Clinica Chimica Acta 383 (2007) 30 – 40
www.elsevier.com/locate/clinchim
Invited critical review
Saliva specimen: A new laboratory tool for diagnostic and basic investigation
Silvia Chiappin a , Giorgia Antonelli a , Rosalba Gatti b , Elio F. De Palo a,⁎
a
Section of Clinical Biochemistry, Department of Medical Diagnostic Sciences and Spec. Ther., University of Padua, c/o ex Istituto di Semeiotica Medica,
Via Ospedale 105-35128, Padova, Italy
b
Azienda Ospedaliera, Divisione di Endocrinologia, Padova, Italy
Received 31 October 2006; received in revised form 30 March 2007; accepted 7 April 2007
Available online 25 April 2007
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.
© 2007 Elsevier B.V. All rights reserved.
Keywords: Saliva; Hormones; Collection; Storage; Stability
Contents
1.
2.
3.
4.
5.
6.
7.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Salivary production and composition . . . . . . . . . . . . . . . . . .
2.1. Inorganic compounds. . . . . . . . . . . . . . . . . . . . . . .
2.2. Organic compounds (non-protein and lipids). . . . . . . . . . .
2.3. Protein/Polypeptide compounds . . . . . . . . . . . . . . . . .
2.4. Hormones . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Saliva collection . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Saliva storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Chemical and biochemical laboratory analyses . . . . . . . . . . . . .
5.1. Interferences . . . . . . . . . . . . . . . . . . . . . . . . . . .
Comparison of saliva and circulating concentrations of compounds . .
Saliva as a diagnostic tool. . . . . . . . . . . . . . . . . . . . . . . .
7.1. Viral and bacterial infections (genomes and antibodies detection)
7.2. Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7.3. Pharmaceutical and abuse drugs . . . . . . . . . . . . . . . . .
7.4. Hormones . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7.5. DNA tests . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7.6. Sialochemistry analysis. . . . . . . . . . . . . . . . . . . . . .
⁎ Corresponding author. Tel.: +39 049 8213016; fax: +39 049 8272485.
E-mail address: [email protected] (E.F. De Palo).
0009-8981/$ - see front matter © 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.cca.2007.04.011
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31
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38
S. Chiappin et al. / Clinica Chimica Acta 383 (2007) 30–40
31
8. Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
1. 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 αamylase 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 ++ sovrasaturation 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.
2. 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 salts/ions [11] and total proteins [5] among glands can
be observed. Salivary proteins are expressed differently
among individual glands, like submandibular and sublingual
glands. For example cystatin C is secreted by the submandibular gland and MUC5B mucin and calgranulin are secreted
by the sublingual gland [5]. Moreover, salivary composition
varies, depending upon whether salivary secretion is basal or
stimulated [11].
Salivary output and composition depend on the activity of
the autonomic nervous system: the serous part of the glands is
under the control of the sympathetic system and the mucous
part of both parasympathetic and sympathetic systems. The
α–β adrenergic and cholinergic stimuli (neural or pharmacological) can modify the quantity; viscosity and ionic and
protein concentrations can vary. Parasympathetic stimulation
results in a high flow of saliva containing low levels of organic
and inorganic compounds. Sympathetic stimulation produces a
low volume of protein-rich and K+ -rich saliva [7]. The
presence of food in the mouth can affect salivary composition
as a stimulus for selective protein release; after a meal an
increase of total proteins and of α-amylase in saliva has been
shown [12].
The clearance of compounds from plasma into saliva may
involve several processes:
(a) ultrafiltration through gap junctions between cells of
secretory units (intercellular nexus). Only molecules with
MW b 1900 Da are involved (water, ions, hormones such
as catecholamines and steroids) and their salivary
concentration is 300–3000 times lower in saliva than in
plasma [13].
(b) transudation of plasma compounds into oral cavity, from
crevicular fluid or directly from oral mucosa. The presence
in the saliva of some typical plasmatic molecules, like
albumin, depends on this mechanism [13].
32
S. Chiappin et al. / Clinica Chimica Acta 383 (2007) 30–40
(c) selective transport through cellular membranes: by passive
diffusion of lipophilic molecules (steroid hormones) or by
active transport through protein channels [13].
A compositional analysis of oral fluid may be important for
its implications in physiology, pathology and diagnosis of oral
and systemic diseases.
The various compounds of saliva are: inorganic, organic
non-protein, protein/polypeptide, hormone and lipid molecules
[14,15].
glucose, amino acids, lipids like cholesterol and mono/diglycerides of fatty acids [13,14,24,25]. In saliva amines, such as
putrescine, cadaverine and indole, are also detectable [26].
