Vitamin K and equine osteocalcin: an enigma explored

Vitamin K and equine osteocalcin: an enigma explored

Vitamin K has received considerably less attention over the past 50 years compared to other fat soluble vitamins. Intakes of vitamin K beyond that required for normal blood coagulation were believed to confer no additional benefits, and therefore rarely investigated. The requirements and physiological role of vitamin K appeared to be secure. This view of vitamin K has changed recently following the delineation of its cofactor role in protein carboxylation, and the growing awareness of its interactions with other essential metabolic functions, beyond that of blood coagulation; there is much to be discovered. At present, seventeen vitamin Kdependent proteins (VKDPs) have been characterised, but the functionality of some still remains to be elucidated. Vitamin K has recently been implicated in osteoarthritis and accumulating evidence from human studies supports a protective role for vitamin K. To date however, little research into the role vitamin K may play in equine bone development exists.

Developmental orthopaedic disease (DOD) is a term that encompasses a number of bone related conditions in the horse. It is a significant cause of lameness and wastage in a number of breeds, in particular Thoroughbreds and Warmbloods. It has clinical features in common with that of osteoporosis and osteoarthritis in the elderly. The VKDP osteocalcin is one of the few that has been extensively studied in humans and rodents. Located within bone, this protein plays a vital role in bone metabolism, facilitating the binding of bone minerals with protein. Decreased functionality of this protein has been found to be associated with increased fracture risk and osteoporosis in humans.

The studies in this program were initiated to establish the relationship between vitamin K status and the development of DOD in foals. As bone development begins in utero, the first step was to examine placental transfer of the vitamin and then determine availability to the foal through milk. It was found in the initial experiments (Chapter 3), that foals at birth, had negligible concentrations of vitamin K1 (<0.01ng/mL) in both umbilical cord and foal plasma. The results clearly demonstrated that there is limited trans-placental transfer of vitamin K in the horse. It was also shown that milk can be enriched with vitamin K by supplementation of the mare with the vitamin. These studies confirmed that plasma concentrations were not a reliable measure of overall vitamin K status.

Circulating osteocalcin and, its carboxylation status, has been proposed as one of the indicators of vitamin K status and a ‘biomarker’ of bone metabolism. Current assays available to measure osteocalcin, however, do not provide information about the degree of osteocalcin carboxylation. The aim of the subsequent studies, was to exploit the recent advances in the use of mass spectrometry (MS) based techniques and investigate the feasibility of a method to accurately measure the carboxylation status of circulating osteocalcin in equine plasma, after vitamin K supplementation.

The development of a MS-based method to quantify circulating carboxylated osteocalcin in plasma was investigated in Chapter 5. In this study, equine osteocalcin was in-silico digested into peptides and synthetically synthesised. A targeted MS-analysis, by LC-MS/MS MRM method, was developed based on these osteocalcin specific peptides. The method was not sensitive enough to detect native osteocalcin in plasma samples collected in Chapter 3. Studies conducted in Chapter’s 6 and 7 aimed to validate the use of a proteomics based enrichment strategy, to increase the sensitivity of this assay for detection of osteocalcin in plasma and other carboxylated VKDPs.

The use of hydroxyapatite was investigated in Chapter 6 as an as an enrichment strategy to detect osteocalcin in plasma. A method was successfully developed and validated with the osteocalcin synthetic standards; Carboxylated Analysis by Ceramic Hydroxyapatite Enrichment (CACHE). The studies conducted in Chapter 7 characterised and investigated the applicability of the CACHE protocol on biological samples with known elevation in vitamin K using foal plasma. Plasma samples taken from the foals at day 14 (Chapter 3) that showed a statistically significant difference between the control group and the supplemented group (P<0.05) were chosen for this study. In this study a number of unique proteins were identified in the enriched elute plasma fraction however, no carboxylated VKDPs, other than prothrombin, were detected. It is anticipated that further optimisation of the CACHE method will facilitate the detection other carboxylated VKDPs.

When the findings of these Chapters are considered, further examination of the quantification and role of osteocalcin is warranted. A key outcome of this thesis was the development of a novel method: CACHE. With further developments in the fields of proteomics explored in this thesis, it is anticipated that new insights into the metabolism and mechanisms of vitamin K, and its intrinsic relationship to osteocalcin and other VKDPs will be uncovered. It should then be possible to delineate the relationship of vitamin K status to DOD development in the foal.