged quaternary amine group and has been shown to achieve an lower limit of quantification (LLOQ) for 1,25(OH)2D3 of 2 pg/mL using a starting serum volume of 200 μL—achieving an estimated 10-fold greater signal-to-noise ratio compared with 1,25(OH)2D3-PTAD adducts measured in parallel as a part of the same study [61]. Although the potential benefits are obvious for metabolites that circulate in the pg/mL range; Amplifex has also been used for simultaneous measurement of other vitamin D metabolites including 25(OH) D and 24,25(OH)2D3 [70,71]. A major drawback of derivatization is the added number of sample handing steps and sample preparation time; thus derivatization is not conducive to highthroughout applications and is difficult to automate, despite the improved sensitivity achieved. The decision to derivatize or not to derivatize will depend on the analyst’s objectives and the balance between the sensitivity and throughput required.

2+

WdĂĚĚƵĐƚŽĨ ϭ͕ϮϱͲ;K,ͿϮϯ

2 ϳ

ϲ

1 1

ŵͬnj ϯϭϰ

ŵͬnj ϱϳϰ → ϯϭϰ 1

2 2+

+2

2

Wd

1 1

ϱ 1

2 2

WͲd

1 1

1

1 2 2

DYͲd 1

1

1

2

1

2

1

2

2

2

ŵƉůŝĨĞdž

1 1

1 1

915

2 1

2

FIGURE 50.4  Structures of different Cookson-type reagents used for derivatization of vitamin D metabolites. This figure presents a subset of Cookson-type reagents, depicting different variations to the simplest and most commonly used reagent, PTAD, to enhance the sensitivity of LC-MS/MS–based vitamin D metabolite assays in positive-ion mode by the addition of proton-affinitive functional groups. The structure of the PTAD adduct of 1,25-(OH)2D3 depicts a commonly observed C6–C7 cleavage fragment which forms the basis of many MRM transitions reported in Table 50.3. LC-MS/MS, liquid chromatography tandem mass spectrometry; MRM, multiple reaction monitoring. DAP-TAD, 4-(4'-dimethylaminophenyl)-1,2,4triazoline-3,5-dione; DMEQ-TAD, 4-[2-(6,7-dimethoxy-4-methyl3-oxo-3,4-dihydroquinoxalyl)ethyl]-1,2,4-triazoline-3,5-dione; PTAD, 4-phenyl-1,2,4-triazoline-3,5-dione.

of two characteristic peaks in conserved proportions can act as a useful qualitative tool to assess signal specificity. Unlike the large number of fragments produced by native vitamin D metabolites during collision-induced dissociation, 1,2,4-triazoline-3,5-dione (TAD) adducts yield a limited number of fragments, often including a structurally informative ion arising from C6–C7 cleavage, critical for maximizing sensitivity in multiple reaction monitoring (MRM) [69](Fig. 50.4). A number of groups have demonstrated the utility of Cookson-type reagents in extending the range of vitamin D metabolites detected in a single assay beyond that of 25(OH)D, including 24,25(OH)2D, as well as 1,25(OH)2D [65,63,53,62,57]. A myriad

Sample Separation The major recent advance in separations techniques used for LC-MS/MS has been the development of ultra-high performance liquid chromatography (UHPLC) in 2004 [72]; chromatography systems have been designed to operate at the high back pressures required for highly efficient separations on <2-μm particle size columns. The technique effectively improves signal-to-noise by sharpening chromatographic peaks while reducing run time—critical for high-throughput LC-MS/ MS applications. Superficially porous particle (SPP)-based columns have also been used to improve the efficiency achieved with UHPLC. Because changing the particle porosity affects the required backpressure less so than reducing the particle size, SPP columns can be used to achieve UHPLC-like separations with standard HPLC equipment [73]. Jumaah and colleagues recently reported the use of supercritical fluid chromatography/mass spectrometry in the analysis of serum vitamin D metabolites [59]. Supercritical fluids are advantageous due to their solvating power and diffusivity, potentially enabling highly efficient separations in a fraction of the run time normally associated with HPLC, and offers the analyst ability to tune the solvent density as yet another tool to optimize the chromatographic system [74]. Supercritical carbon dioxide is typically used as the mobile phase in combination with methanol and possesses chemical properties similar to heptane. The fact that SFC is based on normal-phase chromatography is particularly appealing because separation of vitamin D metabolites is

