Method Development and Validation of Vitamin D2 and Vitamin D3 Using Mass Spectrometry Devon Victoria Riley A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science University of Washington 2016 Committee: Andrew Hoofnagle Geoffrey Baird Dina Greene Program Authorized to Offer Degree: Laboratory Medicine ©Copyright 2016 Devon V. Riley ii University of Washington Abstract Method Development and Validation of Vitamin D2 and Vitamin D3 Using Mass Spectrometry Devon V. Riley Chair of the Supervisory Committee: Associate Professor Andrew Hoofnagle, MD, PhD Vitamin D has long been known to maintain bone health by regulating calcium and phosphorous homeostasis. In recent years, scientists have discovered additional physiological roles for vitamin D. The complex interaction between the active vitamin D hormone and its metabolic precursors continues to be a rich area of research. Fundamental to this research is the availability of accurate and precise assays. Few published assays for vitamins D2 and D3 have contained sufficient details on method validation or performance characteristics. The liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay developed for this thesis has undergone a rigorous validation and proven to yield a sensitive and specific method that exceeds the capabilities of all previously published methods. Developing and validating a novel assay is often complicated by the lack of established acceptability standards. This thesis explores this challenge, specifically for establishing meaningful interpretations and qualification standards of the lower limit of the measuring interval. Altogether, future research focused on vitamins D2, D3 and the Vitamin D pathway can benefit from this robust LC-MS/MS assay and the associated quality parameters outlined in this thesis. iii Table of Contents Page List of Figures…………………………………………………………………...……... v List of Tables…………………………………………………………………………... vi Chapter 1: Introduction………………………………………………………………… 1 Chapter 2: Quantification of Vitamin D2 and Vitamin D3 in Human Serum to Evaluate Exposure……………………………………………………………………………….. 13 Introduction……………………………………………………………………. 14 Materials and methods………………………………………………………… 17 Results…………………………………………………………………………. 26 Discussion……………………………………………………………………... 41 Chapter 3: Lower Limit of the Measuring Interval……………………………………. 44 Chapter 4: Conclusion…………………………………………………………………. 56 Acknowledgements……………………………………………………………………. 60 References……………………………………………………………………………... 61 iv List of Figures Figure Number Page 1.1…………………………………………………………………………….……..…. 4 1.2………………………………………………………………………….…..………. 8 2.1……………………………………………………………………………………… 17 2.2……………………………………………………………………………………… 18 2.3……………………………………………………………………………………… 27 2.4……………………………………………………………………………………… 29 2.5……………………………………………………………………………………… 30 2.6……………………………………………………………………………………… 31 2.7……………………………………………………………………………………… 32 2.8……………………………………………………………………………………… 33 2.9……………………………………………………………………………………… 34 2.10…………………………………………………………………………………….. 35 3.1……………………………………………………………………………………… 50 3.2……………………………………………………………………………………… 51 v List of Tables Table Number Page 1.1………………………………………………………………………..….………… 5 2.1……………………………………………………………………………...……… 20 2.2………………………………………………………………………………...…… 21 2.3……………………………………………………………………………………… 28 2.4……………………………………………………………………………………… 36 2.5……………………………………………………………………………………… 37 2.6……………………………………………………………………………………… 37 2.7……………………………………………………………………………………… 38 2.8……………………………………………………………………………………… 39 2.9……………………………………………………………………………………… 39 2.10…………………………………………………………………………………….. 40 2.11…………………………………………………………………………………….. 40 3.1……………………………………………………………………………………… 49 vi Chapter 1: Introduction 1 Publications focusing on the association of vitamin D deficiency and various disease pathways have increased significantly over the last few years. The concentrations that define sufficiency (or deficiency) for the active hormone, its precursors, and its metabolites are still debated. For example, 25-hydroxyvitamin D (25(OHD)) is the precursor to the active hormone, 1,25-dihydroxyvitamin D (1,25(OH)2D), and is often used as the measure of vitamin D status because it has the longest half-life. The concentration defining sufficiency for 25(OH)D varies between medical institutions and reference laboratories, ranging from >20 ng/mL1 to >30 ng/mL2. 