Assessing a method and reference material for quantification of vitamin D binding protein during pregnancy

Abstract

Vitamin D plays a vital role in successful pregnancy outcomes for both the mother and fetus. Vitamin D is bound to vitamin D binding protein (VDBP) in blood and is carried to the liver, kidneys and other target tissues. Accurate measurements of the clinically measured metabolite of vitamin D, 25-hydroxyvitamin D [25(OH)D], depend on complete removal from the binding protein. It has been found that VDBP concentrations increase in maternal serum during pregnancy, obfuscating the accuracy of 25(OH)D concentration measurements in pregnant women. Additionally, measurements of VDBP concentrations during pregnancy have been performed using immunoassays, which suffer from variations due to differences in antibody epitopes, making clinical comparisons difficult. Quantification of VDBP is also of interest because changes in VDBP expression levels may indicate negative outcomes during pregnancy, such as preterm delivery and restricted fetal growth. To address the need for accurate measurement of VDBP during pregnancy, a method using liquid chromatography-isotope dilution mass spectrometry (LC-IDMS) was developed to quantify VDBP using isotopically labeled peptides as internal standards. This method was used to quantify VDBP in Standard Reference Material® (SRM) 1949 Frozen Human Prenatal Serum, which was prepared from separate serum pools of women who were not pregnant and women during each trimester of pregnancy. VDBP concentrations were found to be lowest in the serum pool from non-pregnant women and increased in each trimester. These data had good repeatability and were found to be suitable for reference value assignment of VDBP in SRM 1949.

Keywords: 25(OH)D, 25-hydroxyvitamin D; AAA, amino acid analysis; CV, coefficient of variation; GC-globulin; Isotope-dilution; LC-IDMS, liquid chromatography-isotope dilution mass spectrometry; LC-MS/MS; LC-MS/MS, liquid-chromatography tandem mass spectrometry; MRM; MRM, multiple reaction monitoring; NIST, National Institute of Standards and Technology; PARs, peak area ratios; Pregnancy; Quantification; SRM, Standard Reference Material®; TCEP, tris(2-carboxyethyl)phosphine hydrochloride; TFA, trifluoroacetic acid; TFE, trifluoroethanol; VDBP, vitamin D binding protein; Vitamin D binding protein.

Fig. 1

Total ion chromatograms of the two MRM transitions for TSALSAK and VLEPTLK collected during analyses of digested samples of SRM 1949. The non-pregnant serum pool is shown.

Fig. 2

One-way ANOVA analysis of the serum pooled from non-pregnant women prepared in triplicate on three different days. Aliquots of the three vials were removed on day 1 and prepared after one freeze-thaw cycle for days 2 and 3. Diamonds indicate the 95% confidence interval. Mid green line is the mean of each set and the remaining lines define the quartiles. Grey line is the grand mean. The star indicates a significant difference according to a post hoc Tukey test.