Review For reprint orders, please contact [email protected] 9 Review Saliva: a reliable sample matrix in bioanalytics Bioanalysis Saliva is gaining increasing attention as a bioanalytical sample matrix. Mostly because Michael Gröschl of the easy and noninvasive collection, it is not only beneficial in endocrinological Celerion Switzerland AG, Allmendstrasse and behavioral science, but also in pediatrics. Saliva also has the advantage of being 32, CH-8320 Fehraltorf, Switzerland Author for correspondence: the only body fluid which can be collected even during physical exercise, for example, Tel.: +41 43 355 7619 during sportive activities, and there are physiological characteristics that make it [email protected] superior to serum/plasma or urine for specific scientific questions. This review provides an insight into the physiology of saliva formation, explaining how certain compounds enter this bodily fluid, and gives advice for collection, storage and analytical methods. Finally, it presents a number of reliable and proven applications for saliva analysis from scientific fields including endocrinology, sports medicine, forensics and immunology. First draft submitted: 17 January 2017; Accepted for publication: 2 March 2017; Published online: 15 May 2017 Keywords: bioanalytics • endocrinology • forensics • immunology • noninvasive • saliva • sports medicine Traditional biological matrices for the analysis achieved for the majority of parameters. There- of diagnostic substances, such as hormones, fore, it is still state of the art to assess saliva drugs or immunoglobulins, are serum, plasma concentrations in combination with results and urine. Although serum and plasma pro- obtained from matched plasma samples. vide information about the currently circu- This review provides possibilities of saliva lating concentration of the analyte, urine analysis in various fields of research and to provides an overview of the accumulated show potentials for diagnostics and clinical 8 excretory metabolites since the last act of uri- trials. First, information is given on physiol- nation. Over years, the analysis of saliva has ogy of the salivary glands and saliva secretion. attracted increased attention, in particular, Subsequently, the methodological aspects for among clinicians and researchers who con- sampling and measurement of saliva are dis- 2017 sider saliva as a noninvasive and stress-free cussed. Finally, the diversity of saliva analysis alternative to blood sampling. is demonstrated with examples from the fields Numerous publications have demonstrated of endocrinology, forensic chemistry, thera- the analysis of saliva to be a useful alterna- peutic drug monitoring, sports medicine, tive for determining many endocrine param- metabolic diseases, immunology, and oral eters since saliva lacks the one main problem and gastrointestinal diseases. of blood collection, which is the invasiveness of the venipuncture resulting an increased Anatomy & physiology of the response of stress hormones, namely glucocor- salivary glands ticoids [1,2] and catecholamines [3]. To date, a The major part (65%) of saliva is produced definitive interpretation of salivary concentra- by the submandibular glands. The parotid tions for daily routine purpose has not been glands contribute between 20 and 50% to part of 10.4155/bio-2017-0010 © 2017 Future Science Ltd Bioanalysis (Epub ahead of print) ISSN 1757-6180 Review Gröschl the total saliva volume, depending on stimulation [4,5]. Furthermore, saliva contains factors of the immune Only a minor fraction of 5% is secreted by the sublin- response, such as secretory IgA [12], cathelicidin [13] or gual glands. The remaining 10% derive from numer- adrenomedullin [14], controlling the microbial growth ous small glands embedded in the oral cavity [5,6]. For on the epithelia. routine applications, open-field studies or outpatient The acini are surrounded by blood capillaries that sampling, mixed saliva is the only suitable sample enable the passage of substances from the circulation material, while gland-specific collection at the exit into the salivary glands (Figure 1) [15] . of the excretory ducts with cannulas or pipettes is The concentration- or speed-limiting step of hor- extremely intricate and is only performed for specific mone transfer from blood into saliva is the passage otorhinolaryngologic investigations. This mixed saliva through the capillary wall and the membrane of the contains, to a small portion, exudate from the gingiva glandular epithelial cells. Transfer of substances from and peeled oral epithelial cells. the capillaries into the acini is driven by diverse mech- Saliva is produced in secretory end pieces of the anisms resulting in a complex fluid. These transport glands, called