VI.  DIAGNOSIS AND MANAGEMENT

916

50.  MASS SPECTROMETRY ASSAYS OF VITAMIN D METABOLITES

known to be best achieved on normal phase systems [75]; but in clinical studies, this has been superseded by the necessity to deliver analytes to the mass spectrometer in reversed-phase solvents. The relatively inferior ability of reversed-phase systems to separate vitamin D metabolites has been mitigated by recent advances in LC systems however. Jumaah reported limit of quantification (LOQ) for 24,25-(OH)2D3 and 1,25(OH)2D3 of 1.19 and 7 ng/mL, therefore not amenable for in vivo 1,25(OH)2D3 quantification as yet. The group noted that MS detection was done using a QTOF instrument in MS mode, raising the question that more sensitivity might be gained by switching to MS/ MS mode or MRM on a triple quadrupole instrument. An inherent limitation of mass spectrometry is the inability to differentiate between isomers; thus the specificity of LC-MS/ MS-based measurement of vitamin D metabolites relies heavily on the LC step. Considerable attention has been given to the separation of 3-epi-25(OH)D3 from measurements of 25(OH) D3 (Fig. 50.1). Most RIA-based measurements of 25(OH)D3 are not confounded by the presence of 3-epi-25(OH)D3 because this metabolite does not bind the antibody used. While 3-epi25(OH)D3 is present at 4% of 25(OH)D3 levels in adults, and at up to 60 of the levels of 25(OH)D3 in infancy, overestimation of LC-MS/MS-based 25(OH)D3 measurements will occur if the two metabolites were not chromatographically separated [76,77]. Use of pentafluorophenyl columns has been the most common strategy to chromatographically resolve these two metabolites [76,78], presumably due to the faster separations possible in comparison to cyano columns [79,80]. A wider range of column chemistries including phenyl and C18 are also effective at resolving certain TAD adducts of 3-epi25(OH)D3 and 25(OH)D3 due to the change in selectivity provided by the TAD moiety [53,57]. In multimetabolite assays focusing on lower abundance metabolites, attention must be given to the number of dihydroxylated vitamin D metabolites present in the circulation including 24,25(OH)2D3, 3-epi24,25(OH)2D3, 23,25(OH)2D3, 25,26-(OH)2D3, 1,25-(OH)2D3, and 4,25-(OH)2D3. In the development of a candidate reference method for 24,25(OH)2D3, Tai and colleagues report that an elution time of 30 min was needed to sufficiently resolve this metabolite from 23,25(OH)2D3, 25,26(OH)2D3, 1,25(OH)2D3, as well the 3-epi form, on an SPP C18 column without derivatization [36]. A longer LC gradient using 2-D LC was also used by Casetta et al. [64] to ensure selectivity of their native 1,25(OH)3D3 measurements over contamination from 24,25(OH)2D3 and 25,26(OH)2D3, an assay that was more recently used to selectively measure 24,25(OH)D in IIH patients [81]. Methods involving shorter run time used for assaying dihydroxylated vitamin D metabolites have been reported [58], including methods that exploit the altered chromatographic properties of TAD adducts. Ketha et al. [56] recently assessed 24,25-(OH)2D3 levels in IIH patients using PTAD adducts and noted contamination of the 6S isomer of PTAD-24,25-(OH)2D3 with that of 25,26(OH)2D3 and decided to use the well-resolved 6R peak instead; taking advantage of the presence of two chromatographic peaks for 24,25(OH)2D3 while keeping the run time at 7 min. An assay developed by Kaufmann and colleagues adapted for studying serum vitamin D metabolites using DMEQ-TAD derivatization demonstrated the importance of separating 24,25(OH)2D3

