The ratio of serum 24,25-dihydroxyvitamin D3 to 25-hydroxyvitamin D3 is predictive of 25-hydroxyvitamin D3 response to vitamin D3 supplementation

doi:10.1016/j.jsbmb.2011.05.003

The Journal of Steroid Biochemistry and Molecular Biology

Volume 126, Issues 3–5, September 2011, Pages 72-77

https://doi.org/10.1016/j.jsbmb.2011.05.003 Get rights and content

Abstract

24,25-Dihydroxyvitamin D (24,25VD) is a major catabolite of 25-hydroxyvitamin D (25VD) metabolism, and may be physiologically active. Our objectives were to: (1) characterize the response of serum 24,25VD₃ to vitamin D₃ (VD₃) supplementation; (2) test the hypothesis that a higher 24,25VD₃ to 25VD₃ ratio (24,25:25VD₃) predicts 25VD₃ response.

Serum samples (n = 160) from wk 2 and wk 6 of a placebo-controlled, randomized clinical trial of VD₃ (28,000 IU/wk) were analyzed for serum 24,25VD₃ and 25VD₃ by mass spectrometry.

Serum 24,25VD₃ was highly correlated with 25VD₃ in placebo- and VD₃-treated subjects at each time point (p < 0.0001). At wk 2, the 24,25:25VD₃ ratio was lower with VD₃ than with placebo (p = 0.035). From wk 2 to wk 6, the 24,25:25VD₃ ratio increased with the VD₃ supplement (p < 0.001) but not with placebo, such that at wk 6 this ratio did not significantly differ between groups. After correcting for potential confounders, we found that 24,25:25VD₃ at wk 2 was inversely correlated to the 25VD₃ increment by wk 6 in the supplemented group (r = −0.32, p = 0.02) but not the controls.

There is a strong correlation between 24,25VD₃ and 25VD₃ that is only modestly affected by VD₃ supplementation. This indicates that the catabolism of 25VD₃ to 24,25VD₃ rises with increasing 25VD₃. Furthermore, the initial ratio of serum 24,25VD₃ to 25VD₃ predicted the increase in 25VD₃. The 24,25:25VD₃ ratio may therefore have clinical utility as a marker for VD₃ catabolism and a predictor of serum 25VD₃ response to VD₃ supplementation.

Highlights

► Serum 24,25-dihydroxyvitamin D₃ (24,25VD₃) response to vitamin D₃ (VD₃) supplement. ► Serum 24,25VD₃ correlated strongly with 25-hydroxyvitamin D₃ (25VD₃). ► Initial ratio of serum 24,25VD₃ to 25VD₃ predicted the increase in 25VD₃. ► 24,25:25VD₃ ratio reflects 25VD₃ catabolism and response to VD₃ supplement.

Introduction

Vitamin D (VD) has received considerable attention because of associations between low VD status and increased risk for several diseases, including osteoporosis, cancers, multiple sclerosis, diabetes, cardiovascular disease, and microbial infections [1], [2], [3], [4], [5], [6], [7]. The determinants of serum 25-hydroxyvitamin D (25VD), the classic measure of VD status, include environmental (e.g. season, latitude, sunlight, diet) [8], [9], demographic [e.g. ethnicity, body mass index (BMI)] [10], and genetic factors (e.g. polymorphisms in metabolism and transport genes) [11], [12], [13]. However, the factors that modify response to VD supplementation warrant further study, especially in view of the large inter-individual variation that has been reported in serum 25VD response to supplementation with identical doses of VD [12], [14], [15]. An analysis of 24,25-dihydroxyvitamin D (24,25VD), the major metabolite of 25VD, could provide clinically relevant information that may shed light on these inter-individual differences.

24,25VD is produced via 24-hydroxylation of 25VD by the cytochrome P450 24-hydroxylase enzyme (CYP24A1; V_max = 0.088 mol/min/mol P450, K_m = 160 nM) [16]. In addition, CYP24A1 catalyzes the side-chain metabolism of 1,25-dihydroxyvitamin D (1,25VD), considered to be the primary active metabolite. CYP24A1 is expressed in many tissues [17], [18], [19], [20] but the biological activity of 24,25VD remains controversial. The general view is that 24,25VD production is the first step to inactivate 25-hydroxylated metabolites of VD, thus regulating synthesis of 1,25VD [21], [22]. However, there is considerable evidence demonstrating that 24,25VD has unique biological properties, including physiological roles in embryogenesis, cartilage development, and fracture repair [23], [24], [25], [26], [27], [28], [29]. Recently, Larsson et al. demonstrated that 24,25VD binds to catalase, suggesting that 24,25VD-mediated signal transduction may occur through modulating hydrogen peroxide production [30].

