Journal of Indian Academy of Oral Medicine and Radiology, April-June 2013;25(2):00-00

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Sialochemistry: A Key to Investigation for Oral Diagnosis
JIAOMR
REVIEW ARTICLE
Sialochemistry: A Key to Investigation for Oral Diagnosis
Parvathi Devi MK, Shrinivas Koppal, Thriveni R, Amit Byatnal, Rajshekar Patil, Usha C
ABSTRACT
As a diagnostic fluid, saliva offers distinctive advantages over
serum because individuals with modest training can collect it
non-invasively. Saliva may provide a cost-effective approach
for the screening of large populations. Gland specific saliva can
be used for diagnosis of pathology specific to one of the major
salivary glands. Analysis of saliva may be useful for the diagnosis
of hereditary disorders, autoimmune diseases, malignant and
infectious diseases, and endocrine disorders, as well as in the
assessment of therapeutic levels of drugs and the monitoring
of illicit drug use.
Keywords: Saliva, Sialochemistry, Sjogrens syndrome.
How to cite this article: Devi MK P, Koppal S, Thriveni R,
Byatnal A, Patil R, Usha C. Sialochemistry: A Key to Investigation
for Oral Diagnosis. J Indian Aca Oral Med Radiol 2013;25(2):0-0.
Source of support: Nil
Conflict of interest: None declared
INTRODUCTION
A large number of diagnostic analyses have been shown to
be present in saliva, including steroid hormones
1
and the
HIV antibody.
2
For the past two decades, oral health
researchers have been developing salivary diagnostic tools
to monitor oral diseases (including periodontal diseases)
3,4
as well as for caries risk assessment.
5
These diagnostic
advances range from genetic susceptibility analysis of
interleukin-1 (IL-1) genetic alleles to the analysis of oral
pathogens identified via lectin staining for caries risk
assessment.
6
The current development of diagnostic
biomarkers (via proteomic and genomic approaches) in
conjunction with technological developments in salivary
diagnostics will lead to the development of robust diagnostic
tools for dentists to use in making clinical decisions and
predicting treatment outcomes.
7
SALIVA COLLECTION
Whole saliva is a product of secretion of 3 major glands
(parotid, submandibular, sublingual) and many minor glands
(labial, buccal, palatal). Unstimulated saliva is usually
obtained as the patient spits out every 60 sec or by forward
bended head the patient allows saliva to drip off the lower
lip into a cylinder. By collection of saliva in the tube the
flow rate per unit time can be measured. When volume
measurement is not required the saliva can be collected on
cotton rolls, gauze or filter paper. For evaluating salivary
gland function or when large volumes of saliva are required
10.5005/jp-journals-10011-......................
for analytic purposes, stimulated whole saliva is used.
Method of collection is the same as for unstimulated
saliva.8The usual masticatory stimuli are paraffin wax or a
washed rubber band. A standard gustatory stimulus is
obtained by 2% citric acid applied directly to the tongue
every 15 to 60 sec. Parotid saliva can be collected by
aspiration from the duct opening with a micropipette. Parotid
saliva is best collected with Lashleys vacuum chamber
( Figs 1 and 2).
9
Submandibular and sublingual saliva can be collected
by cannulation of the duct with micropipette, but in practice
this is both uncomfortable for the patients and technically
difficult since the duct orifice is mobile and has a strong
sphincter (Figs 3 and 4). Because of that, alginate and
silicone impression material is used for retention of the
collecting tube. As alternative and simple technique is to
block off secretion from the parotid glands with absorbent
swabs and collect mixed submandibular and sublingual
saliva by pipette from the floor of the mouth. Saliva from
labial and palatal glands can be collected by filter paper
disc or disc of other synthetic materials.
10
SIALOCHEMISTRY
The diagnostic use of saliva has attracted the attention of
numerous investigators because of the noninvasive nature
and relative simplicity of collection. The presence of six
enzymes was established in parotid saliva: acid phosphatase,
total esterases, cholinesterase, lipase, beta-glucuronidase,
and lysozyme. Broth cultures used for this study with whole
saliva indicated that all but sulfatase and lysozyme were
produced by the oral flora.