from 3-epi-24,25(OH)2D3 and 1,25(OH)2D3, described in the following section. [53,82]. Of importance to 1,25(OH)2D3 assays is potential interference of 4,25(OH)2D3 which can circulate at concentrations similar to 1,25(OH)2D3, arising from metabolism of 25(OH)D3 by CYP3A4 [83,84]. The group identified this metabolite partly on the basis that it eluted close to the PTAD adduct of 1,25(OH)2D3, giving a structurally informative A-ring cleavage fragment of m/z 314 and was sensitive to periodate treatment, which indicated the presence of hydroxyl groups at adjacent carbons. This highlighted the importance of careful separation of 1,25-(OH)2D3 from 4β,25(OH)2D3 in clinical assays, as well as the utility of using offline approaches such as periodate sensitivity to structurally differentiate dihydroxylated vitamin D metabolites, and as a way of studying interferences in method development [83,85]. A common approach to 1,25(OH)2D3 measurement involves immunoextraction before LC-MS/MS analysis. Although analyte-specific antibodies are a common strategy to remove matrix interferences, Laha and colleagues have characterized the cross-reactivity of an antibody used for 1,25(OH)2D3 immunoextraction with other vitamin D metabolites and found that 4β,25(OH)2D3 is poorly recovered, thus providing an effective means to remove certain isobaric as well as matrix interferences. On the other hand, metabolites such as 24,25(OH)2D3 and 25(OH)D exhibited significant recovery from extraction with the same antibody, allowing vitamin D metabolite multiplexing on the basis of the poor specificity of the anti-1,25 antibody [68,86]. This work points to the important role of the liquid chromatography step in ensuring the specificity of LC-MS/MS assays.

Mass Spectrometry To enable detection by the mass spectrometer, analytes are ionized by one of the two common techniques: ESI or APCI. ESI has been suggested to have greater sensitivity but greater variation versus APCI [87]. Furthermore, APCI has been recommended for analysis of certain steroids which share structural resemblance and nonpolar characteristics with vitamin D. Quantification of vitamin D metabolites by mass spectrometry most often involves MRM or SRM data acquisition modes available on low mass resolution instruments such as triple quadrupole or linear ion trap instruments equipped with ESI or APCI sources. The relatively poor specificity of selecting a parent ion (e.g., m/z 401, Table 50.3) using low–mass resolution instruments is circumvented by monitoring a highly specific fragment ion (e.g., m/z 383, Table 50.3) in the second stage, arising from collision-induced dissociation of the selected parent. It is common practice to monitor water-loss fragment ions, as it offers adequate signal intensity; however, this approach may be associated with reduced specificity and greater imprecision [87]. Use of high-resolution mass spectrometers in either scanning or fragmentation mode for analysis of 25(OH)D has been shown to be well correlated to measurements from triple quadrupole instruments with LOQs down to ∼2 ng/mL [60,88]. In a semiquantitative study, Jumaah and colleagues demonstrated that 24,25(OH)2D3 could be quantitated in serum using a time-offlight instrument in scanning mode, coupled to supercritical fluid chromatography [59], but it remains to be seen if accurate

VI.  DIAGNOSIS AND MANAGEMENT

Application of LC-MS/MS to the Study of Vitamin D Metabolism in Animal Models

mass detectors offer sufficient sensitivity for 1,25(OH)D quantification. The major advantage of accurate mass detectors is the specificity of the scanning mode, which enables broad-based metabolomics studies including vitamin D, without being limited to predefined MRM transitions. Taken together, it is the combination of sample preparation, chromatographic separation, and mass spectrometry-based detection & quantification that together help to overcome many of the challenges of LC-MS/MS–based studies of vitamin D.

APPLICATION OF LC-MS/MS TO THE STUDY OF VITAMIN D METABOLISM IN ANIMAL MODELS The successful use of LC-MS/MS for measuring serum metabolites of vitamin D in clinical studies has opened up the possibility of using this technique to study vitamin D metabolism in animal models, where diet and genotype can be controlled. The major technical considerations that differ between clinical and animal studies comprise limited sample volumes available, and differences in circulating concentrations of certain vitamin D metabolites, presumably due to a combination of species-based differences in vitamin D-associated cytochromes P450, VDR, as well as pharmacokinetic parameters. We have applied our LC-MS/MS methodology [53] to study vitamin D metabolism in a variety of knockout mouse models, some of which have been treated with rescue diets as well as transgenes. Quantitative data from the studies are presented in Table 50.4, and representative chromatographic profiles from selected models are shown in Fig. 50.5. This section highlights the important contribution of LC-MS/MS of multiple metabolites of vitamin D to the study of knockout mouse models of vitamin D-related disease states. Use of low serum volumes from mice needs to be carefully prioritized between vitamin D metabolites and other physiological biomarkers, which has often necessitated the need for sample pooling. Furthermore, RIA-based measurements of 25(OH)D requiring serum volumes of at least 50 μL enable the measurement of total 25(OH)D, but not other vitamin D metabolites, which would require separate individual assays, if sufficient samples volumes exist. Our current LC-MS/ MS assay using a typical starting volume of 100 μL serum enables simultaneous measurement of DMEQ-TAD adducts of 25(OH)D3, 3-epi-25(OH)D3, 24,25(OH)2D3, and 25(OH)D326,23-lactone over triplicate injections into the LC-MS/MS system, allowing serum to be measured from individual animals rather than pooled samples [53]. Starting serum volumes can easily be reduced to 25 μL if a single analytical injection is used. In wild-type mice, 24,25(OH)2D3 circulates at concentrations of 2- to 8-fold greater than healthy human subjects, up to 8–10 ng/mL. In addition, the metabolite 25(OH)D3-26,23lactone, which is barely detectable in human samples, can be detected in wild-type mouse serum in upwards of 6 ng/mL [85]. It should be noted that deuterated internal standards of 25(OH)D3-26,23-lactone are not currently available, and measurements are based on the recovery of d6-24,25(OH)2D3 and the calibration line for 24,25(OH)2D3. The relatively high

