Saliva is often neglected as a body fluid of diagnostic or prognostic value, even though generally well accepted by the patients. This is due to lack of a standardized collection procedure. The aim of this study was to identify the ideal saliva collection technique and develop new sensitive methods to detect and analyse markers related to pain in healthy pain-free subjects. Plasma and five different saliva collection approached was evaluated during strictly controlled conditions. Levels of nerve growth factor (NGF), calcitonin gene-related peptide (CGRP) and brain derived neurotropic factor (BDNF) were determined using novel western blotting based technology. Glutamate and substance P (SP) was determined using commercial available methods. Several new isoforms were found for NGF, CGRP and BDNF in saliva. The isoform pattern showed significant variation in both expression and chemiluminescence levels between different collection methods. New sensitive methods to study pain related markers in saliva were developed in this study. Furthermore, we are first to demonstrate a correlation between the Glutamate concentration in stimulated whole saliva and blood. However, the fundamental conclusion drawn is the importance of consistency in the collection method.

Determination of the variability in the salivary proteome is a prerequisite for the development of saliva as a diagnostic and prognostic tool in particular physiological states. In this context, it is important that technical variability induced by sample collection and processing is kept at minimum to be able to reproducibly assess variability in states of health and disease. In the current study, the proteome profile in unstimulated and stimulated whole, parotid and sublingual saliva was investigated using two-dimensional gel electrophoresis. Saliva samples were structurally collected from ten examined and characterized healthy individuals during the exactly same conditions. The results demonstrated that different collection methods provide clear differences in the snapshot of the salivary proteome and also in the relative amount of specific proteins. The variable nature of the salivary proteome suggests that different approaches may have to be adopted when studying its composition or its possible role as an indicator for particular physiological states. The results emphasize the importance of consistency when collecting saliva samples for proteomic analysis.

Background Different pain syndromes may be characterized by different profiles of mediators reflecting pathophysiological differences, and these alterations may be measured in a simple saliva sample. The aims of the current study were to compare concentration of glutamate, serotonin (5-HT), nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and substance P (SP) in saliva and plasma from a well-defined group of patients with chronic temporomandibular disorders myalgia (TMD-myalgia) with a group of pain-free controls, and further investigate the relationship between these markers and clinical characteristics. Methods Patients diagnosed according to the diagnostic criteria for TMD ( n = 39), and matched healthy pain-free controls (n = 39) were included. Stimulated whole saliva and plasma samples were collected in the morning. Glutamate was analysed using a colorimetric assay, and 5-HT and SP were analysed by commercially available ELISA. Levels of NGF and BDNF were determined using multiplex electrochemiluminescence assay panel. Results Patients expressed higher salivary and plasma levels of glutamate (saliva: 40.22 ± 13.23 μmol/L; plasma: 30.31 ± 18.73 μmol/L) than controls (saliva: 33.24 ± 11.27 μmol/L; plasma: 20.41 ± 15.96 μmol/L) ( p < 0.05). Salivary NGF (0.319 ± 0.261 pg/ml) and BDNF (3.57 ± 1.47 pg/ml) were lower in patients compared to controls (NGF: 0.528 ± 0.477 pg/ml; BDNF 4.62 ± 2.51 pg/ml)(p’s < 0.05). Contrary, plasma BDNF, was higher in patients (263.33 ± 245.13 pg/ml) than controls (151.81 ± 125.90 pg/ml) ( p < 0.05). 5-HT was undetectable in saliva. Neither plasma 5-HT, nor SP levels differed between groups. BDNF and NGF concentrations correlated to levels of psychological distress ( p < 0.0005). Conclusion The higher levels of salivary and plasma glutamate in patients with TMD-myalgia compared to controls strengthens its importance in the pathophysiology of TMD-myalgia. However, the lack of correlation to pain levels question its role as a putative biomarker. Patients with TMD-myalgia further had lower levels of salivary NGF and BDNF, but higher plasma BDNF. These results and their correlations to psychological distress warrant further investigations.

The aim of this study was to compare the salivary cortisol level, pain intensity and psychological factors between patients with chronic and acute oro-facial pain (OP) and pain-free subjects. Twenty-seven females with chronic OP (a diagnosis of myofascial pain according to the Research Diagnostic Criteria for Temporomandibular Disorders with at least 6 months duration), 24 females with acute OP (<10 days duration) and 27 pain-free females participated. Morning saliva was collected from all participants for analyses of the cortisol level. The pain intensity was assessed on a 0-10 numeric rating scale. The participants were evaluated by the Symptom Checklist 90-revised for levels of depression and somatisation, and the Perceived Stress Scale. The cortisol levels among the three patient groups were similar with no significant group differences. The median (interquartile range) current pain level did not differ between chronic and acute OP and was, respectively, 5 (4) and 5 (3). Patients with chronic OP showed significantly higher scores for depression, somatisation and perceived stress compared with patients with acute OP (Ps < 0.001), but there were no significant differences between acute OP and controls. To conclude, there were no differences in cortisol level between groups, despite significant higher levels of depression, somatisation and perceived stress in patients with chronic OP. This shows that psychological distress has a more important role in chronic than in acute OP. However, the relation between pain, adreno-cortical activity and psychological distress is complex and warrants further investigation.

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.