Daytime changes of salivary biomarkers involved in pain

The study aimed to investigate salivary levels of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), substance P (SP) and glutamate at five time points from morning to afternoon in a well-characterised healthy and pain-free individuals. Ten young adults were included. Unstimulated and stimulated whole saliva were collected from each participant repeatedly across the day. Blood samples were drawn in connection with the first and last saliva sample as reference standard. Levels of NGF and BDNF were determined using gel-free Western blot technology, glutamate levels were analysed using a colorimetric assay, and SP was determined using a commercially available ELISA. Salivary NGF and BDNF showed significant differences between the different collection times in both unstimulated (NGF; P = .006; BDNF; P = .026) and stimulated whole saliva (NGF; P = .006; BDNF; P = .019). The highest concentrations of the neuropeptides were expressed in the early morning, and they thereafter decreased across the day. In contrast, the expression of salivary glutamate and SP did not show any significant changes across the day. Plasma levels of NGF were higher in the evening sample (P = .028); otherwise, there were no significant differences for any of the other markers between morning and evening samples. NGF and BDNF in whole saliva showed a significant variation across the day. On the contrary, no variation in the levels of SP and glutamate was detected. These findings highlight the importance of consistency in the collection time and approach in biomarker studies using saliva.