Few clinical studies have reported circulating 24,25VD concentrations [31], [32], [33], [34], [35], [36], [37], likely because its measurement is technically challenging and its physiological role is unclear. Liquid chromatography–tandem mass spectrometry (LC–MS/MS) has received increased attention because it is capable of measuring 25VD₂ and 25VD₃ separately, but the capability for measuring 24,25VD has not been widely exploited. Furthermore, the effects of VD₃ supplementation on serum 24,25VD₃ concentrations in humans are unknown. Here, we characterize the biochemical response of serum 24,25VD₃ to VD₃ supplementation in healthy adults using a highly sensitive and specific LC–MS/MS assay for simultaneous determination of serum 25VD₃ and 24,25VD₃ concentrations. We hypothesized that a higher 24,25VD₃ to 25VD₃ ratio (24,25:25VD₃) would predict a smaller serum 25VD₃ response to an increased VD₃ intake because a relatively higher 24,25VD₃ would indicate higher catabolism.

Section snippets

Study samples

Human serum samples (n = 160) were obtained from a randomized, double-blind, placebo-controlled clinical trial carried out in Toronto, Canada (latitude 43°N). Healthy young adults, half of whom were female, received either 28,000 IU VD₃/wk as a supplement or fortified cheese of equivalent bioavailability (n = 60), or a placebo (n = 20), for 8 weeks during the winter months [38]. Serum aliquots were stored at −80 °C until analysis. Under these storage conditions, VD metabolites are stable in serum or

LC–MS/MS method evaluation

All data were normally distributed, as indicated by the Kolmogorov–Smirnov test. LC–MS/MS assay performance characteristics are shown in Table 1. Total imprecision for all VD metabolites (CV = 7.3–14%) was comparable to immunoassays (5–15%) [40]. Linearity was confirmed across the analytical measurement range for all VD metabolites. The functional sensitivity for all VD metabolites (≤1 nmol/L) was lower (i.e. higher sensitivity) than immunoassays (≤10 nmol/L). 25VD₂ was not detected in any sample.

Discussion

Our data suggest a new clinical indication utility for measuring serum 24,25VD₃, the major metabolite of 25VD₃, by a novel LC–MS/MS assay for simultaneous determination of 25VD₃ and 24,25VD₃. The developed LC–MS/MS method was highly sensitive, specific, and the first to quantify 24,25VD₃ in serum. Investigators should therefore exploit the capability of LC–MS/MS methods to measure both serum 24,25VD and 25VD simultaneously. Indeed, 24,25VD is the most abundant 25VD metabolite and its roles in

Acknowledgments

This work was supported by grants from the Dairy Farmers of Canada and the Natural Sciences and Engineering Research Council of Canada (NSERC). We thank the laboratory of Dr. Eleftherios P. Diamandis (University Health Network, Toronto, Canada) for performing the LC–MS/MS analyses. We also thank DiaSorin for the in kind donation of the LIAISON 25 OH Vitamin D TOTAL kits.

References (45)

J.M. Lappe et al.
Vitamin D and calcium supplementation reduces cancer risk: results of a randomized trial
Am. J. Clin. Nutr.
(2007)
M. Urashima et al.
Randomized trial of vitamin D supplementation to prevent seasonal influenza A in school children
Am. J. Clin. Nutr.
(2010)
K. Brock et al.
Low vitamin D status is associated with physical inactivity, obesity and low vitamin D intake in a large US sample of healthy middle-aged men and women
J. Steroid Biochem. Mol. Biol.
(2010)
L.M. Hall et al.
Vitamin D intake needed to maintain target serum 25-hydroxyvitamin D concentrations in participants with low sun exposure and dark skin pigmentation is substantially higher than current recommendations
J. Nutr.
(2010)
L. Fu et al.
Common genetic variants of the vitamin D binding protein (DBP) predict differences in response of serum 25-hydroxyvitamin D [25(OH)D] to vitamin D supplementation
Clin. Biochem.
(2009)
T.J. Wang et al.
Common genetic determinants of vitamin D insufficiency: a genome-wide association study
Lancet
(2010)
H.J. Armbrecht et al.
Expression of 25-hydroxyvitamin D 24-hydroxylase cytochrome P450 in kidney and intestine. Effect of 1,25-dihydroxyvitamin D and age
FEBS Lett.
(1991)
J.L. Omdahl et al.
The 25-hydroxyvitamin D 24-hydroxylase
A.W. Norman et al.
Update on biological actions of 1alpha, 25(OH)2-vitamin D3 (rapid effects) and 24R,25(OH)2-vitamin D3
Mol. Cell Endocrinol.
(2002)
S. Masuda et al.
A novel high-performance liquid chromatographic assay for vitamin D metabolites using a coulometric electrochemical detector
J. Pharm. Biomed. Anal.
(1997)

