Introduction
The diagnosis of probable Alzheimer’s disease (AD) and other common neurodegenerative disorders remains primarily reliant on a clinical assessment outside the specialist clinic. However, an AD diagnosis can now be supported by positron emission tomography (PET) and cerebrospinal fluid (CSF) biomarkers that detect the hallmark-underlying pathologies of amyloid-β (Aβ) [
1] and tau [
2]. One of the many challenges that the dementia community face is the detection of the pre-symptomatic phase of the AD using non-invasive, widely accessible and disease relevant biomarkers. In recent times, blood biomarkers have taken centre stage, with measurements of Aβ species [
3‐
6], the axonal injury marker neurofilament light (NfL) [
7,
8] and phosphorylated tau on threonine 181 (P-tau181) [
9] showing much promise. There are now international efforts underway to progress these biomarkers to be applicable for clinical use [
10]. Without question, a blood biomarker is far more attainable for population screening than PET or CSF; however, it still faces certain logistical limitations. Saliva has been proposed as a potential easily collectable source of biomarkers for the diagnosis and risk assessment for a range of pathological conditions occurring not only in the mouth but also systemically [
11]. Disorders that have been targeted include periodontal and oral mucosal diseases, oral, pancreatic, lung and breast cancer, together with diabetes and hepatitis C infection [
12]. The major salivary glands secrete saliva in response to cholinergic innervation from cranial nerves VII and IX, which are monitored by the autonomic nervous system (ANS) [
13]. This relation to the nervous system suggests that these gland secretions may represent various physiologies of the nervous system. Indeed, central nervous system (CNS) proteins are secreted into the saliva in an age-dependent manner [
14,
15]. Furthermore, via passive diffusion, active transport or microfiltration proteins can pass from the blood into the saliva [
13,
16]. For these reasons, saliva may contain novel biomarkers for CNS injury or be an alternative and more accessible source in sampling AD-related biomarkers that are currently being eagerly pursued in blood. In this review, we summarise the current evidence for salivary biomarkers in detecting AD and related disorders, while considering important factors related to saliva production, composition and collection in older adults. This article is based on previously conducted studies and does not contain any studies with human participants or animals performed by any of the authors.
Production of Saliva and Impacts of Aging, Local and Systemic Pathology
Saliva collection generally represents a pooled sample of the products from three pairs of major salivary glands (submandibular, sublingual and parotid), supplemented by numerous minor salivary glands. In addition, this material includes microorganisms, their by-products, host cells from epithelial surfaces, and other components released from the gingival crevices around teeth (gingival crevicular fluid). Therefore, it is important to understand the processes of production and regulation of saliva, and how this may differ in populations, especially older adults, since any variations may impact on the relative validity of proposed biomarkers.
Saliva production varies between different glands, not only in production volume but also in composition [
17]. The exocrine glands contain secreting epithelial cells located in structures called acini as the terminal element of the ductal tree within the gland. Acinar cells will produce either dilute saliva with low levels of mucins or mucin-rich secretion. Whilst the parotid glands largely contain non-mucinous acinar cells, submandibular glands are mixed, whereas the sublingual glands and the minor glands located throughout the mouth are largely mucin forming. The ducts and acini are surrounded by myoepithelial cells, a rich blood supply and dense innervation by parasympathetic and sympathetic nerves. Consequently, the steady unstimulated saliva flow occurring throughout the day is made up primarily of glands producing mucinous saliva; 68% from submandibular and sublingual, and around 4% from numerous minor glands. However, when salivary output is stimulated (e.g. during mastication via mucosal and periodontal mechanoreceptors, activated taste bud receptors, smells or thermal changes [
18‐
21]), the percentage contribution from parotid saliva rises from around 1/4 to over 1/2 [
22] even though other glands also increase their production. Autonomic innervation is supplied to many (not just acinar) salivary grand cells, although there is some variation between glands in the extent of sympathetic input [
23,
24]. In animal models sympathetic stimulation produces a different protein-rich saliva, and mucin production from mucous glands seems to be initiated by parasympathetic stimulation [
25]. As a result, whole salivary composition is believed to be influenced by stimulation and the rate of secretion, although this may be mainly related to dilution effects of molecules from non-glandular sources. Many components of freshly secreted saliva are actively secreted by acinar and ductal cells and so these will not be altered, with the exception of sodium, chloride and bicarbonate salts, which are more concentrated in stimulated saliva following autonomic stimulation and changes in flow rates [
26]. Transport mechanisms include exocytosis of protein storage granules, ion pumps, transport proteins and vesicular proteins [
17]. Whole saliva will additionally contain some blood components (including immunoglobulins such as IgG) which have entered as a transudate within the gland system as well as the supplemental materials listed above.
Saliva flow is generally considered to be frequently reduced in older individuals. However, there has been some disagreement on the role of direct age-related changes compared to indirect effects such as the influence of medications that are commonly experienced by older adults. Medications which are known to increase the risk of hyposalivation include anticholinergics, proton pump inhibitors, antidepressants, phenothiazines, benzodiazepines, antihistamines, diuretics and various antihypertensives [
27,
28]. Statins have also been reported as being responsible for oral dryness in a small interventional study [
29]. Hence salivary volume and composition could further be influenced as an outcome, complication or side effect from systemic disease and treatment (including local radiotherapy and systemic medications). These effects may be direct or indirect, mediated by other pathways such as dehydration, which may itself be related to impaired fluid intake, emesis, diarrhoea or polyuria.