acini. These acini drain into the sali- mechanisms are either passive diffusion, ultrafiltration vary ducts, with small ‘striated’ ducts opening into through pores or active and energy dependent against wider intercalated and excretory ducts (Figure 1). The concentration gradients [16] . daily total volume ranges between 500 and 1500 ml, In passive diffusion, lipid-soluble materials cross the depending on water balance and stimulation [7,8]. cell membranes of capillaries and acini. The salivary con- Various enzymes like amylase and lipase, prepar- centrations of lipid-soluble, unconjugated steroids such ing food for digestion, are supplied by saliva [9–11] . as cortisol and testosterone approximate the unbound Protein-bound steroid or amine Lipophilic substances transported from blood Capillary Hydrophilic substances transported from blood Steroid after conversion Peptides produced by salivary glands Tunnel or transporter protein Acinus Glandular enzymes Acinus Striated duct Capillary Intercalated duct Expression and secretion Active transport (energy dependent) Passive diffusion Blood flow Saliva flow Figure 1. Transport mechanisms of substances into saliva. Small lipophilic compounds, such as steroids, enter the glands by passive diffusion. Hydrophilic compounds (electrolytes and many proteins) are actively transported from the circulation to the glands. Enzymatic conversion has been reported (e.g., androstenedione to testosterone by 17-hydroxysteroid oxidoreductase). Peptides are either actively transported from blood to saliva (e.g., insulin) or are produced by the glands themselves (e.g., EGF). 10.4155/bio-2017-0010 Bioanalysis (Epub ahead of print) future science group Saliva: a reliable sample matrix in bioanalytics Review plasma concentrations, while hydrophilic, conjugated Serum (IU/ml) steroids such as dehydroepiandrosterone sulfate only 50 reach 1% of the unbound plasma concentrations [17] . Saliva (IU/ml) Ultrafiltration drives the transport of small polar 40 molecules into saliva through tight junctions at the apical pole of the acini. Mazariegos et al. [18] found 30 these tight junctions in the resting rat parotid gland 20 impermeable to tracers of molecular weights >2 kDa. IU/ml An active transport mechanism clearly applies for 10 many electrolytes [19,20] and for some peptides such as insulin [21], but was also proven for some drugs [16] . 0 Borzelleca and Cherrick [22] had already investigated the presence of antibiotics in saliva in 1965. The secre- 01530456075 90 105 120 tion of penicillin and tetracycline appeared to correlate Min with the concentration in blood. Since the secretion of penicillin by the salivary glands and by the kidney was Figure 2. Curse of insulin in matched serum and saliva prevented by probenecid, an inhibitor of renal activity, (n = 8 each) after oral glucose administration. In saliva, penicillin secretion in saliva is subjected to an active the CMax is delayed by approximately 30 min, reaching transport. Zuidema and Van Ginneken [23] confirmed only lower concentrations compared with serum. this finding. Another inhibitor of renal activity, pro- benecid, showed no effect on the salivary secretion of broad antiviral activity, since hepatitis B virus remains diprophylline. These data suggested that the secretory infectious in mixed saliva [29]. mechanism in the kidney and in the salivary gland is A prerequisite for conducting pharmacokinetic not identical. studies using saliva is to avoid residues of orally admin- It is noteworthy that an active transport is not only istered drugs in the oral cavity, as shown for amphet- energy-consuming, but also time-consuming, and can amines capsuled in gelatin [30]. Usually, the problem lead to a shift in the appearance of the compound, can be solved by rinsing the mouth with water before when matched plasma and saliva samples are plotted starting the collection, but the possibility of dilution together, as indicated in Figure 2 for human insulin. effects needs to be considered [31] . The contribution of The movement of marker substances from blood crevicular fluid from the gingiva to mixed saliva can be to saliva and vice versa was investigated [24], describ- limited by avoiding tooth brushing prior to sampling ing permeability barriers in the glands that allow for and by rinsing the mouth with water prior to sam- some substances to pass readily into the saliva while pling [32,33]. To ensure robust and reproducible profiles, other substances were held back. Some pharmaceuti- circadian rhythms as well in salivary gland activity [34] cals enhance saliva secretion by dilation of the tight and production of compounds being transferred into junctions of the secretory end pieces [25], while some saliva (e.g., steroid hormones