New Approaches to Assessing Vitamin D Status.

Introduction: Vitamin D is routinely assessed by measuring total 25-hydroxyvitamin D (25OHD). Measuring the free fraction of 25OHD may reflect the vitamin D status better in conditions where binding proteins are altered, as most of 25OHD is tightly bound to vitamin D binding protein or loosely bound to albumin and the free fraction is less than 1%. Free 25OHD can be measured by calculation or directly by immunoassay. Saliva is also thought to be a source of free 25OHD. Aims: To develop a salivary 25OHD method; to assess whether saliva contains binding proteins; and to investigate whether measurements of free 25OHD remain unchanged during an inflammatory insult despite a decrease in total 25OHD. Methods: Liquid chromatography mass spectrometry method development was performed for salivary 25OHD. Saliva was also assessed for the presence of binding proteins. 22 participants undergoing surgery for total hip or knee replacement was assessed for total and free levels of vitamin D. Results: 25OHD was able to be measured as low as 6.7pmol/L, however the recovery of 25OHD in saliva was unsatisfactory. No vitamin D binding protein or albumin were detected in saliva. Total 25OHD, vitamin D binding protein and albumin decreased by 23% (p=0.001), 20% (p<0.001) and 20% (p=0.001) following arthroplasty, whereas free 25OHD remained stable. Conclusion and future work: The salivary 25OHD method is promising, however there was inadequate recovery of 25OHD in salivary samples so the method requires improvement. The free fraction was shown to be more stable than total 25OHD during an acute inflammatory response, and this could be due to the free fraction not being as affected by the levels of binding proteins. Future research aims to assess whether salivary 25OHD also remains stable during an acute inflammatory response and therefore could be used as a surrogate marker of serum free 25OHD.