Fatty acids, too, are measurable in saliva and in particular αlinoleic acid and arachidonic acid can be measured in human
saliva: their concentration seems to correlate with a dietary fatty
acid intake [14]. In saliva lactate is also measurable and its
concentration shows a high correlation with capillary blood
lactate concentration [6].
2.3. Protein/Polypeptide compounds
2.1. Inorganic compounds
Whole saliva contains mainly water, strong and weak ions
(Na+, K+, Mg2+, Ca2+, Cl−, HCO3−, HPO32−) which can generate
buffer capacity. The primary secretion from salivary glands is
plasma ultrafiltrate (isotonic compared to plasma) but in
salivary ducts there is energy-dependent reabsorption of Na+
and Cl− resulting in a hypotonic fluid secretion, with a lower ion
concentration compared to plasma. In salivary gland ducts
mineralocorticoid receptors are present, so salivary glands are
mineralocorticoid-responsive [16]: for this reason salivary K+
concentration is higher than the plasma concentration (25 vs.
4 mmol/l) and Na+ concentration is lower in saliva compared
with that in plasma (2 vs. 145 mmol/l) [7]. Several factors may
modify the salivary ionic concentration, furthermore, the
composition of unstimulated saliva is different from stimulated
saliva (which is more similar in composition to plasma). For
example, an increase in the salivary flow rate, obtained by
stimulation with acidic food, increases the concentrations of
sodium, chloride and bicarbonate and decreases the concentrations of salivary potassium and phosphate, compared with
unstimulated saliva [17] (Table 1).
Similar to other biological fluids, salivary ionic concentration is usually measured by ion selective electrodes, atomic
absorption (or emission) [18] and the traditional spectrophotometric methods [19,20].
2.2. Organic compounds (non-protein and lipids)
Small amounts of organic non-protein compounds can be
detected in saliva. Uric acid is one of the most important
antioxidant compounds in saliva [21,22], bilirubin and creatinine are also detectable [8,23]. Saliva moreover contains
Table 1
Comparison of inorganic compounds between saliva and plasma
Inorganic compounds
(mmol/l)
Whole unstimulated
saliva
Whole stimulated
saliva
Plasma
Na+
K+
Cl−
Ca2+
HCO−3
Mg2+
NH3
5
22
15
1–4
5
0.2
6
20–80
20
30–100
1–4
15–80
0.2
3
145
4
120
2.2
25
1.2
0.05
Adapted from Aps JKM and Martens LC [7].
The salivary levels of total protein increase also through βsympathetic activity in the salivary glands, since saliva
secretion is mainly evoked by the action of adrenergic mediators
[6]. Saliva contains a large number of protein compounds, of
which the structure and function have been studied with
traditional biochemical techniques, including liquid chromatography, gel electrophoresis, capillary electrophoresis (CE),
nuclear magnetic resonance, mass spectrometry, immunoassays
(RIA, IRMA, EIA, ELISA) and lectin probe analysis [27,28].
Most of these analyses were performed on the investigation of a
specific group of salivary proteins, but some efforts were made
to obtain a complete pattern of all salivary proteins with
proteomic techniques.
Usually, in a proteomic experiment, proteins are first
separated with a two dimension electrophoresis (2-DE) and
then detected by a suitable stain. For example, to specifically
identify a saliva protein, a spot obtained by 2-DE is excised and
digested by tryptic enzymes. The resulting fragments are
analysed by MALDI-MS [Matrix Assisted Laser Desorption–
Mass Spectrometry] to measure molecular masses of peptides
[27]. With this method about 40 proteins were originally
identified from human whole saliva but, adding a shotgun
proteomics method as a complementary technique, about 300
salivary proteins have now been identified [27]. Recently Guo et
al. studied the salivary proteome using a capillary isoelectrofocusing coupled with electrospray-ionizaton tandem mass
spectrometry [29]. Hannig et al. obtained a chromatographic
pattern of salivary proteins using reverse phase HPLC and the
presence of 5 typical salivary proteins was demonstrated [30].
Tanaka et al. used a microchip CE for separation of salivary
compounds with fluorescein isothiocyanate derivatization [31].
Comparing the results performed with the different techniques
mentioned, low overlaps are obtained because often, in
proteomic experiments, a false-positive identification of peptides sequence in proteomic databases occurs. By using these
different techniques it has been possible to recognize the most
abundant salivary proteins (Table 2) [32–35].
Among the salivary proteins obtained by classical protein
analysis methods, those deriving from salivary gland production
can be recognized (of which the most abundant are amylase,
PRPs secretory IgA (s-IgA) and carbonic anydrase). Other
proteins can be derived from plasma leakage (albumin,
transferrin, IgG) [36].
