LC-MS/MS for Identifying Patients with CYP24A1 Mutations

Ketha, Hemamalini; Kumar, Rajiv; Singh, Ravinder J.

doi:10.1373/clinchem.2015.244459

Cited by 49 publications

(30 citation statements)

References 25 publications

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“…The existence of two adduct peaks from 24,25‐(OH) 2 D 3 with DMEQ‐TAD allowed for alternative methods for quantifying the components and, importantly, the 25‐OH‐D 3 :24,25‐(OH) 2 D 3 ratio for IIH patients and their relatives. However, unlike other analysts who had a similar problem separating 4‐phenyl‐1,2,4‐triazoline‐3,5‐dione (PTAD) adducts of 24,25‐(OH) 2 D 3 and 25,26‐(OH) 2 D 3 , we chose the larger of the two adduct peaks (11.20 min) to quantitate 24,25‐(OH) 2 D 3 and determine the 25‐OH‐D 3 :24,25‐(OH) 2 D 3 ratios used in this study (Table ). Whereas the rapid chromatographic approach fails to resolve 24,25‐(OH) 2 D 3 and 25,26‐(OH) 2 D 3 and therefore overestimates 24,25‐(OH) 2 D 3 values and falsely lowers the 25‐OH‐D 3 :24,25‐(OH) 2 D 3 ratio, the extended chromatography provides a fully resolved 24,25‐(OH) 2 D 3 peak and a more accurate 25‐OH‐D 3 :24,25‐(OH) 2 D 3 ratio than with the published method (Fig.…”

Section: Resultsmentioning

confidence: 99%

Improved Screening Test for Idiopathic Infantile Hypercalcemia Confirms Residual Levels of Serum 24,25-(OH)2D3 in Affected Patients

Kaufmann

Morse

Molloy

et al. 2017

Journal of Bone and Mineral Research

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CYP24A1 mutations are now accepted as a cause of idiopathic infantile hypercalcemia (IIH). A rapid liquid-chromatography tandem mass spectrometry (LC-MS/MS)-based blood test enabling measurement of the 25-OH-D :24,25-(OH) D ratio (R) can identify IIH patients on the basis of reduced C24-hydroxylation of 25-OH-D by CYP24A1 in vivo. Although values of this ratio are significantly elevated in IIH, somewhat surprisingly, serum 24,25-(OH) D remains detectable. The current study explores possible explanations for this including: residual CYP24A1 enzyme activity in individuals with certain CYP24A1 genotypes, expression of alternative C24-hydroxylases, and the possibility of isobaric contamination of the 24,25-(OH) D peak on LC-MS/MS. We employed an extended 20-min run time on LC-MS/MS to study serum vitamin D metabolites in patients with IIH due to mutations of CYP24A1 or SLC34A1; in unaffected heterozygotes and dialysis patients; in patients with vitamin D deficiency; as well as in normal subjects exhibiting a broad range of 25-OH-D levels. We identified 25,26-(OH) D as a contaminant of the 24,25-(OH) D peak. In normals, the concentration of 24,25-(OH) D greatly exceeds 25,26-(OH) D ; however, 25,26-(OH) D becomes more significant in IIH with CYP24A1 mutations and in dialysis patients, where 24,25-(OH) D levels are low when CYP24A1 function is compromised. Mean R in 30 IIH-CYP24A1 patients was 700 (range, 166 to 2168; cutoff = 140) as compared with 31 in 163 controls. Furthermore, patients possessing CYP24A1 L409S alleles exhibited higher 24,25-(OH) D levels and lower R (mean R = 268; n = 8) than patients with other mutations. We conclude that a chromatographic approach which resolves 24,25-(OH) D from 25,26-(OH) D produces a more accurate R that can be used to differentiate pathological states where CYP24A1 activity is altered. The origin of the residual serum 24,25-(OH) D in IIH patients appears to be multifactorial. © 2017 American Society for Bone and Mineral Research.