The current opinion based on primary research and systematic reviews [
30,
31] is that there is an age-related decrease in salivary gland function and thus in xerostomia, and that medication merely enhances this further. However, the primary research is somewhat contradictory for age-related changes in both unstimulated [
32‐
35] and in stimulated [
35‐
38] salivary flow rate, and large inter-individual differences between participants within study groups have been reported. Nagler and Hershkovich [
39] have shown how there is not only a change in volume but also composition (including ions, immunoglobulins and other proteins) with aging, although their participants were taking a range of medications to maintain health. However, this was confirmed in a large cross-sectional study by Dodds et al. [
40]. Consequently, it would seem that saliva flow is impaired in older populations, and that aging can also modify the composition of saliva produced. It would seem necessary to accommodate this into study design and data analysis if possible when determining the value of biomarkers in populations of differing or mixed ages.
Finally, psychogenic causes, such as depression, anxiety or stress, can also result in xerostomia mediated by sympathetic stimulation. In cases of AD, stroke or other neurological pathology, patients may complain of dry mouth in the presence of reduced or normal salivary secretion due to altered production [
41]. Hence self-reported dryness in some patients may not by itself be an exclusion factor for attempted salivary sampling—it would seem wise to confirm this with intraoral examination even if ultimately collection is unsatisfactory. The impact of these challenges is illustrated by the observations recently reported by Galloway et al. [
42] that approximately 1/3 of participants in the recent UK Biobank study were unable to produce an adequate saliva sample, and that this was noticeably increased amongst those with a range of systemic diseases. Indeed only 57.7% of participants with a diagnosis including dementia, AD or Parkinson’s diseases (PD) were able to produce an adequate sample for archiving (Table
1). Therefore, it appears that results from salivary analysis, whilst offering excellent potential as a research and management tool, may have to be considered carefully allowing for age, concurrent medication and other risk factors. Each of these factors may independently have an impact on data and these variables should at least be recorded in clinical studies. Likewise, there may be issues with obtaining adequate samples for some participants, especially the elderly and/or those with significant systemic disease.
Table 1
Number of UK Biobank participants with selected health conditions (non-cancerous) who attempted to produce a saliva sample at baseline assessment (selected data, adapted from Galloway et al. [
42])
All participants in UK BioBanks | 120,175 | 84,721 (70.5) |
Diabetes mellitus | 743 | 4787 (64.4) |
Cerebrovascular disease | 2723 | 1725 (63.3) |
Ischemic heart disease | 6839 | 4474 (65.4) |
Alzheimer’s and Parkinson’s disease | 284 | 164 (57.7) |
Clinical depression | 7632 | 5006 (65.6) |
Chronic obstructive pulmonary disease | 1245 | 739 (59.4) |
Asthma | 14,442 | 10,040 (69.5) |
Inflammatory bowel disease | 3524 | 2354 (66.8) |
Rheumatoid arthritis | 1447 | 919 (63.5) |
Osteoporosis | 2652 | 1721 (64.9) |
Acute renal failure/Chronic kidney disease | 679 | 415 (61.1) |
Conclusions
In the last decade, there has been considerable advancement in the detection of AD-related biomarkers in non-invasive peripheral materials, such as blood. This remarkable and rapid success has posed a new challenge—can other peripheral sources be utilised for AD-related biomarkers, namely saliva? The data currently available shows that Aβ42, Aβ40 and multiple tau species are all detectable in saliva using conventional immunoassays. While salivary Aβ40 and T-tau are not related to clinical AD disease, increases in Aβ42, phosphorylated tau species and decreases in lactoferrin may have potential. Yet, how salivary Aβ42 relates to brain amyloid metabolism, to CSF or plasma levels of Aβ42, or to brain amyloidosis evaluated by Aβ PET status is not known. On the other hand, the antimicrobial peptide lactoferrin has been shown to correlate well with AD CSF biomarkers and to detect preclinical AD. In PD, multiple forms of α-syn have been investigated, with oligomeric α-syn being the most promising for disease diagnosis. Although the non-invasive nature of saliva is an attractive attribute there remain important considerations. Firstly, standardisation in collection (e.g. stimulated versus unstimulated), pre-processing (e.g. centrifugation speeds) and storage (e.g. addition of sodium azide) is currently lacking. While the majority of studies included in this review analysed unstimulated whole saliva, there are very clear distinctions in proteome content between unstimulated and stimulated collections from different glands. It is currently not known how the concentrations of Aβ, tau and other biomarkers discussed in this review differ between sublingual, parotid, whole unstimulated and stimulated saliva or if their presence in saliva is simply from blood. Secondly, while the successful collection of blood and CSF from a consenting participant is driven by a physician, the access to unstimulated saliva is solely dependent on the participant. This is likely to create large variabilities in the material collected and therefore stimulated saliva could be a preferred method due to the simplicity of the collection method. As previously mentioned, there is substantial evidence available to demonstrate that elderly patients, particularly suffering with dementia, find it difficult to produce an adequate saliva sample. For these reasons, a saliva biomarker would be more suitable for the preclinical phase of disease and not for individuals with significant cognitive impairment. At this moment in time, the limited data in large well-characterised and, in some cases, conflicting reports means we are unable to conclude this with any real certainty. Nonetheless, the current research reviewed in this article cautiously indicates the potential of saliva as a non-invasive biomarker source at the preclinical phase of the disease which should be further investigated to determine the reliability of such biomarkers in detecting disease pathology or monitoring disease progression.