8
Abnormal proteins are also
Fig. 1: Lashleys vacuum chamber
Parvathi Devi MK et al
2
hormones (cortisol, progesterone, estriol, testosterone, etc.)
and drugs (diazepam, caffeine, lithium, theophylline,
tolbutamide, methotrexate, antibiotics, anticonvulsants, etc.)
is becoming a conventional procedure. Salivary analysis
can be valuable to discriminate and monitor swellings of
major salivary glands such as chronic recurrent parotitis,
where the blood-saliva barrier is violated in the
inflammatory flare-ups and characterized by the leakage of
serum components (albumin) and lactoferrin into saliva
(Tabak et al., 1978).
9
Saliva has also been of value in systemic diseases such
as cystic fibrosis, where a decrease in flow rate and a
significant increase in sodium concentration has
been reported to occur in minor salivary glands (Wiesmann
et al., 1972).
7
Saliva is a fluid that can be easily collected, contains
locally-derived and systemically-derived markers of
periodontal disease, and hence may offer the basis for a
patient specific diagnostic test for periodontitis. Higher
enzyme activities were found in the adult periodontitis
patients compared to the healthy controls for alkaline
phosphatase, esterase, -glucuronidase, -glucosidase, and
other aminopeptidases. Saliva from patients with localized
juvenile periodontitis contained the highest levels of
butyrate esterase and cystiene aminopeptidase.
12
Sialochemistry can reveal the differentiation between
normal and abnormal function of the glands, information
about gland dysfunction and its impact on the oral
environment, clues to homeostatic fluctuations as a result
of circulatory, innervatory or hormonal adjustments. The
development of microchips for salivary components offers
great possibilities to use oral fluid for point-of-care testing.
1
Saliva can be used to detect recent use of illicit drugs,
for monitoring patient compliance with psychiatric
medications.
4
Saliva is also useful for the monitoring of
anti-epileptic drugs. Salivary carbamazepine levels were
found to be 38% of serum carbamazepine levels, and a
positive correlation between salivary and serum
carbamazepine levels was observed. Salivary theophylline
concentration demonstrated a better correlation with serum
concentration of free theophylline than with serum
concentration of total theophylline.
7
Saliva may be used for monitoring levels of anti-cancer
drugs and a reliable alternative to serum for the monitoring
of irinotecan levels.
8
Salivary ethanol concentration used
as an index of the blood ethanol concentration, provided
that the salivary sample is obtained at least 20 min following
ingestion allowing for absorption and distribution of alcohol,
and prevent a falsely elevated reading due to the oral route
of consumption.
11
Other recreational drugs that can be
Fig. 2: Lashleys cupcollection of saliva from parotid duct
Fig. 3: Collection of saliva
Fig. 4: Collection of saliva in floor of the mouth by suction
produced under exceptional conditions, such as the
development of tumours and nutritional deficiency. Low
a-amylase concentrations are seen in cases of starvation and
after destruction and degeneration of the acinar cells.
Elevated a-amylase is seen in abnormal ductal water loss.
Furthermore, acute inflammation of the glands produces a
rise in plasma and urine amylase due to gross glandular
leakage. This will be seen in mumps as well as in the
presence of a salivary calculus.
11
Another fertile area of
application for salivary analysis is in laboratory medicine,
where determining and monitoring levels of various
Journal of Indian Academy of Oral Medicine and Radiology, April-June 2013;25(2):00-00
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Sialochemistry: A Key to Investigation for Oral Diagnosis
JIAOMR
identified in saliva are amphetamines, barbiturates,
benzodiazepines, cocaine, phencyclidine (PCP) and opioids.
2
Saliva increasingly being used as an investigational aid
in the diagnosis of systemic diseases that affect the function
of the salivary glands and the composition of the saliva,
such as Sjgrens syndrome, alcoholic cirrhosis, cystic
fibrosis, sarcoidosis, diabetes mellitus and diseases of the
adrenal cortex, the onset and severity of infectious diseases
can be determined by monitoring the presence of antibodies
to the microorganisms found in saliva.
12
In disease diagnosis, saliva has many advantages over
both serum and urine. For example, salivary assays for anti-
bodies (to viruses and bacteria), unconjugated steroid
hormones (e.g., estrogen, testosterone and progesterone),
environmental toxins (e.g., cadmium, lead and mercury),
tobacco (cotinine) and certain drugs (e.g., ethanol, theophylline
and lithium) are sufficiently sensitive to accurately reflect
the blood concentrations of these substances.