917

concentrations of both 24,25(OH)2D3 and 25(OH)D3-26,23lactone in mice can be described as catch-22: These metabolites provide two measurements of CYP24A1 function within the same assay, and their high concentration is predicted to permit further reduction of the minimum starting volume of serum to as low as 10 μL. However, caution should be exercised when interpreting RIA-based measurements of 25(OH)D in mice because cross-reactivity of certain antibodies against 25(OH) D with 24,25(OH)2D3 [and perhaps 25(OH)D3-26,23-lactone] previously reported in clinical studies [92] is exacerbated in rodent models where these metabolites circulate in significantly greater concentrations than human subjects. Zhu and coworkers investigated circulating 25(OH)D levels in CYP2R1 mice using RIA, HPLC, and LC-MS/MS–based techniques [89]. The work showed that CYP2R1-null animals exhibited more than 50% reduction in serum 25(OH)D to 5–7 ng/mL compared with 10–17 ng/mL in wild-type animals based on LC-MS/MS and HPLC analysis of 10-week old animals, a finding that parallels low 25(OH)D levels in VDDR-1B rickets patients possessing inactivating mutations in CYP2R1 [93,94,6]. As shown in Table 50.4 and Fig. 50.5, circulating 24,25(OH)2D3 and 25(OH)D3-26,23-lactone were appropriately reduced, suggesting normal vitamin D catabolism in the CYP2R1-null animals. RIA-based measurements of 25(OH)D in this study were 2- to 3-fold greater than HPLC and LC-MS/MS, with 25(OH)D3 in the 10-week CYP2R1-null animals reading 17.8 ng/mL. When comparing the sum of 25(OH) D + 24,25(OH)2D3 and 25(OH)D3-26,23-lactone using LC-MS/ MS, to RIA of 25(OH)D3, the RIA measurement was only 1.2fold greater than LC-MS/MS suggesting significant cross-contamination of 25(OH)D measurement with other vitamin D metabolites when using RIA [89]. The dramatic increase in circulating 25(OH)D3 in the CYP27A1 (−/−) animals is explained by upregulated CYP2R1 mRNA expression and not dysregulated catabolism because 24,25(OH)2D3 and 25(OH)D3-26,23lactone appeared normal relative to circulating 25(OH)D3 [89]. Given that the significant 25(OH)D remaining in CYP2R1-null animals was able to sustain normal serum 1,25(OH)2D3 (as measured by luciferase reporter assay) and the mice did not develop rickets, the authors concluded that another enzyme must be involved in the 25-hydroxylation step in vivo [89]. Studies of vitamin D metabolism in the CYP24A1 knockout mouse have shown that the plasma half-life of exogenous doses of 3H-1,25(OH)2D3 is 10-fold greater than wild-type animals [85,95,96]. The physiological importance of CYP24A1 was demonstrated by the fact that half of the mice die due to hypercalcemia caused by an uncatabolized excess of 1,25(OH)2D3 and rationalized the role of CYP24A1 mutation in the pathogenesis of IIH [42,53]. We recently re-visited vitamin D metabolism in the CYP24A1 knockout mouse, this time by studying endogenous serum metabolites using LC-MS/ MS (Table 50.4, Fig. 50.5). Serum 24,25(OH)2D3 was strikingly reduced from 6–8 ng/mL to 1.2 ng/mL in the CYP24A1(−/−) animals, resembling that of IIH patients [97]. Furthermore, 25(OH)D3-26,23-lactone was also reduced from approximately 4 to 0.1 ng/mL in the nulls. The most distinguishing feature of metabolic profiles of CYP24A1-null mice compared with IIH patients is the dramatic increase in 25(OH)D3 observed in the

