Euro Weight Loss-2015
Frankfurt, Germany
August 18 – 20, 2015
Ana Rodrigues Costa
How saliva can be
useful in weight
management programs
Ana Rodrigues Costa
University of Évora, Portugal
Biologic
functions of
saliva
Moistening and
preprocessing of food
Bicarbonate and phosphate
buffer systems, Carbonic
anhydrases, Urea, Sialin
Gustin, Salivary proline-rich
proteins, Amylases, Lipase,
Carbonic anhydrase VI
Taste
and food
perception
Secretory
Buffering
immunoglobulin A (IgA),
lysozyme, lactoferrin,
Cystatins,
Anti viral and
Histatins, Mucins,
anti bacterial
Salivary
Peroxidases
Amylases, Mucins,
Lipase
Digestion
Functions
Histatins
Anti
fungal
Aiding in deglutition
Tissue
Coating
Protection of the oral cavity
Amylases, Cystatins, Mucins,
Proline-Rich Proteins,
Statherins
Mineralization Cystatins,
Histatins, ProlineRich Proteins,
Statherins
Lubricati
on &
Viscosity
Mucins, Statherins
SALIVA – a complex body fluid
Exocrine contributions:
- From the three pairs of “major” salivar glands
- From the “minor” salivar glands
Markers derived from
sympathetic or parasympathetic
salivar secretion stimulation
How is saliva
produced?
•
Sympathetic stimulation
Secretion of a small amount of saliva
with increased protein concentration
Non-exocrine components:
- Micro-organisms
- Desquamated epithelial cells
- Leukocytes
- Gingival crevicular fluid
- Mucosal transudation
•
Parasympathetic stimulation
Production of a high volume of saliva
with low protein concentration
Genetic markers
Markers derived from the
circulation
https://www.google.pt/search?q=salivary+glands&rlz=1C1SFXN_enPT498PT508&espv=2&biw=1366&bih=643&tbm=isch&tbo=u&source=univ&sa=X&ved=0CB4QsARqFQoTCLO50J2eoccC
FQKzFAodS8oJIQ#imgdii=BOhm7SDx2EybuM%3A%3BBOhm7SDx2EybuM%3A%3BaaH3BzbivbMU9M%3A&imgrc=BOhm7SDx2EybuM%3A
 Saliva offers distinctive advantages over blood collection - it is a
readily available biofluid that meets the demands for a collection:
Advantages of
saliva analysis
 Non invasiveness
 Stress-free
 Inexpensive
 Relatively to urine, the great advantage is:
 Saliva can reflect real-time levels of biomarkers
DNA or RNA
Which saliva
components
can be
measured?
Hormones
Total
protein
17a-hydroprogesterone
Aldosterone
Androstenedione
Enzymes
Chromogranin A
Cortisol
a-amylase
DHEA
carbonic anhydrase
Estradiol
lypase
Estriol
Estrone
Melatonin
Progesterone
Testosterone
Insuline
Inorganic
Leptine
componentes Ghreline
Na+, K+
Cl-, Mg2HPO42-, HCO3H+
Metabolic
Inflammation
markers
C-Reactive Protein
Interleukin-1 b
Interleukin-6
Neopterin
Secretory Immunoglobulin A
TNF-a
Glycose
Cholesterol
LDH-cholesterol
Triglycerides
Stress related markers
In the context
of weight
management
programs …
 Cortisol
 DHEA
 a amylase
Metabolism related
markers






Glycose
Cholesterol + HDL/cholesterol
Triglycerides
Insulin
Leptin
Ghrelin
Physical exercise markers





Testosterone
Cortisol
DHEA
a amylase
IgA, Lysozyme
Taste related markers
Saliva Proteomic analysis
 a amylase
 Carbonic anhydrase
 Lipase
Unstimulated whole saliva
Standard
protocols for
whole saliva
collection
Passive drool
Spitting
Saliva is allowed to drip off
the lower lip and is collected
in a vial
Saliva which spontaneously
appears in the mouth is
spitted in to a vial
Oral Swab Method
Stimulated whole saliva
Salivary flow is stimulated by
mastication for 10 min with
~1g of paraffin wax (Parafilm).
