Invited critical review
Saliva specimen: A new laboratory tool for diagnostic and basic investigation

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Abstract

The assay of saliva is an increasing area of research with implications for basic and clinical purposes. Although this biological fluid is easy to manipulate and collect, careful attention must be directed to limit variation in specimen integrity. Recently, the use of saliva has provided a substantial addition to the diagnostic armamentarium as an investigative tool for disease processes and disorders. In addition to its oral indications, the analysis of saliva provides important information about the functioning of various organs within the body. In this respect, endocrine research certainly occupies a central role. The present review considers the laboratory aspects of salivary assays with respect to the different analytes including ions, drugs and various non-protein/protein compounds such as hormones and immunoglobulins. This review also examines the consequences of preanalytical variation with respect to collection strategy and subsequent storage conditions. It is likely that the use of saliva in assays will continue to expand thus providing a new instrument of investigation for physiologic as well as pathophysiologic states.

Introduction

Saliva in humans is a mouth fluid possessing several functions involved in oral health and homeostasis, with an active protective role in maintaining oral healthiness. Saliva helps bolus formation by moistening food, protects the oral mucosa against mechanical damage and plays a role in the preliminary digestion of food through the presence of α-amylase and other enzymes. It also facilitates taste perception, allowing soluble food-derived molecules to reach the gustative papillae and buffer the acid components of food with the bicarbonates (originating from salivary gland carbonic anhydrase). Saliva also has a role in maintaining teeth enamel mineralization: several proteins (statherin, proline rich proteins – (PRPs) and mucins) allow Ca++ sovra-saturation in saliva to be maintained [1]. Saliva has defence functions against pathogen microorganisms, in the presence of defence proteins that react in specific (immunoglobulins) or non-specific (lysozyme, peroxydase, cystatins, lactoferrin, hystatins and others) ways, inhibiting microorganisms growth [2], [3].

In humans, oral fluid originates mainly from three pairs of major salivary glands (parotid, sublingual and submandibular) and from a large number of minor salivary glands. Parotid glands are entirely serous glands since their secretion lacks mucin, whereas submandibular and sublingual glands are mixed sero-mucous. Minor salivary glands are mainly Von Ebner glands (entirely serous organs situated in the connective tissue below the circumvallatae papillae) and Blandin-Nühm mucous glands [4].

Salivary composition varies in relation to the serous or mucous component of the glands [5]; the relative contribution of each type of gland to total unstimulated saliva secretion varies from 65%, 23%, 8% to 4% for submandibular, parotid, Von Ebner and sublingual glands respectively [6].

Saliva components have also a non-glandular origin, so oral fluid cannot be considered as the only production of salivary glands, because it also contains fluids originating from oropharingeal mucosae (oral mucosal transudate cells, bacteria, fungi, virus, upper airways secretions, gastrointestinal reflux) [7], [8]. Saliva contains also crevicular fluid, an extracellular fluid-derived from the epithelia of the gingival crevice. Crevicular fluid is produced at approximately 2–3 μl/h per tooth and it can be considered as a plasma transudate [9]. Oral fluid may also contain food debris and blood-derived compounds (actively or passively transferred), such as plasmatic proteins, erythrocytes and leucocytes in case of oral inflammation or mucosal lesions [7].

The aim of the present review is to update various aspects related to the laboratory investigation, taking into account the principal compounds present in this body fluid and in particular considering the most important substances. The proteoma and related compounds are described, taking into consideration the matrix effects and therefore the composition of this fluid. The importance of proper collection and storage of the saliva samples is also discussed. Salivary hormones and their connection with the circulating ones, which might be linked, could be an important aspect.

Section snippets

Salivary production and composition

Healthy adult subjects normally produce 500–1500 ml of saliva per day, at a rate of approximately 0.5 ml/min [6] but several physiological and pathological conditions can modify saliva production quantitatively and qualitatively, e.g., smell and taste stimulation, chewing, psychological and hormonal status, drugs, age, hereditary influences, oral hygiene [7] and physical exercise [6], [10]. Each type of salivary gland secretes a characteristic type of saliva. Differences in the concentration of

Saliva collection

Saliva can be easily collected from humans. The patient must receive detailed information about the collection protocol: the importance of the exact timing of the samples, to exclude brushing teeth before the collection, and to avoid food and fluid (apart from water) ingestion or chewing gum for at least 30 min before collection, and to rinse the mouth with water (preferably distilled). They should also be informed on the procedures for storing samples. If necessary, inhibitors or specific

Saliva storage

Saliva specimens, after collection, should preferably be kept on ice, aliquoted and frozen as soon as possible to maintain the sample integrity. The refrigeration prevents the degradation of some molecules in saliva and, when necessary, bacterial growth must also be prevented. Moreover, saliva contains bacterial protease enzymes which can degrade several salivary proteins: this can affect protein compound investigation. Nurkka's et al. investigation suggested that s-IgA can be degraded at room

Chemical and biochemical laboratory analyses

Saliva is a biological matrix still less used then plasma in clinical setting, even though it possesses several advantages mainly regarding the collection and storage steps.

Whole oral fluid compounds have been examined with a large number of methods/techniques: colorimetric/spectrophotometric, solid phase extraction and HPLC or CE with UV detection and immunoassays. In effect salivary analyses are more often performed with the aim to study a group of molecules (e.g., proteins) or to detect and

Comparison of saliva and circulating concentrations of compounds

The concentration of the biochemical compounds in the circulation is, in general, well documented and, together with the standardized steady-state condition, defines the range of variation and the reference values. Plasma concentrations of the more investigated components for diagnostic purposes define narrow ranges, whereas oral fluid composition exhibits wide variation, both quantitatively and qualitatively [7]. This large variability, with respect to the composition of oral fluid, challenges

Saliva as a diagnostic tool

Recently there has been increasing interest in diagnosis based on saliva analyses, because saliva has a simple and non-invasive collection method. Oral fluid sampling is safe for the operator and the patient, and has easy and low-cost storage. These characteristics make it possible to monitor several biomarkers in infants, children, elderly and non-collaborative subjects, and in many circumstances in which blood and urine sampling is not available. Another reason that makes saliva interesting

Conclusions

The saliva matrix is an upcoming area of research for basic and clinical application purposes, with considerable potential for growth and progress.

Saliva is a really useful specimen when a qualitative answer is required (for example in toxicology). It is also usable for quantitative measurements of several analytes, particularly when a stable correlation between plasmatic and salivary levels can be achieved. Nevertheless, to date salivary assays are still little used compared with plasma

Acknowledgements

Authors would like to thank Mrs. Julia Stevens for her excellent and patient help with the English grammar. The authors thank also doctor Linda M. Castell [Cellular Nutrition Research Group—Nuffield Department of Anaesthetics, University of Oxford, Radcliffe Infirmary Woodstock Road, Oxford (UK)] for her important and helpful supervision and suggestions of the manuscript.

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