VI.  DIAGNOSIS AND MANAGEMENT

TABLE 50.4  Quantitative Comparison of Serum Vitamin D Metabolite Profiles in Selected Knockout Mouse Models, Each Studied Using the Same LC-MS/MS Method [53] 25(OH)D3:25(OH)D326,23-Lactone, Ratio

1,25-(OH)2D3, pg/mL

References

5.8 ± 0.6

2.9 ± 0.2

<300b

[89]

1.2 ± 0.1

1.7 ± 0.4

4.1 ± 0.9

<300

44.9 ± 2.7

1.6 ± 0.2

18.0 ± 3.0

4.0 ± 0.7

<300

10.9 ± 1.5

10.7 ± 2.1

1.0 ± 0.1

2.5 ± 0.4

4.0 ± 0.8

<300

4

12.1 ± 1.1

6.0 ± 0.9

2.1 ± 0.3

4.3 ± 0.1

6.7 ± 1.1

<300

CYP24A1 (±)

8

15.0 ± 1.4

8.0 ± 0.9

1.9 ± 0.1

4.1 ± 0.4

3.7 ± 0.5

<300

CYP24A1 (−/−)

7

102.6 ± 9.2

1.2 ± 0.1

84.8 ± 9.2

0.1 ± <0.1

852.7 ± 82.7

<300

CYP27B1 (−/−)

4

79.8 ± 5.5

1.2 ± 0.1

69.1 ± 3.7

0.1 ± <0.1

619.2 ± 57.2

<300

Wild type

9

20.2 ± 3.2

9.6 ± 2.1

2.2 ± 0.2

3.0 ± 1.1

3.6 ± 1.7

<300

VDR (−/−), 2 mo

8

8.2 ± 0.9

0.3 ± <0.1

47.8 ± 8.1

0.1 ± <0.1

98.0 ± 37.0

3140 ± 470

VDR (−/−) + RD,d 2 mo

16

10.1 ± 1.8

0.1 ± 0.1

110.4 ± 30.0

0.1 ± <0.1

170.0 ± 55.1

3420 ± 630

VDR (−/−) + RD, 4/6 mo

4

10.5 ± 0.7

0.9 ± 0.2

12.3 ± 2.0

2.7 ± 0.4

3.9 ± 0.4

<300

VDR (−/−)/Tg mVDRe

7

18.4 ± 4.7

8.5 ± 1.8

2.1 ± 0.2

3.4 ± 0.5

5.4 ± 0.8

<300

VDR (−/−)/Tg hVDRe

3

22.2 ± 1.2

8.9 ± 1.6

1.9 ± 0.2

2.0 ± 0.3

5.9 ± 1.1

<300

VDR (−/−)/Tg L233S hVDRe

5

6.7 ± 0.8

0.1 ± <0.1

62.3 ± 0.3

<0.1

139.5 ± 16.4

3303 ± 656

Wild type ↓P↓Df

5

11.5 ± 1.7

4.5 ± 0.6

2.5 ± 0.3

1.7 ± 0.4

6.7 ± 1.1

44.9 ± 11.9g

Wild type ↑P↓Df

5

16.0 ± 2.7

7.2 ± 2.0

2.4 ± 0.4

5.6 ± 1.3

3.1 ± 0.8

35.2 ± 7.5

SLC34A1 (−/−) ↓P↓Df

6

12.8 ± 1.6

5.1 ± 0.6

2.5 ± 0.3

1.8 ± 0.3

7.2 ± 1.4

65.9 ± 18.9

SLC34A1 (−/−) ↑P↓Df

6

16.0 ± 2.7

8.5 ± 1.7

1.9 ± 0.2

4.3 ± 0.7

3.7 ± 0.4

30.3 ± 0.4

Genotype

24,25-(OH)2D3, ng/mL

25(OH)D3:24,25(OH)2D3, Ratio

N

25(OH)D3, ng/mL

Wild type

4

16.9 ± 1.4a

12.1 ± 0.9

1.4 ± 0.1

CYP2R1 (−/−)