Human saliva proteins can have a wide range of functional
properties. They can be related to the immune response and oral
S. Chiappin et al. / Clinica Chimica Acta 383 (2007) 30–40
Table 2
Salivary proteins
Origin
Functions
Concentrations and
references
Total proteins
0.47 ± 0.19 mg/ml [10]
0.9 ± 0.2 mg/ml [32]
4.3–710.0 mg/dl [33]
2.67 ± 0.54 mg/ml [29]
α-Amylase
Starch digestion
3257 ± 1682 U/ml [32]
1080.0 ± 135.6 IU/l [8]
476 ± 191 μg/ml [29]
Albumin
Plasma
Mainly from
0.2 ± 0.1 mg/ml [32]
plasma leakage
0.8–192.0 mg/dl [33]
Cystatins
SM N SL
Antimicrobial
14.3 kDa form
group
(cistein-proteinase 58 ± 25 μg/ml
inhibitor)
14.2 kDa form
91 ± 46 μg/ml [29]
Hystatin
P
Antifungal
1190 ± 313 μg/ml [29]
Secretory-IgA B lymphocytes Antimicrobial
124.3–335.3 μg/ml [34]
Lactoferrin
Mucous N serous Antimicrobial
3.7 ± 2.5 μg/ml [32]
Lysozyme
SL N SM, P
Antimicrobial
3.5–92.0 μg/ml [33]
21.8 ± 2.5 mg/dl [8]
59.7–1062.3 μg/ml [34]
Mucins group Mucous glands Lubrication
MUC5B: 2.4 ±
1.7 U/ml [32]
PRPs
P
Binding to bacteria Acidic PRP: 456 ±
and with dietary
139 μg/ml
tannins
Basic PRP: 165 ±
69 μg/ml [29]
Statherin
Ca++ binding
4.93 ± 0.61 μmol/l [35]
36 ± 18 μg/ml [29]
Transferrin
Plasma
0.58 ± 0.20 mg/dl [8]
SM = submandibular; SL = sublingual; P = parotid.
defence, like lysozyme, lactoferrin, lactoperoxydase, immunoglobulins, agglutinins, chitinases and mucins which in saliva
participate in the protection of the oral tissues; other proteins
possess bacteria-killing properties, like histatins and defensins.
The salivary immunoglobulins are mainly s-IgA (N85%) and
are produced directly by the B lymphocytes present near the
salivary glands. s-IgA is secreted in the interstitial fluid, taken
up by the acinar and ductal cells of the salivary glands and
subsequently secreted into the saliva. The remaining 5–15% of
salivary immunoglobulins are mainly IgG and IgM, derived
from crevicular fluid or from plasma leakage [2]. All these
proteins and peptides have a broad spectrum of antimicrobial
activity, so their functions overlap, but the meaning of this
redundancy is still not clear. The physiological conditions under
which saliva is collected can have a significant influence on the
concentration of immunoglobulins; furthermore, Gleason's
review underlines the immune response and risk of illness in
athletes [37]. Other proteins are also present: for example,
enzymes and enzyme inhibitors, hormones such as growth
factors, and cytokines such as interleukin-8 [38,39].
Further functional properties of human salivary proteins/
peptides are:
1. Inhibition of calcium precipitation: PRPs and statherins
promote remineralization of the enamel by the binding of
calcium ions [1].
2. Taste perception (carbonic anhydrase) [1].
33
3. Digestion (amylase, involved in digestion of starch, Von
Ebner gland proteins with endonuclease activity) [1].
4. Inhibition of proteinase (cystatin, inhibitor of serine
proteinases, tissue inhibitors of metalloproteinases) [1].
5. Other functions: transcription, cell proliferation, signal
transduction, chemotaxis and cell motility.
2.4. Hormones
Some hormones commonly measured in plasma, such as
steroids, non-steroids, peptide and protein hormones, can be
detected in the oral fluid. Before any significance can be
attached to the measurement of a hormone in saliva, the
existence of a connection between circulating levels and mouth
areas must be defined. Where a hormone both originates and is
active in the mouth, it possesses a very specific role. It may
derive also from the circulation by passive diffusion or active
transport, or originate partly from both sources.
This information about the salivary hormone origin allows a
definition of its role and connection with pathological and
physiological states to enable a suitable analysis. At first sight, it
appears to be a useful index of systemic bioactivity; the quantitative measurements of hormones in saliva must be studied to
prove a correlation with levels in serum. Thus the connection
between the free and the total circulating fractions, together with
the more or less specific binding proteins, should be considered.