2
The concentration of IgA was somewhat elevated
(commensurate with reduced flow rate); the levels of IgG,
IgM, and albumin were normal. The major functional
abnormality of the parotid gland in Sjgrens syndrome
appears to be luminal transport in the ductal region; leakage
of serum components is minimal. Sialochemistry can be
helpful in differentiating Sjgrens disease from other
diseases of the salivary gland and in assessing degree of
pathologic change.
3
p53 antibody is a tumor suppressor protein which is
produced in cells exposed to various types of DNA-
damaging stress can also be detected in the saliva of patients
diagnosed with oral squamous cell carcinoma (SCC) and
can thus assist in the early detection and screening for this
tumor (Tavassoli et al., 1998). Higher concentrations of
salivary defensin-1 were detected in patients with oral SCC
in comparison with the defensin-1 concentration in the saliva
of patients with adenocarcinoma and in healthy controls.
4
In one of the studies the activity of CK (creatinine
kinase) LDH, ACP (acidic phosphatase) GGT (gamma
glutamyl transferase) in saliva from patients with periodontal
disease before & after treatment (experimental Group
30 samples) & in saliva from periodontally healthy patients
(20 samples). The results showed the elevated levels of these
enzymes before the treatment in periodontal patients when
compared with healthy patients.
5
The significant decrease
of sodium in sialadenosis, and increase of sodium and total
protein in sialadenitis.
6
Lal et al found that Histatins were decreasing in AIDS
patients. However Atkinson (1990) found that the saliva of
HIV-positive patients contained increased levels of histatins
in stimulated submandibular saliva Lactoferrin output was
shown to be significantly reduced in HIV-positive individuals.
However, when examining the stimulated submandibular
saliva. Atkinson et al found that saliva of HIV positive
patients contained increased levels of lactoferrin.
10
Significantly lower levels of lactoferrin were found in
resting saliva of children with chronic protein-energy
malnutrition. Non- insulin dependent diabetics were shown
to have increased levels of lactoferrin. Lactoferrin content
was also increased in parotid saliva of irradiated patients.
9
Parotid and submandibular gland secretions collected
from patients with rheumatoid arthritis or systemic sclerosis
have been analyzed and the results compared with those
obtained from a matched group of healthy individuals. The
saliva samples assayed for amylase, kallikrein, protein, and
salivary IgA concentration.
3
Acute hepatitis A (HAV) and hepatitis B (HBV) were
diagnosed based on the presence of IgM antibodies in saliva.
The ratio of IgM to IgG anti-HAV antibody correlated with
the time interval from onset of infection (Parry et al., 1989).
Further, salivary antibody levels were used for the detection
of infected individuals. Comparison of serum and saliva
levels of antibody to HAV revealed excellent agreement
(sensitivity =98.7% and specificity =99.6%; Ochnio et al.,
1997). Analysis of oral fluid samples collected with Orasure
provided an excellent method for the diagnosis of viral
hepatitis B and C. Sensitivity and specificity of 100%
for the detection of antibodies for both diseases in oral
fluid in comparison with serum antibodies were reported
(Thieme et al., 1992). Sialocemistry has been used for
screening for hepatitis B surface antigen (HbsAg) in
epidemiological studies.
11
ORAL FLUID NANOSENSOR TEST
High-impact diseases, including cancer, cardiovascular
disease, and neurological disease, are challenging to
diagnose without supplementing clinical evaluation with
laboratory testing. Even with laboratory tools, definitive
diagnosis often remains elusive. The Oral Fluid NanoSensor
Test (OFNASET) technology platform combines cutting-
edge technologies, such as self-assembled monolayers
(SAM), bionanotechnology, cyclic enzymatic amplification,
and microfluidics, with several well-established techniques
including microinjection molding, hybridization-based
detection, and molecular purification. The use of the
OFNASET helps multiplex detection of salivary biomarkers
for oral cancer.
3
A nanoparticle that signals when cells are undergoing
apoptosis the kind of cell death triggered by cancer therapies.
The new nanoparticles could finally provide oncologists
with a rapid assay that could tell them that a given therapy
is working.
Parvathi Devi MK et al
4
The apoptosis detector phosphatidylserine (PS), is found
normally on the inside of the cell membrane, but this
molecule moves to the outside layer of the cell membrane
when a cell begins apoptosis. To detect this early indicator
of apoptosis, the researchers constructed an artificial small,
fluorescent protein that would bind to four different
molecules of PS. Test tube experiments showed this
construct did indeed bind to PS but not to other membrane
components; however, when the investigators tried to use
it to detect apoptotic cells, the experiments were a failure.