4

6.8 ± 1.0

5.6 ± 0.7

CYP27A1 (−/−)

4

70.5 ± 11.3

CYP2R1/CYP27A1(−/−)c

5

Wild type

25(OH)D3-26,23Lactone, ng/mL

25(OH)D, 25-hydroxyvitamin D; LC-MS/MS, liquid chromatography tandem mass spectrometry; LLE, liquid–liquid extraction; VDR, vitamin D receptor. aMean ± SD. bLOD of 1,25-(OH) D assay based on LLE is ∼300 pg/mL. 2 3 cDenotes CYP2R1 and CYP27A1 double-knockout mouse. dRescue diet (RD) containing high phosphate and high calcium. eVDR (−/−) transgenic animals rescued with mouse, human, or L233S variant of human VDR. fDenotes low phosphate/low vitamin D diet, or high phosphate/low vitamin D diet. 3 3 g1,25-(OH) D assay based on immunoextraction before LC-MS/MS analysis. 2 3

[85,90]

[82]

[91]

919

Application of LC-MS/MS to the Study of Vitamin D Metabolism in Animal Models

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FIGURE 50.5  Overlay of vitamin D metabolite profiles in selected knockout mouse models. Profiles arising from specific MRM transitions of DMEQ-TAD adducts are shown for (A) 25(OH)D3, m/z 746 → 468 (B) 24,25(OH)2D3, m/z 762 → 468 and (C), 25(OH)D3-26,23-lactone, m/z 774 → 468. This figure emphasizes the chromatographic separation of each epimeric form, as the separation of 1,25(OH)2D3 from 24,25(OH)2D3 in the VDR knockout mouse model [82]. A profile for a wild-type mouse from a selected study is shown [85]. Retention times have been normalized to account for variability of the chromatography over the time these studies were conducted from 2013 to 2016. Quantitation of metabolites is shown in Table 50.4. MRM, multiple reaction monitoring; VDR, vitamin D receptor. DMEQ-TAD, 4-[2-(6,7-dimethoxy-4-methyl-3-oxo-3,4-dihydroquinoxalyl)ethyl]-1,2,4-triazoline3,5-dione; RD, rescue diet.

animal model, due to lack of catabolism to 24,25(OH)2D3 and 25(OH)D3-26,23-lactone. This apparent accumulation 25(OH) D3 is not observed in all IIH patients, presumably due to a combination of genetic polymorphisms, and individual variation in dietary vitamin D intakes and sun exposure. Both accumulation of 25(OH)D3 and reduced concentration of serum catabolites manifest as an increase in 25(OH)D3:24,25(OH)2D3 ratio from 2 to 85, and increase in 25(OH)D3:25(OH)D3-26,23lactone 3.7–6.7 to 852 in the knockout animals [85]. A similar vitamin D metabolite profile was observed when CYP27B1 was ablated, because of a lack of CYP24A1 upregulation when 1,25(OH)2D is absent [90,98]. The vitamin D metabolite profile was remarkably similar to that of a patient with VDDR-1 possessing inactivating mutations to CYP27B1 [90]. VDR knockout mice have rickets due to a lack of expression of vitamin D-dependent gene expression [99,100]. VDRmediated genomic action is also required for self-regulation of the vitamin D endocrine system via CYP24A1 expression to catabolize 1,25(OH)2D3 [101,102] and to suppress 1α-hydroxylation of 25(OH)D3 by CYP27B1 [103]. As such, serum metabolite profiles of VDR-null mice are characterized by low 24,25(OH)2D3 and 25(OH)D3-26,23-lactone, as well as superphysiological concentrations of 1,25(OH)2D3, in addition to elevated parathyroid hormone (PTH) [82,99,100]. Mineral ion homeostasis and the aberrant vitamin D metabolite profile were corrected by introducing wild-type human or mouse transgenes, due to normalization of vitamin D-dependent