Also the correlation of the saliva levels with the free bioactive
circulating form, if present, is of great importance.
Catecholamines can be recognized in saliva ranging from
250 to 800 pg/ml [13], but their source is still uncertain. They
seem to originate by diffusion from serum, but there is also an
amount of salivary catecholamines derived by direct release
from sympathetic nervous terminations, so their concentration
is poorly correlated with that of plasma. The increase in plasma
catecholamines after physical exercise is not accompanied by a
salivary catecholamine elevation. Salivary catecholamine is
therefore not considered a useful index of general sympatethic
tone. The concentration of catecholamine metabolites (like
dihydroxyphenylglycol) instead shows a good correlation with
plasma levels [40].
There is little information about thyroxin and triiodothyronine levels in saliva: in preliminary studies they were detected
in saliva and their salivary levels (1.10 ± 0.07 nmol/l for T4 in
healthy subjects) seem to correlate with plasma levels [41].
Steroid detection is perhaps the most interesting application
in salivary hormonal studies. Steroids have often been studied
because salivary-free steroid hormones can give good information on serum-free levels [42] (for further details: see Section 6).
The most commonly assayed biomarkers in saliva are cortisol,
testosterone, dehydroepiandrosterone (DHEA) [43–46], 17hydroxyprogesterone, progesterone [46,47] and aldosterone
[48]. Table 3 outlines steroid hormones measurable in saliva
specimens.
Salivary cortisol measurement is today a widely accepted
alternative to the determination in plasma or serum: since the
adrenal cortex is responsive to stress, venipuncture for blood
collection can lead to an iatrogenic increase of plasma gluco-
34
S. Chiappin et al. / Clinica Chimica Acta 383 (2007) 30–40
Table 3
Salivary steroid hormones
Compounds
Methods
Analytical aspects
Cortisol
Testosterone
DHEA-s
Progesterone
DELFIA RIA EIA
ELISA
ELISA
RIA
Specific for saliva
Specific for saliva; decrease with storage at room temperature
Specific for saliva; no effect with storage at room temperature
Modified from serum assay; antibodies may cross react with other steroids
Concentrations and references
Estradiol
Aldosterone
RIA
RIA
Immediately after awakening: 3.6–35.1 nmol/l [43]
140.30 ± 154.15 pg/ml [44]
291.21 ± 294.81 pg/ml [44]
Luteal phase: 436 ± 34 pmol/l [46]
Follicular phase: 22.1 ± 2.7 pmol/l
Modified from serum assay; antibodies may cross react with other steroids Luteal phase: 20.6 ± 0.4 pmol/l [46]
Modified from serum assay
138–475 pmol/l [47]
corticoid levels. From this perspective, the stress-free salivary
collection for cortisol measurement has an advantage compared
to plasma, especially when a cortisol measurement has to be
achieved in children [49]. Salivary-free cortisol concentrations
do not seem to be dependent on salivary flow rate [6].
The protein polypeptide hormones are perhaps a new
analytical approach of the medicine laboratory but, at the
present time, there are still too few investigations about protein
and polypeptide hormone levels in saliva. For example
Rantonen et al., using an IRMA [Immuno Radio Metric
Assay], detected growth hormone (GH) in human saliva, at
levels of 8.6 ± 11.1 μU/l (100–1000 times less than the
physiological serum levels), even in samples free from blood
contamination, and found a linear correlation between serum
and salivary GH levels. This finding suggests a passive
diffusion of GH from blood to saliva [50]. In saliva also other
protein hormones have been detected, such as prolactin [28],
insulin-like growth factor I (IGF-I) [51] and melatonin.
Melatonin levels in saliva reached 500 pmol/l at nighttime
and were found to correlate significantly with plasmatic
concentrations [52].
3. 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 additives are
to be included in the specimen before the samples are collected/
analysed, otherwise it can be frozen until the laboratory
analyses.
This collection method may have a diagnostic and therapeutic
aim. The chemical and biochemical laboratory measurements
can therefore be carried out to obtain monitoring and follow-up
analyses with repeated sampling over the course of minutes,
hours, days or longer, for example in studies on the circadian or
seasonal rhythm of several biological parameters (e.g., steroids).
Recent reports suggest, however, that compliance of patients
with fixed time sampling protocols may be problematic
especially if saliva collection is self-made, and a low compliance
can bias results [53,54].
The standardization of salivary collection has a great
importance in saliva analysis, because several factors may
affect salivary flux and composition.
Whole saliva, glandular-duct saliva, crevicular fluid and
mucosal transudate are achievable specimens for which
specially designed collecting methods are possible. Today
several methods and devices are available. Among them, the
easiest and the most feasible method is the collection of whole
oral fluid.