J.M. Mata-Granados et al.

Inappropriate serum levels of retinol, alpha-tocopherol, 25 hydroxyvitamin D3 and 24,25 dihydroxyvitamin D3 levels in healthy Spanish adults: simultaneous assessment by HPLC

Clin. Biochem.

(2008)

I. Schoenmakers et al.

Interrelation of parathyroid hormone and vitamin D metabolites in adolescents from the UK and The Gambia

J. Steroid Biochem. Mol. Biol.

(2010)

D. Wagner et al.

The bioavailability of vitamin D from fortified cheeses and supplements is equivalent in adults

J. Nutr.

(2008)

D. Wagner et al.

An evaluation of automated methods for measurement of serum 25-hydroxyvitamin D

Clin. Biochem.

(2009)

Y.R. Lou et al.

25-Hydroxyvitamin D(3) is an agonistic vitamin D receptor ligand

J. Steroid Biochem. Mol. Biol.

(2010)

R. St-Arnaud

CYP24A1-deficient mice as a tool to uncover a biological activity for vitamin D metabolites hydroxylated at position 24

J. Steroid Biochem. Mol. Biol.

(2010)

H.A. Bischoff-Ferrari et al.

Fracture prevention with vitamin D supplementation: a meta-analysis of randomized controlled trials

JAMA

(2005)

E. Giovannucci et al.

25-Hydroxyvitamin D and risk of myocardial infarction in men: a prospective study

Arch. Intern. Med.

(2008)

P.T. Liu et al.

Toll-like receptor triggering of a vitamin D-mediated human antimicrobial response

Science

(2006)

K.L. Munger et al.

Serum 25-hydroxyvitamin D levels and risk of multiple sclerosis

JAMA

(2006)

P.R. von Hurst et al.

Vitamin D supplementation reduces insulin resistance in South Asian women living in New Zealand who are insulin resistant and vitamin D deficient—a randomised, placebo-controlled trial

Br. J. Nutr.

(2010)

R. Jorde et al.

Cross-sectional and longitudinal relation between serum 25-hydroxyvitamin D and body mass index: the Tromso study

Eur. J. Nutr.

(2010)

Cited by (136)