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Section: Resultsmentioning

confidence: 99%

Improved Screening Test for Idiopathic Infantile Hypercalcemia Confirms Residual Levels of Serum 24,25-(OH)2D3 in Affected Patients

Kaufmann

Morse

Molloy

et al. 2017

Journal of Bone and Mineral Research

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“…Mutations in CYP24A1 are causative for idiopathic infantile hypercalcemia and vitamin D prophylaxis . Analytical methods have used a 25(OH)D/24,25(OH) 2 D ratio for determining patients with CYP24A1 mutations …”

Section: Cyp24a1 and Vitamin D Catabolismmentioning

confidence: 99%

The vitamin D metabolome: An update on analysis and function

Jenkinson

2019

Cell Biochemistry & Function

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Current understanding of vitamin D tends to be focussed on the measurement of the major circulating form 25‐hydroxyvitamin D3 (25OHD3) and its conversion to the active hormonal form, 1α,25‐dihydroxyvitamin D3 (1α,25(OH)2D3) via the enzyme 25‐hydroxyvitamin D‐1α‐hydroxylase (CYP27B1). However, whilst these metabolites form the endocrine backbone of vitamin D physiology, it is important to recognise that there are other metabolic and catabolic pathways that are now recognised as being crucially important to vitamin D function. These pathways include C3‐epimerization, CYP24A1 hydroxylase, CYP11A1 alternative metabolism of vitamin D3, and phase II metabolism. Endogenous metabolites beyond 25OHD3 are usually present at low endogenous levels and may only be functional in specific target tissues rather than in the general circulation. However, the technologies available to measure these metabolites have also improved, so that measurement of alternative vitamin D metabolic pathways may become more routine in the near future. The aim of this review is to provide a comprehensive overview of the various pathways of vitamin D metabolism, as well as describe the analytical techniques currently available to measure these vitamin D metabolites.

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“…Serum 24,25-dihydroxycholecalciferol (24,25(OH) 2 D 3 ) was measured via a LC-MS/MS assay we recently developed and validated [53]. The limit of detection was 0.03 ng/mL; the corresponding limit of quantification was 0.1 ng/mL.…”

Section: 0 Study Assessmentsmentioning

confidence: 99%

Effects of cholecalciferol supplementation on serum and urinary vitamin D metabolites and binding protein in HIV-infected youth

Eckard

Thierry-Palmer

Silvestrov

et al. 2017

The Journal of Steroid Biochemistry and Molecular Biology

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Vitamin D insufficiency is widespread in HIV-infected patients. HIV and/or antiretroviral therapy (ART), particularly efavirenz (EFV), may interfere with vitamin D metabolism. However, few data from randomized, controlled trials exist. Here, we investigate changes in vitamin D metabolites and binding protein (VDBP) after 6 months of supplementation in a randomized, active-control, double-blind trial investigating 2 different monthly cholecalciferol (vitamin D3) doses [60,000 (medium) or 120,000 (high) IU/month] vs. a control arm of 18,000 IU/month in 8–25 year old HIV-infected youth on ART with HIV-1 RNA <1000 copies/mL and baseline 25-hydroxycholecalciferol (25(OH)D3) ≤30 ng/mL. A matched healthy uninfected group was enrolled in a similar parallel study for comparison. Changes after 6 months were analyzed as intent-to-treat within/between groups [control group (low dose) vs. combined supplementation doses (medium+high)]. At 6 months, 55% vs. 82% of subjects in control and supplementation groups, respectively, reached 25(OH)D3 ≥30 ng/mL (P=0.01) with no difference between medium and high doses (both 82% ≥30 ng/mL). There were few differences for those on EFV vs. no-EFV, except serum VDBP decreased in EFV-treated subjects (both within- and between-groups P≤0.01). There were no significant differences between the HIV-infected vs. healthy uninfected groups. The major finding of the present study is that cholecalciferol supplementation (60,000 or 120,000 IU/month) effectively raises serum 25(OH)D3 in the majority of HIV-infected subjects, regardless of EFV use. Notably, response to supplementation was similar to that of uninfected subjects.

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LC-MS/MS for Identifying Patients with CYP24A1 Mutations

Cited by 49 publications

References 25 publications

Improved Screening Test for Idiopathic Infantile Hypercalcemia Confirms Residual Levels of Serum 24,25-(OH)2D3 in Affected Patients

Improved Screening Test for Idiopathic Infantile Hypercalcemia Confirms Residual Levels of Serum 24,25-(OH)2D3 in Affected Patients

The vitamin D metabolome: An update on analysis and function

Effects of cholecalciferol supplementation on serum and urinary vitamin D metabolites and binding protein in HIV-infected youth

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