Evidently, their test construct did not bind strongly enough
and for a long enough time to apoptotic cells to be detectable.
Later a magnetic iron oxide nanoparticle that has been
developing for imaging tumor metastasis. They reasoned
that they could attach an average of 41 of the PS-binding
molecules to each nanoparticle, which should increase
dramatically the ability of the resulting construct to not only
bind to PS, but remain stuck to the apoptotic cell through a
process called cooperative binding. Cooperative binding is
what gives such enormous sticking power to the hundreds
of tiny loops and hooks on matching pieces of Velcro the
nanoparticle-based PS-binder to cells triggered to undergo
apoptosis, they were able to easily identify the apoptotic
cells using a fluorescent microscope or a standard cell
sorting apparatus. Their new apoptosis detector holds
significant promise for use in cancer imaging applications.
4
The UCLA (University of California at Los Angeles)
laboratory recently discovered that discriminatory and
diagnostic human mRNAs are present in the saliva of healthy
people and people with disease. The salivary transcriptome
offers an additional valuable resource for disease
diagnostics. The first report of the salivary transcriptome
demonstrated that the normal salivary transcriptome consists
of about 3,000 mRNAs.
7
Of particular importance is that of
the 3,000 mRNAs, 180 are common between healthy subjects,
constituting the normal salivary transcriptome core (NSTC).
To demonstrate the diagnostic and translational potential
of the salivary transcriptome, the UCLA group profiled and
analyzed saliva from patients with oral cancer. Four genes
from the NSTC (IL-8, ornithine decarboxylase, spermidine
acetyltransferase and IL-1) were able to discriminate and
predict whether a saliva sample was from a patient with
cancer or from a healthy subject, with a sensitivity and
specificity of 91 percent each (receiver operator
characteristic [ROC] =0.95) (Figs 5 and 6). The group has
validated these salivary transcriptome biomarkers for oral
cancer detection in approximately 300 subjects. The
behavior of these salivary transcriptome biomarkers is
consistent that is, their levels are significantly higher in the
saliva of patients with oral cancer than in the saliva of
matched control subjects.
5
CONCLUSION
The immune and non-immune antimicrobial salivary factors
have been measured in different disease states to observe
the variations in their levels in an attempt to understand the
pathogenesis of some systemic diseases and to formulate
better lines of treatment particularly for diseases that are
either difficult to cure or that are not treatable yet. The highly
sensitive test procedures that are now commonplace makes
it practical to quantitate despite very low concentrations a
large number of hormones and drugs in saliva. The saliva
has found use as a diagnostic aid in an increasing number
of clinical situations and in systemic diseases that can affect
salivary gland function and composition such as Sjogrens
syndrome, cystic fibrosis and diseases of adrenal cortex.
Sialochemistry does not accommodate classical
nosology. Different diseases that have inflammatory
processes in common will show the same changes in their
sialochemical patterns. These patterns are very sensitive.
At the same time the specificity regarding classified diseases
is low. Therefore a correct diagnosis will always require a
full clinical and laboratory investigation. However
sialochemistry is a useful means of chronologically monitoring
quantitative changes.
Fig. 5: Apoptosis detector
Fig. 6: Nanosensor detector
Journal of Indian Academy of Oral Medicine and Radiology, April-June 2013;25(2):00-00
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Sialochemistry: A Key to Investigation for Oral Diagnosis
JIAOMR
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ABOUT THE AUTHORS
Parvathi Devi MK (Corresponding Author)
Assistant Professor, Department of Oral Medicine and Radiology
AMES Dental College and Hospital, Raichur, Karnataka, India
e-mail: drparoomr@yahoo.co.in
Shrinivas Koppal
Reader, Department of Oral Medicine and Radiology, AMES Dental
College and Hospital, Karnataka, India
Thriveni R
Professor and Head, Department of Oral Medicine and Radiology
AMES Dental College and Hospital, Raichur, Karnataka, India
Amit Byatnal
Assistant Professor, Department of Oral Medicine and Radiology
AMES Dental College and Hospital, Raichur, Karnataka
American Samoa
Rajshekar Patil
Assistant Professor, Department of Oral Medicine and Radiology
AMES Dental College and Hospital, Raichur, Karnataka, India
Usha C
Assistant Professor, Department of Oral Medicine and Radiology
Sharavathi Dental College and Hospital, Shimoga, Karnataka
American Samoa