A more specific specimen can be obtained with less easy
collection procedures. The following procedures are available
to collect a single constituent of the whole saliva:
(a) Saliva produced by a single salivary gland can be
collected by cannulation of a single salivary duct [53],
or by a metal or acrylic cup placed over the Stenson's duct
for the collection of pure parotid saliva [5].
(b) Crevicular fluid can be collected inserting directly in the
gingival crevice an appropriate device [9], such as filter
paper, a commercial micropipette or a thin tube. It
provides excellent sampling of a serum transudate but it is
unlikely that commercial tests would be developed for
this fluid due to the difficulty in applying the collector and
the small volume of fluid collected [55,56].
(c) Pipette suction from under the tongue [57].
(d) Pipette suction from the paragingival gutter [8,57].
(e) Saliva from submandibular glands can be collected by
placing the tip of a collection device at the orifice of the
Wharton's duct, after placing sterile cotton sponges in the
floor of the mouth and over the buccal mucosal areas to
occlude the parotid and sublingual ducts [5].
These methods allow differences in composition among
salivary gland secretions and other sources of oral fluid
constituent to be studied but, from a practical point of view,
tests based on this type of sampling could not be widely
marketed because of the high degree of training required for the
specialist collecting the specimen. For these reasons these
sampling methods are not commonly used [58].
Whole saliva (or whole oral fluid) is easy to collect, is more
representative of the oral milieu, and collection does not need to
be performed by a trained specialist but can be made by the
patient or study participant.
Unstimulated whole saliva can be collected with several oral
fluid collector devices and commercial devices are available:
S. Chiappin et al. / Clinica Chimica Acta 383 (2007) 30–40
1. Passive drooling (no oral movements): allowing saliva to
drain off the lower lip into a plastic vial [57,59,60].
2. Spitting directly into a collector vial: specimens collected by
spitting contain up to 14 times more bacteria than those
collected by drooling and this can affect storage and further
analysis of several compounds [57].
Stimulated whole saliva can be obtained with oral movements such as gentle mastication or with the use of citric acid.
Citric acid has the potential to stimulate salivary secretion and it
lowers the sample pH (b 3.0), but it may also affect analysis
results in hormonal immunoassay by interfering with antibody
binding [61]. Citric acid may also interfere with the measurement of some analytes, such as testosterone [62].
The most commonly used devices are sterile cotton dental
rolls as Salivette (Sarsted, Newton, NC). The cotton roll is
placed into the subject's mouth and is gently chewed for about
1–2 min, then placed into the vial. Saliva is obtained by
expressing the saturated cotton using a needleless syringe or
better by centrifugation. Cotton wool sampling may induce
variations in salivary immunoassays: testosterone, DHEA,
estradiol, 17-OH hydroxyprogesterone [63] may be artificially
high and s-IgA levels may be artificially low. Salivary levels of
other steroid hormones (cortisol, DHEA-S and cotinine) do not
seem to be affected by cotton wool sampling [64], but our
laboratory data (unpublished) demonstrate cortisol binding to
cotton wool does occur.
To avoid this type of analyte interference, non-cotton based
sampling are available:
1. Polystyrene foam swabs [60].
2. Rayon balls, such as Orapette (Trinity Biotech, Dublin,
Ireland) [63].
3. Polyester Salivette (Sarsted, Newton, NC) [64].
However, polystyrene and polyethylene vials and tubes were
found to bind and remove up to 87% of the progesterone from
saliva [65].
Stimulated saliva may also be obtained using non absorbing
methods:
1. Chewing a piece of paraffin wax of standardized size
[66,67].
2. Chewing neutral gum base [59].
3. Chewing parafilm or rubber bands [20].
4. Keeping in mouth powdered drink crystals [68] or other
foodstuff containing citric acid [17].
4. 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.
35
investigation suggested that s-IgA can be degraded at room
temperature from bacterial proteases [57]. Ng et al. reported a
decrease of 10% in s-IgA levels after 8 months of storage at
− 30 °C of plain saliva [34]. Morris et al. observed that IgG
levels measured by antibody capture ELISA in saliva samples,
did not decrease stored at room temperature (10–20 °C) for a
week [69]. Other saliva substances, such as catecholamines, can
have a short biological half-life and a rapid degradation [40].
The method of specimen storage can therefore influence the
concentration of some compounds modifying their levels.
With regard to saliva storage and cortisol, this hormone
seems to decrease its concentration by 9.2% per month at room
temperature, but at 5 °C no effect on salivary cortisol
concentration for up to 3 months was demonstrated [70].
