Objectives:
The UK Biobank (UKB) is a national study of the health of 500,000 people, aged 40-69 yrs, who completed health-related tests, a questionnaire and gave samples of blood and urine. Salivas from 120,000 of these subjects were collected, transported at 4oC and placed in ultra-low temperature archives by 24 hr following collection. The present study assessed the impact of holding saliva at 4oC for 24hr on a range of potential biomarkers.
Methods:
Unstimulated whole mouth saliva (UWMS) was collected from 23 volunteers aged 45-69 years. Salivas were divided into three aliquots and one aliquot was immediately frozen at -80°C, a ribonuclease inhibitor was added to aliquot 2 before freezing whilst the remaining aliquot was stored at 4°C for 24 hours and then frozen at -80°C (mimicking the UKB protocol). Samples were thawed and analysed.
Results:
Protein electrophoresis and Western blotting showed some loss of β-Actin under UKB conditions whilst cystatin S and MUC5B appeared to be unaffected. Immunoassay of a range of cytokines and C-Reactive Protein showed no reduction under UKB conditions. Assessment of mRNA by real-time PCR revealed no difference between UKB and immediately preserved samples. The oral microbiome, as revealed by 454 sequencing of tRNA, showed variations between subjects but paired samples within subjects were very similar. Levels of nitrite in saliva were reduced by 30% under UKB conditions but the steroid hormones cortisol and dehydroepiandrosterone were maintained.
Conclusions:
Concentrations of many potential biomarker molecules are not significantly reduced in salivas collected and stored under UKB conditions, which indicates that the frozen archive of 120,000 samples will be of great value in the phenotypic characterization of disease cohorts and the identification of potential prognostic markers of disease.
Acknowledgement:
The authors are grateful for funding from UKBiobank and NIHR Comprehensive Biomedical Research Centre at Guy's & St Thomas' Foundation Trust.