Liquid chromatography-tandem mass spectrometry in fat-soluble vitamin deficiency
2023, Clinica Chimica Acta
Fat-soluble vitamins, including vitamins A, D, E, and K, are essential for maintaining normal body function and metabolism. Fat-soluble vitamin deficiency may lead to bone diseases, anemia, bleeding, xerophthalmia, etc. Early detection and timely interventions are significant for preventing vitamin deficiency-related diseases. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is developing into a potent instrument for the precise detection of fat-soluble vitamins due to its high sensitivity, high specificity, and high resolution.
Determination of 24,25-dihydroxyvitamin D<inf>3</inf> in Vitamin D External Quality Assessment Scheme samples using a reference measurement procedure
2023, Journal of Steroid Biochemistry and Molecular Biology
Ninety archived human serum samples from the Vitamin D External Quality Assessment Scheme (DEQAS) were analyzed using a reference measurement procedure (RMP) based on isotope dilution liquid chromatography – tandem mass spectrometry (ID LC-MS/MS) for the determination of 24,25-dihydroxyvitamin D₃ [24,25(OH)₂D₃]. These 24,25(OH)₂D₃ results, in conjunction with concentration values assigned using RMPs for 25-hydroxyvitamin D₂ [25(OH)D₂] and 25-hydroxyvitamin D₃ [25(OH)D₃], provide a valuable resource for assessing the accuracy of measurements for 24,25(OH)₂D₃ and for investigating the relationship between 24,25(OH)₂D₃ and 25(OH)D₃. Results for 24,25(OH)₂D₃ using the RMP were compared to DEQAS consensus values demonstrating that the consensus values were not sufficient to assess the accuracy of measurements among different laboratories and methods. A multivariable regression analysis approach using historical DEQAS consensus values for various total 25(OH)D assays was used to assess the contribution of 24,25(OH)₂D₃ concentration on the assay response. The response of several ligand binding assays for total 25(OH)D was shown to be impacted by the presence of 24,25(OH)₂D₃.
Pharmacology and pharmacokinetics of vitamin D
2023, Feldman and Pike's Vitamin D: Volume One: Biochemistry, Physiology and Diagnostics
This chapter describes the metabolic parameters of vitamin D and its metabolites according to the framework of pharmacokinetics. The pharmacokinetics of vitamin D is considerably more complex than a standard pharmacological agent, because of the required stepwise hydroxylation into the active form, 1,25(OH)₂D, its endogenous, cutaneous synthesis, and presence of vitamin D and its metabolites in the circulation and tissues. Also, its binding to the vitamin D–binding protein (DBP) influences the properties of the vitamin D metabolites. These aspects are discussed throughout this chapter. The absorption, distribution, dose-response, metabolism, excretion, and toxicity of vitamin D and its metabolites are reviewed. In addition, we present the current knowledge of factors influencing 25-hydroxyvitamin D half-life, the influence of DBP on vitamin D metabolism, and the differences in pharmacokinetics between bound and free 25-hydroxyvitamin D.
Mass spectrometry assays of vitamin D metabolites
2023, Feldman and Pike's Vitamin D: Volume One: Biochemistry, Physiology and Diagnostics
Liquid chromatography-tandem mass spectrometry (LC–MS/MS) has emerged as the gold standard technique with the capability for reliable, accurate, and high-throughput quantification of circulating vitamin D metabolites. The complexity of vitamin D metabolism has driven the continuous development of the basic steps involved in LC–MS/MS methodology, including sample extraction, derivatization, separation, and mass spectrometry, used to interrogate the role of vitamin D nutrition and metabolism in human health and disease states. From the perspective of both the high-throughput clinical chemistry laboratory as well as basic research applications, this chapter describes how LC–MS/MS can be optimized to provide sensitive and specific measurements of 25-hydroxyvitamin D, along with other low-abundance metabolites including 24,25-dihydroxyvitamin D and 1,25-dihydroxyvitamin D, simultaneously. Furthermore, we describe the advantages of using LC–MS/MS in studying mouse models of vitamin D metabolism, and how this work has contributed to an improved understanding of the pathophysiology of certain vitamin D–related disease states.
23,25-Dihydroxyvitamin D<inf>3</inf> is liberated as a major vitamin D<inf>3</inf> metabolite in human urine after treatment with β-glucuronidase: Quantitative comparison with 24,25-dihydroxyvitamin D<inf>3</inf> by LC/MS/MS
2022, Journal of Steroid Biochemistry and Molecular Biology
The complete understanding of the excretion of surplus 25-hydroxyvitamin D₃ [25(OH)D₃] in humans remains to be accomplished. In our previous study, 24,25-dihydroxyvitamin D₃ [24,25(OH)₂D₃] 24-glucuronide was identified as a major urinary vitamin D₃ metabolite, while the glucuronide of 23,25-dihydroxyvitamin D₃ [23,25(OH)₂D₃] is another metabolite of interest but has not been sufficiently evaluated. Although the quantitative analysis of 24,25(OH)₂D₃ liberated in urine by the treatment with β-glucuronidase (GUS) has been conducted, no information was provided about the amount of the glucuronidated 23,25(OH)₂D₃ in the urine. In this study, we first developed and validated a liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS)-based method for the simultaneous quantification of 23,25(OH)₂D₃ and 24,25(OH)₂D₃ liberated in urine by GUS. The analysis of the urine samples revealed that the amount of 23,25(OH)₂D₃ was almost as much as that of 24,25(OH)₂D₃, in contrast to the fact that the plasma concentration of 23,25(OH)₂D₃ was much lower than that of 24,25(OH)₂D₃. These results strongly suggested that 23,25(OH)₂D₃ is more susceptible to glucuronidation and more promptly excreted into urine than 24,25(OH)₂D₃. Furthermore, the amount ratios of 23,25(OH)₂D₃ to 24,25(OH)₂D₃ in the urine samples did not markedly vary during the day (morning/evening) and even by a week-long vitamin D₃ supplementation (1000 IU/body/day). We concluded that the C-23 hydroxylation plays a crucial role in the urinary excretion of surplus 25(OH)D₃.
Clinical and biomarker modifiers of vitamin D treatment response: The Multi-Ethnic Study of Atherosclerosis
2022, American Journal of Clinical Nutrition
Different 25-hydroxyvitamin D [25(OH)D] thresholds for treatment with vitamin D supplementation have been suggested and are derived almost exclusively from observational studies. Whether other characteristics, including race/ethnicity, BMI, and estimated glomerular filtration rate (eGFR), should also influence the threshold for treatment is unknown.
The aim was to identify clinical and biomarker characteristics that modify the response to vitamin D supplementation.
A total of 666 older adults in the Multi-Ethnic Study of Atherosclerosis (MESA) were randomly assigned to 16 wk of oral vitamin D₃ (2000 IU/d; n = 499) or placebo (n = 167). Primary outcomes were changes in serum parathyroid hormone (PTH) and 1,25-dihydroxyvitamin D [1,25(OH)₂D] concentrations from baseline to 16 wk.
Among 666 participants randomly assigned (mean age: 72 y; 53% female; 66% racial/ethnic minority), 611 (92%) completed the study. The mean (SD) change in PTH was −3 (16) pg/mL with vitamin D₃ compared with 2 (18) pg/mL with placebo (estimated mean difference: –5; 95% CI: –8, –2 pg/mL). Within the vitamin D₃ group, lower baseline 25-hydroxyvitamin D [25(OH)D] was associated with a larger decline in PTH in a nonlinear fashion. With baseline 25(OH)D ≥30 ng/mL as the reference, 25(OH)D <20 ng/mL was associated with a larger decline in PTH with vitamin D₃ supplementation (–10; 95% CI: –15, –6 pg/mL), whereas 25(OH)D of 20–30 ng/mL was not (–2; 95% CI: –6, 1 pg/mL). A segmented threshold model identified a baseline 25(OH)D concentration of 21 (95% CI: 13, 31) ng/mL as an inflection point for difference in change in PTH. Race/ethnicity, BMI, and eGFR did not modify vitamin D treatment response. There was no significant change in 1,25(OH)₂D in either treatment group.
Of characteristics most commonly associated with vitamin D metabolism, only baseline 25(OH)D <20 ng/mL modified the PTH response to vitamin D supplementation, providing support from a clinical trial to use this threshold to define insufficiency. This trial was registered at clinicaltrials.gov as NCT02925195.