Furthermore, Garde and Hansen demonstrated that repeated
cycles of freezing and thawing did not affect the concentration
of salivary cortisol [70]. Saliva progesterone concentration was
stable for 3 months at room temperature [65].
Storage procedure and time from the collection mainly affect
the analysis of the biochemical variables characterized by
temperature instability and bacterial growth. Some salivary
compounds can have a very short half-life so the sample to be
analysed needs a narrow range of time after collection; other
substances can remain stable in saliva for a longer time and may
be detected and quantified after a long time [57]. For this reason,
the choice of different storage procedure before the analysis
depends on the type of molecule, taking into account its stability.
We propose, as a general approach to avoid degradation of
salivary compounds, that the specimens should be stored taking
into account the following outline:
1. Immediately store saliva aliquots without any processing.
Specimens can often be stored at room temperature (when
analysis is carried out immediately or in 30–90 min from
collection), at +4 °C (when analysis is carried out in 3–6 h
from collection), at − 20 °C and better at − 80 °C (when
analysis is carried out days to months after collection).
2. Snap-freezing of saliva in liquid nitrogen: mix each saliva
aliquots with an equal volume of 80% glycerol in H2O, then
dip the sample in liquid nitrogen. This storage procedure
aims to inhibit the bacterial protease activity degrading some
salivary protein compounds, such as s-IgA [57].
3. Inhibition of the enzyme activity present in saliva: mix each
saliva aliquot with enzyme inhibitors 10:1 (leupeptin,
aprotinin and 4-[2-aminoethyl] benzenesulfonyl fluoride)
[57]. In our laboratory a mixture of protease inhibitors and
stabilizing substances is used (aprotinin, leupeptin, antipain,
pepstatin A, phenyl methyl sulfonyl fluoride, EDTA and
thimerosal).
4. Addition of sodium azide (NaN3) to saliva specimens in
attempt to retard bacterial growth. The use of sodium azide
does not influence the measurement of salivary markers
when serum-based immunoradioassays are modified for
saliva, not even if these methods involve separation or
extraction steps. But the possible interference of sodiumazide with horseradish peroxydase, a common component of
enzyme immunoassays, must be taken into account [45].
36
S. Chiappin et al. / Clinica Chimica Acta 383 (2007) 30–40
5. Addition of trifluor acetate at 10% water solution, to
denature salivary enzymes that could degrade several
salivary compounds, such as proteins and steroid hormones
[49].
5. 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 measure a single
compound in oral fluid. Most reports do not use the suggested
procedure with the use of commercially available reagents, but
frequently adapt the commercial kits, designed for serum or
plasma assay, modifying the procedures/techniques to obtain a
method suitable for saliva. In these cases the analysis results
should be well defined with method performance data and
reference ranges. Moreover, in some cases, the oral fluid
analysis is to be preferred having a better performance and
efficiency than the plasma analysis.
Among immunoassays, to date, several commercial kits are
available for plasma, serum or urine analysis of a wide range of
compounds, and they may be adapted for saliva analyses with
slight modifications [34,49,51]. Recently an increasing number
of kits especially designed for salivary assays have become
available [71] such as salivary melatonin and steroids (cortisol,
testosterone, estriol etc.).
5.1. Interferences
Blood contamination could be a problem in saliva assays
because, when some blood compounds are present in the oral
mucosa, quantitative estimates of salivary molecules may be
compromised. This aspect is relevant because the correlation
between saliva and serum concentrations is necessary to use oral
fluid as an alternative to plasma matrix (for further details: see
Section 6). Schwartz et al. proposed salivary transferrin as an
index of blood sample contamination [72]. Huang observed that
some proteins (hemoglobin β-chain, apolipoprotein A-1,
thioredoxin peroxiredoxin B, a protein highly expressed in
erythrocytes) increase their levels in whole saliva when oral
bleeding occurs. These proteins have also been proposed as
possible markers of blood contamination of saliva [28].
Some studies indicate that food products may contain
substances which can potentially cross-react with steroid
antibodies used in salivary immunoassays for testosterone
(e.g., bovine hormones in dairy products) [64], but some other
studies did not find any effect of food intake on the
measurement of cortisol and 17OH-progesterone and progesterone with RIA methods [49].
As previously described, sodium azide can interfere in EIA
and ELISA.
6. 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 the
development of bioanalytical technologies and the statement of
salivary compound reference values in healthy subjects. This is
the major difference between oral fluid and plasma. These
physiological variations in oral fluid may cause difficulties in the
standardization of salivary analyses, making plasma assays
preferable to oral fluid because of their better stability.