gene expression. Transgenic animals containing an L233S variant of human VDR which does not bind 1,25(OH)2D3 failed to rescue the rickets or the abnormal metabolic profile [104]. When 2-month old VDR-null animals were given a high calcium, high-lactose rescue diet [105,106], serum calcium and phosphate returned to appropriate levels; however, it took from 4 to 6 months on the rescue diet for normalization of PTH to occur in most of the VDR-null animals [82]. Normalization of PTH was concomitant with restoration of 1,25(OH)2D3, 24,25(OH)2D3, and 25(OH)D3-26,23-lactone to appropriate serum levels in the absence of VDR, which suggested a role for PTH in the suppression of CYP24A1 and the presence of VDRindependent regulation of basal CYP24A1 expression in the kidney. Although 24,25(OH)2D3 was restored to only 1/10 of wild-type levels among the diet-rescued animals, the LC-MS/ MS–based approach revealed that most of the 24-hydroxylated metabolites present comprised, 3-epi-24,25(OH)2D3, identified on the basis of co-chromatography with an in vitro product of 3-epi-25(OH)D3 produced in a CYP24A1-overexpressing cell line. Why serum metabolite profiles of the diet-rescued VDR knockout mouse possess abundant 3-epi-24,25(OH)2D3 and 3-epi-25(OH)D3-26,23-lactone remains unknown. Mutations in the sodium/phosphate cotransporter 2A (SLC34A1) have been identified as another cause of IIH where patients present with hypercalcemia renal phosphate wasting and elevated 1,25(OH)D [91]. The critical role of dysregulated phosphate homeostasis in SLC34A1-mediated hypercalcemia

VI.  DIAGNOSIS AND MANAGEMENT

920

50.  MASS SPECTROMETRY ASSAYS OF VITAMIN D METABOLITES

was demonstrated by normalization of serum phosphate, FGF23, 1,25(OH)D (Table 50.4), and calcium when the SLC34A1 knockout mice were placed on a high-phosphate diet, a result that paralleled successful treatment of an IIH-SLC34A1 patient with phosphate supplementation [91]. Taken together, LC-MS/MS has proven to be a sensitive and selective method that can be easily adapted from clinical to animal studies, enabling quantitation of multiple vitamin D metabolites simultaneously in individual animals where sample volume can be limited. Furthermore, cross-reactivity of RIA-based measurements of 25(OH)D with other metabolites is exacerbated in rodent models where 24,25(OH)2D3 and 25(OH) D3-26,23-lactone circulate in greater concentrations compared with human subjects. Selective assays for 25(OH)D proved critical in assessing the contribution of CYP2R1 and CYP27A1 to circulating 25(OH)D. LC-MS/MS also enables assessment of endogenous vitamin D metabolites, reducing reliance on techniques which involve studying clearance of radiolabeled vitamin D metabolites given as a bolus dose. Where possible, use of the same LC-MS/MS platform to study clinical disease states and animal models plays an important role in validating proposed pathogenic mechanisms and identifying species-based differences in vitamin D metabolism and physiology.

may become integrated into larger scale quantitative comprehensive metabolomics profiles to elucidate or stratify a diverse array of clinical phenotypes [40,109,110].

References













FUTURE DIRECTIONS To date, 25(OH)D measurements remain the focus of clinical laboratories. Because of the high demand for nutrition-based measurements, simultaneous assessment of multiple vitamin D metabolites including 1,25(OH)2D and 24,25-(OH)2D and 25(OH)D is not the default assay and is only rationalized for a small number of patients because of the low sample throughput associated with derivatization steps needed to achieve appropriate LOQs. Clinical research studies in combination with animal models, however, have demonstrated the utility of multimetabolite assays as a means to interrogate diseases of calcium/phosphate homeostasis including IIH. Furthermore, the multimetabolite assays have identified specific vitamin D metabolite profiles associated with certain diseases, allowing the assay to aid in the differential diagnosis of hypercalcemia, ultimately enabling timely treatment that is appropriate to the cause. Although IIH due to CYP24A1 mutations is rare, a broader population may benefit from multimetabolite testing including those individual with hypercalcemia of unknown cause, or those with a family history of hypercalcemia, or even possibly those at risk for vitamin D deficiency. We anticipate that as the interplay between advances in LC-MS/MS technology and clinical research continues, measurement of a greater number of metabolites in a single assay will be possible, which will expand the scope of unique vitamin D metabolite profiles associated with specific disease states [107,108]. Whether such multimetabolite assays become the preferred vitamin D test in the clinical chemistry laboratory will ultimately depend on how effectively advances in LC-MS/MS and sample preparation technology can narrow the gap between assay sensitivity and sample throughput. Ultimately, vitamin D metabolites























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VI.  DIAGNOSIS AND MANAGEMENT