25(OH)D and 1,25(OH)2D are commonly tested in the clinical laboratory and numerous testing methods for these compounds have been documented, some of which are immunoassays, high performance liquid chromatography (HPLC), and liquid chromatography-tandem mass spectrometry (LC-MS/MS)3. In contrast, there are limited publications focused on quantifying the metabolites upstream of 25(OH)D, specifically vitamin D2 and D3. None of the documented vitamin D2/D3 assays utilize mass spectrometry. The objective of this thesis was to develop and validate an LC-MS/MS assay that quantifies vitamin D2 and D3. Vitamin D is complex and consists of several important precursors in addition to the active hormone (see Figure 1.1 and Table 1.1). Vitamin D3, also called cholecalciferol (hereafter referred to as vitamin D3), is synthesized in humans from 7-dehydrocholesterol (a cholesterol precursor) in the skin or consumed through the diet. 7-dehydrocholesterol absorbs UVB radiation, specifically between 280 and 320 nm, and is transformed to 4 previtamin D3. Previtamin D3 undergoes thermal isomerization to vitamin D3. If the skin is exposed to excessive sunlight, previtamin D3 can undergo transformation to inactive 2 5 metabolites, some of which are lumisterol3 and tachysterol3 (discussed in chapter 2). Vitamin D2, also called ergocalciferol (hereafter referred to as vitamin D2), is produced in plants and consumed through the diet. Vitamin D2 is produced in plants from the phototransformation of ergosterol after sunlight exposure. The conversion of ergosterol to vitamin D2 is analogous to the conversion of 7-dehydrocholesterol to previtamin D3 in human skin. Vitamin D2 and D3 are transported to the liver by vitamin D-binding protein where 25-hydroxylase catalyzes the reaction of vitamin D2 and vitamin D3 to 25- hydroxyvitamin D2 and 25-hydroxyvitamin D3 (collectively referred to as 25(OH)D), respectively. 25-hydroxylase is a microsomal cytochrome P450 (CYP) enzyme of which there are several isoforms. Each of these isoforms can hydroxylate vitamin D2 and 6 vitamin D3. 25(OH)D has a longer half-life and higher concentration compared to vitamin D2, vitamin D3, and active hormones. The active vitamin D hormones are 1,25- dihydroxyvitamin D2 and 1,25-dihydroxyvitamin D3 (collectively referred to as 1,25(OH)2D) and are produced from an additional hydroxylation by 1α-hydroxylase, mainly in the kidneys.7 The proximal convoluted tubules in the kidneys are the main source of this enzyme; however, other tissues and cell types, like keratinocytes, have the 4 ability to produce 1,25(OH)2D. 25(OH)D and 1,25(OH)2D are both catabolized by 24- hydroxylase (CYP24A1) to inactive metabolites, such as 24,25-dihydroxyvitamin D2 and 24,25-dihydroxyvitamin D3 [24,25(OH)2D], and 1,24,25-trihydroxyvitamin D2 and 8 1,24,25-trihydroxyvitamin D3 [1,24,25(OH)3D]. 3 Figure 1.1. Vitamin D Pathway. Vitamin D3 is produced after sunlight exposure and vitamin D2 is consumed through the diet. They are hydroxylated in the liver to become 25(OH)D. The active hormone (1,25(OH)2D) is produced from a second hydroxylation in the kidney. 25(OH)D and 1,25(OH)2D are further hydroxylated to inactive metabolites 24,25(OH)2D and 1,24,25(OH)3D. 4 Abbr./ MS Reference Metabolite Common Indication Assay? Range Name D / Supplementation Vitamin D 2 No 2 Ergocalciferol compliance, diet, N/A D / and sunlight Vitamin D 3 No 3 Cholecalciferol exposure 25(OH)D/ 25-hydroxyvitamin D Yes 2 Calcidiol* Vitamin D status 20-50 ng/mL☨ 25(OH)D/ 25-hydroxyvitamin D Yes 3 Calcidiol* 1,25(OH) D/ 1,25-dihydroxyvitamin D 2 Yes (active hormone) 2 Calcitriol** chronic kidney 17-72 pg/mL☨ 1,25(OH) D/ 1,25-dihydroxyvitamin D 2 Yes disease 3 Calcitriol** Table 1.1. Vitamin D metabolites with their abbreviations and common names. The table includes: whether or not a mass spectrometry assay existed prior to the publication of this thesis, how the metabolites are used, and reference ranges (if established). This table is not an exhaustive list of all vitamin D metabolites. *Collectively referred to as 25(OH)D **Collectively referred to as 1,25(OH)2D ☨Reference Range from UW Medicine1 The classical function of 1,25(OH)2D is to maintain calcium and phosphorous homeostasis through several mechanisms. 1,25(OH)2D stimulates osteoclasts to resorb bone, enhances absorption of calcium and phosphate from the small intestine, and decreases calcium excretion in the kidneys. 1,25(OH)2D also stimulates the endocrine system by binding to the vitamin D receptor (VDR). In addition to bone, intestine, and kidney, VDRs are found in many tissues such as the pancreas, parathyroid, skin, and 7 mammary epithelium. The widespread presence of VDRs suggests that 1,25(OH)2D also has noncalcaemic or non-classical functions. The non-classical functions are not well understood and further research is needed to understand this complex pathway. In a study by Bouillon, et al.,6 VDR-deficient and vitamin D deficient mice showed an increased sensitivity to autoimmune diseases, an increased susceptibility to oncogene- and chemocarcinogenic-induced cancers, and an increased risk of 5 cardiovascular disease. Knockout mice experiments led Bouillon, et