Many salivary compounds can derive from plasma by
passive diffusion or active transport: in this perspective salivary
analysis has a great potential diagnostic role, because several
biochemical and immunological parameters can be measured
both in blood and in saliva (e.g., immunoglobulins). Therefore
to set up saliva as an alternative matrix to plasma for various
assays of biological quantitative parameter, there must be a high
correlation confirmed between plasma and saliva levels. In the
case of passive diffusion from blood, a correlation should be
found between blood and salivary levels, but the lack of this
correlation does not exclude that the measured component has a
blood origin. In effect the diffusion of the investigated molecule
might only take place partially and an active transport or a
reabsorption can affect the correlation between blood and
salivary levels of the analyte. In any case, to reflect systemic
bioactivity usefully, a quantitative assay for those parameters in
saliva should be highly correlated with the levels in serum.
A large amount of salivary compounds are produced directly
by salivary glands and can hardly be detected in plasma. For
example, secretory IgA and lysozyme are present in great
amounts in saliva, but less in blood, so they can be considered as
originating from saliva. Otherwise the concentration of
transferrin, iron, bilirubin, cholesterol, TG, lipoproteins, IgG
and IgM in blood is 4–15 times higher than in saliva. Nagler et
al. found a high correlation between plasma and salivary levels
for blood urea nitrogen, C4 and total bilirubin, suggesting that
the presence of these compounds in saliva may derive from
passive diffusion from plasma [8].
Saliva is currently used in the measurement of steroid
hormones, such as cortisol [73] and cortisone [74], testosterone
[75,76], because it is commonly accepted that the salivary level
of these steroids reflects the free, unbound circulating fraction.
Steroids are also present in urine with their free, unbound
fraction, but urinary steroid metabolites may cross-react with
antibodies used for the steroid immunoassay analyses. Furthermore, urinary-free steroids are usually measured in the 24-h
urine and this way of collection is often not easy.
The majority of steroids in plasma circulates bound to albumin and specific proteins, such as SHBG (sex hormone
binding globulin) and CBG (cortisol binding globulin), while
S. Chiappin et al. / Clinica Chimica Acta 383 (2007) 30–40
only the free fraction (in equilibrium with the bound steroids) is
the bioactive fraction. Free steroids, because of their liposolubility, pass through the cell membrane, and are directly
available for the cell receptors. Thus, salivary steroids are
thought to reflect the concentration of the physiologically
active unbound steroids in serum [66]. Blood leakage into oral
fluid (due to periodontal diseases, injury or vigorous cleaning)
affects the quantitative estimates of salivary-free steroid
concentrations [55].
Also non-steroid hormones can be detected in saliva, such as
melatonin [52], and preliminary studies suggest that thyroid
hormones can also be measured in saliva [41], but it is not clear
if their detection is caused from plasma leakage.
Protein/Polypeptide hormones do not seem to be detectable in
saliva unless they are directly produced by salivary glands: for
example, leptin seems to be directly produced by salivary glands
and it can be detected in saliva even if it is a protein hormone
(16 kDa) [76]. Generally, protein plasma hormones cannot be
detected in saliva because of their large dimensions: i.e., they
cannot pass through the salivary glands by passive diffusion [7].
In this case, their detection in oral fluid may be an index of
plasma leakage from oral lesions, but mechanisms other than
passive diffusion from plasma, such as an active transport and a
primary secretion, might be present. Another example of a
polypeptide hormone recently proposed for measurement in
human saliva is the insulin-like growth factor I [51]. This
hormone might be produced in the mouth as a growth factor, but
it could be also originated by a diffusion (active or not).
37
reason, oral fluids with a higher content of IgG are suitable for
use in the screening of viral infections and immunization. A
correlation between salivary and serum antibodies has previously been reported for HIV (human immunodeficiency
virus) [79]. Blood contamination improves the HIV antibody
detection in saliva [60]: so this phenomenon is not always a
negative fact, because it can help the revealing of antibodies,
drugs, and other molecules, which are normally detected in
higher levels in plasma than in saliva. Correlation between
salivary and serum IgG levels is also present in HCV antibodies
[80], HAV (hepatitis A virus) [81], EBV (Epstein Barr virus)
[82], CMV (Cito Megalo virus) and rubella virus. Salivary
antibodies have also been reported after immunization against
poliovirus, rotavirus and HAV [80]. For these reasons, salivary
testing for specific antibodies can be an important means to
evaluate systemic immunity in diseases or to evaluate immunity
in response to vaccination. Oral fluid collection devices,
designed for this purpose, are commercially available [60].
7.2. Cancer
c-erbB-2 soluble fragments and 15-3 cancer antigen in breast
cancer research have been demonstrated in saliva [38]. Also, the
profiling of salivary RNA transcript with microarray analysis
seems to be able to distinguish several genes exhibiting
significantly different expression levels in saliva comparing
oral squamous cell carcinoma patients with controls [78].
