Vitamin D in human serum and adipose tissue after supplementation

Abstract

Background: Serum 25-hydroxyvitamin D [25(OH)D] concentration is an indicator of vitamin D exposure, but it is also influenced by clinical characteristics that affect 25(OH)D production and clearance. Vitamin D is the precursor to 25(OH)D but is analytically challenging to measure in biological specimens.

Objectives: We aimed to develop and validate a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for quantification of vitamins D3 and D2 in serum and to explore the potential of circulating vitamin D as a biomarker of exposure in supplementation trials.

Methods: The method was validated using guideline C62-A from the Clinical and Laboratory Standards Institute and was applied in 2 pilot clinical trials of oral vitamin D3 supplementation. Pilot study 1 included 22 adults randomly assigned to placebo or 2000 IU/d. Blood was collected at baseline, 1, 3, 6, and 12 mo. Pilot study 2 included 15 adults randomly assigned to 2000 or 4000 IU/d. Blood and subcutaneous (SUBQ) adipose tissue were collected at baseline and 3 mo.

Results: In study 1, mean change (baseline to 3 mo) in serum vitamin D3 was -0.1 ng/mL in the placebo group and 6.8 ng/mL in the 2000 IU/d group (absolute difference: 6.9; 95% CI: 4.5, 9.3 ng/mL). In study 2, mean change (baseline to 3 mo) in serum vitamin D3 was 10.4 ng/mL in the 2000 IU/d group and 22.2 ng/mL in the 4000 IU/d group (fold difference: 2.15; 95% CI: 1.40, 3.37). Serum and adipose tissue vitamin D3 concentrations were correlated, and the dose-response of vitamin D3 in adipose mirrored that in serum.

Conclusions: We validated a sensitive, robust, and high-throughput LC-MS/MS method to quantify vitamins D3 and D2 in serum. Serum and SUBQ adipose tissue vitamin D3 concentrations increased proportionally to dose with 3 mo of daily supplementation.These trials were registered at clinicaltrials.gov as NCT00552409 (pilot study 1) and NCT01477034 (pilot study 2).

Keywords: Clinical and Laboratory Standards Institute; cholecalciferol; ergocalciferol; liquid chromatography–tandem mass spectrometry; liquid-liquid extraction; validation; vitamin D.

© The Author(s) 2020. Published by Oxford University Press on behalf of the American Society for Nutrition.

Figures

FIGURE 1

Time courses of serum vitamin D3 concentration (A) and serum 25(OH)D3 concentration (B) in the placebo and vitamin D3 (2000 IU/d) groups in pilot study 1. Data are means ± SDs; n = 11/group, 104 observations total, and 6 missing observations (2 at month 1, 1 at month 6, and 3 at month 12). SI conversion factors: vitamin D3, 1 ng/mL = 2.6 nmol/L; 25(OH)D3, 1 ng/mL = 2.5 nmol/L. BL, baseline; 25(OH)D3, 25-hydroxyvitamin D3.

FIGURE 2

Serum 25(OH)D3 concentration compared with serum vitamin D3 concentration at baseline and in the placebo group, i.e., in the unsupplemented state (open shapes), and after 1, 3, 6, or 12 mo of supplementation (closed shapes) in 2 pilot clinical trials of vitamin D3 supplementation. Data are individual participant data (placebo, n = 11; 2000 IU/d, n = 19; 4000 IU/d, n = 7), all observations. SI conversion factors: vitamin D3, 1 ng/mL = 2.6 nmol/L; 25(OH)D3, 1 ng/mL = 2.5 nmol/L. BL, baseline; 25(OH)D3, 25-hydroxyvitamin D3.

FIGURE 3

Serum and SUBQ adipose tissue concentrations of vitamin D3 and 25(OH)D3 at baseline (open circles) and after 3 mo of supplementation (closed circles) with 2000 IU/d (n = 8) or 4000 IU/d (n = 7) of vitamin D3 in pilot study 2. For each visit separately, bivariate associations were estimated with r when both variables were normally distributed or rs when ≥1 variable was skewed. (A) Adipose tissue 25(OH)D3 compared with adipose tissue vitamin D3; (B) adipose tissue vitamin D3 compared with serum vitamin D3; (C) adipose tissue 25(OH)D3 compared with serum 25(OH)D3; (D) adipose tissue vitamin D3 compared with serum 25(OH)D3; (E) adipose tissue 25(OH)D3 compared with serum vitamin D3; (F) serum 25(OH)D3 compared with serum vitamin D3. SI conversion factors: vitamin D3, 1 ng/mL = 2.6 nmol/L, 1 pg/mg = 2.6 nmol/kg; 25(OH)D3, 1 ng/mL = 2.5 nmol/L, 1 pg/mg = 2.5 nmol/kg. BL, baseline; SUBQ, subcutaneous; 25(OH)D3, 25-hydroxyvitamin D3.