Development and Application of LC-MS/MS Methodology to Characterise Plasma Fat-Soluble Vitamer Profiles in Australian Children and Adults

Abstract

Vitamers are organic substances generally classified as either fat-soluble or water-soluble. Fat-soluble vitamers (FSV; include A, D, E and K) are biochemically diverse micronutrients with fundamentally different biological activities. The biochemical, medical and nutritional value of FSV are well documented. FSV play integral roles in a number of physiological processes that include immune function, bone health, vision, and coagulation. Suboptimal FSV concentrations are a significant risk factor for cardiometabolic complications, type II diabetes mellitus, cancer, and several immune system disorders, amongst others. There is increasing evidence that vitamer-specific interactions (e.g., between A, D and K group vitamers) may result in deficiencies, or hypervitaminosis, which can affect their bioavailability and metabolism. FSV concentrations in biological fluids (e.g., plasma and serum) represent the intersection of exogenous and endogenous factors and can help predict future health and disease outcomes. Over the past two decades, there has been a significant increase in translational clinical research and laboratory requests for measurements of FSV concentrations. Recent advances in analytical chemistry, including sophisticated and high-accuracy analytical platforms (e.g., orbitrap mass analyser, ultra-high performance liquid chromatography UHPLC), analytical column technology (e.g., sub-2 μm particle columns), and liquid handling automation techniques have enabled the high throughput identification, detection, and quantification of FSV in biological matrixes. In this thesis, I have developed and validated an automated robotic LC-MS/MS method for the multiplexed quantitation of 11 plasma FSV including all four major FSV groups (i.e., A group vitamers; retinol, retinoic acid and retinyl palmitate, D group vitamers 25 hydroxyvitamin D3 [25-OH-D3] and 1-α-25-dihydroxy-D3 [1-α-25-(OH)2-D3, E group vitamers α-tocopherol, γ-tocopherol and α- tocotrienol and K group vitamers phylloquinone [K1], Menatetrenone [MK-4], and menaquinone-7 [MK-7]). This method quantifies these vitamers at their respective physiological concentrations and at levels that indicate clinical deficiencies (e.g., α-tocopherol). The method I developed was used to characterise FSV profiles in plasma samples from the CheckPoint study of the Longitudinal Study of Australian Children’s (LSAC). I observed a strong vitamer-specific parent-child concordance for all FSV tested. My results also highlight that age, BMI, and sex are significant contributors to FSV concentrations in the family setting. Other exogenous factors: physical activity, supplement intake, healthy food consumption, sun exposure and sunscreen use and endogenous factors; volumetric body fat distribution, the bioavailability of the vitamers, differential inter-conversions between metabolically active vitamers, hereditary contributions and inflammatory conditions also appear to affect the epidemiological distributions of FSV concentrations. In conclusion, family, age, sex, and BMI are important parameters characterising FSV plasma levels in a population setting. The associated endogenous and exogenous factors, however, need to be further validated in future studies to fully elucidate how the FSV profile changes in the epidemiological context and to predict the future health and disease outcomes of an individual.