7.3. Pharmaceutical and abuse drugs
7. Saliva as a diagnostic tool
Recently there has been increasing interest in diagnosis
based on saliva analyses, because saliva has a simple and noninvasive 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 for diagnostic purposes is the linkage with traditional
biochemical parameters which appear in the circulation in
various forms.
Saliva analyses have been used mainly in dentistry and for
studies in oral diseases to help assess the risk of caries, by
measuring saliva buffer capacity and bacterial contents [2]. Oral
fluid is mainly utilized for research and diagnostic purposes
concerning systemic diseases that involve the salivary glands
and oral cavity, such as Sjögren syndrome [11,77], Beçhet
syndrome, benignant and malignant oral tumours [78].
Saliva investigation for diagnostic purposes has been
proposed in the following.
7.1. Viral and bacterial infections (genomes and antibodies
detection)
Oral fluid contains a small amount of IgG, mainly derived
from crevicular fluid and from mucosal transudate. For this
Some pharmaceutical drugs (e.g., lithium, digoxine, phenobarbital and others) have a narrow therapeutical index; for this
reason a constant monitoring of their plasma concentrations is
necessary to achieve the best therapeutic effect while reducing
the risk of adverse effects, especially in patients with hepatic or
renal impairment. Therapeutic monitoring of these drugs is
often performed in plasma. Recently Guo et al. developed a
method with liquid chromatography-electrospray tandem mass
spectrometry to measure levetiracetam levels also in saliva [83].
Furthermore, many substances which are commonly abused can
be detected in human saliva including alcohol, amphetamines,
barbiturates, benzodiazepines, cocaine, lysergic acid dietylamide (LSD), opioids, phencyclidine and cotinine for tobacco
smoke [84]. Salivary testing of these substances is particularly
useful where a “yes/no” answer is required, because plasma
leakage does not affect qualitative analysis [38]. The possibilities offered by saliva for protein compound detection have
recently been proposed [51] and this field of research seems to
be a new frontier for sport anti-doping test purposes.
7.4. Hormones
Putignano et al. proposed midnight salivary cortisol
measurement as a screening procedure for patients with
suspected Cushing's syndrome [85]. Abnormal salivary diurnal
cortisol rhythms have been shown to be predictive of disease
progression in metastatic breast cancer patients [86]. In this
38
S. Chiappin et al. / Clinica Chimica Acta 383 (2007) 30–40
respect, salivary concentrations may be a better measure of the
exposure of target organs to the steroids than the serum
concentrations are. The salivary-free estradiol measurement is
an FDA-approved test which can predict preterm labour in
women at risk [38]. The non-steroid hormone detection and
measurement are rarely performed. Also protein polypeptide
hormones have been analysed in the saliva, as previously
mentioned [28,43–48,51,52] and a patient with hormonal
pathologies could be investigated. Therapy monitoring could
also be included.
7.5. DNA tests
DNA in cells present in whole saliva could also be
investigated. The traditional source of genomic DNA is
blood, but recently saliva has increasingly been investigated
as a source of DNA deriving from oral cells. It provides a useful
source for biomarker profiling and forensic identification
[38,87]. DNA tests in saliva could also be carried out for the
detection of HIV infection, recognizing viral sequences in total
salivary DNA amplifying by polymerase chain reaction a
relatively constant region of HIV-1 genome, [88]. In oral fluid
are also detectable by PCR several oral pathogens that may
cause periodontitis [89].
7.6. Sialochemistry analysis
Sialochemistry for environmental heavy metals (cadmium,
lead, mercury) may be useful in monitoring environmental,
atmospheric and occupational pollutants: Gonzalez et al. found
that saliva can be a good milieu for early monitoring of an
exposure to lead and cadmium, since salivary levels of these
elements arise from the diffusible fraction of plasma [20].
Therefore, there is an increasing interest on the diagnostic
role of saliva but there are only very few studies that utilize oral
fluid in the diagnosis of systemic diseases, such as alcoholic
cirrhosis and cystic fibrosis [3,8]. Consequently this research
area must be further developed, because in saliva the
measurements of some markers of systemic diseases, normally
detected in serum/plasma or urines, seem to be possible.
8. 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 assays, even if it is possible to
have a quantitative estimate of hormones and other substances
in saliva.
In conclusion, saliva is a biological fluid that offers several
opportunities in diagnosis, toxicology and in forensic science.
Furthermore, many salivary proteins offer great potential in
clinical and epidemiological research, in oral as well as in
general health studies.
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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