View all citing articles on Scopus

View full text

The ratio of serum 24,25-dihydroxyvitamin D3 to 25-hydroxyvitamin D3 is predictive of 25-hydroxyvitamin D3 response to vitamin D3 supplementation

Abstract

Highlights

Introduction

Section snippets

Study samples

LC–MS/MS method evaluation

Discussion

Acknowledgments

Am. J. Clin. Nutr.

Am. J. Clin. Nutr.

J. Steroid Biochem. Mol. Biol.

J. Nutr.

Clin. Biochem.

Lancet

FEBS Lett.

Mol. Cell Endocrinol.

J. Pharm. Biomed. Anal.

Clin. Biochem.

J. Steroid Biochem. Mol. Biol.

J. Nutr.

Clin. Biochem.

J. Steroid Biochem. Mol. Biol.

J. Steroid Biochem. Mol. Biol.

Fracture prevention with vitamin D supplementation: a meta-analysis of randomized controlled trials

JAMA

25-Hydroxyvitamin D and risk of myocardial infarction in men: a prospective study

Arch. Intern. Med.

Toll-like receptor triggering of a vitamin D-mediated human antimicrobial response

Science

Serum 25-hydroxyvitamin D levels and risk of multiple sclerosis

JAMA

Vitamin D supplementation reduces insulin resistance in South Asian women living in New Zealand who are insulin resistant and vitamin D deficient—a randomised, placebo-controlled trial

Br. J. Nutr.

Cross-sectional and longitudinal relation between serum 25-hydroxyvitamin D and body mass index: the Tromso study

Eur. J. Nutr.

The ratio of serum 24,25-dihydroxyvitamin D₃ to 25-hydroxyvitamin D₃ is predictive of 25-hydroxyvitamin D₃ response to vitamin D₃ supplementation