Salivette®
- Different composition
(parotid gland has a bigger
contribution)
Circadian rhythm must be taken into account
Ideal time for
saliva
collection
Collection of the
saliva at the same
hour of the day
Dawes, C., J. Physiol. (1972), 220, 529-545
 Avoid alcohol for 12h before sample collection
 Avoid eating major meals within 1h of sample collection
Cautions
during sample
collection
30 min
before
• Brush the teeth properly
without toothpaste
• Avoid food or fluid (apart from
water) ingestion
• Avoid chewing gum
• Avoid smoking
 Rinse mouth with drinking water (or distilled) five minutes before
the collection of saliva
 Discard samples with blood traces
 (Donors should not present signs of periodontal disease or caries).
Samples collected in
tubes placed on crushed
ice
Eventual addition of protease
inhibitors (e.g. sodium fluoride)
Sample
processing
1st centrifugation
2.600 g, 15
min, 4ºC
Cells and large particulate
debris free sample
Eventual storage at
-20ºC
2nd centrifugation
18.000 g, 20
min, 4ºC
Alternative to centrifugation – use of denaturing conditions
(buffers containing 4-6M guanidine hydrochloride or
dithiothreitol)
Mucins free sample
(lower viscosity)
Blood-borne constituents
Reliable measurements assumes a
constant saliva/plasma ratio (SPR)
Interpretation
of results
- pitfalls
Concentration in saliva
truthfully follows intra- and
interdindividual variations in
plasma
Only valid for molecules:
 Diffuse passively
 Low molecular weight (MW)
 Suffer ultrafiltration
 Low MW Steroid
hormones (e.g. cortisol)
 Low MW peptidic
hormones (e.g. insulin)
Salivary glands produced
Marker of
sympathetic NS
activity ?
Concentration/
function
evaluation?
Chronic or acute
evaluations?
How to express these
results?
2.5
Salivary flux: 0.45 ± 0.29 mL/min
(0.05 – 2.16)
Salivary flux (mL/min)
2
1.5
81 participants
Saliva collection in 3
different moments
(at same hour and
status)
1
Individual
salivary fluxes
are diferente.
0.5
0
1
3
5
7
9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47 49 51 53 55 57 59 61 63 65 67 69 71 73 75 77 79 81
1st collection - Jan
2nd collection - March
3rd collection - May
3000
Flux
Flux
1
Prot conc 0.03472606
2500
[Protein] (mg/mL)
Salivary
flux
-
2000
Prot conc
1
1500
1000
500
0
00
01
02
Salivary flux (mL/min)
03
Salivary flux
and protein
concentration
are not
correlated.
Concentration
(Western blot)
Enzymatic activity
(U/mL)
Relative Enzymatic
activity
(U/mg protein)
Measuring
concentration
or activity?
Elsa Lamy, Carla Simões, Lénia Rodrigues, Ana Rodrigues Costa, Rui Vitorino, Francisco Amado, Célia Antunes, Isabel do Carmo “Changes in salivary protein profile in morbid obese women with
and without bariatric Surgery”, in press.
When the purpose is to interpret salivary pretein levels in terms of
autonomic nervous system activity, the assessment of secretion rate can be
relevant.
Rate of Enzymatic
secretion (U/min)
Measuring
secretion?
Rate of Enzymatic secretion (U/min) =
Enzymatic activity (U/mL) x
salivary flow rate (mL/min)
 Blood borne constituents of saliva which present a constant
saliva/plasma ration (like cortisol, cholesterol), reflect intra- and
interindividual variations in plasma. Its interpretations is clear.
Final remarks
 Salivary glands produced constituents (like a-amylase) sometimes
may result in tricky interpretation, but can also be very useful as
biomarkers for use in weight management programs
Acknowledgements
Elsa Lamy
Fernando Capela e Silva
Cristina Pinheiro
Alfredo Pereira
Lénia Rodrigues (Student)
Célia Antunes
Daniela Moreira (Student)
Nuno Batalha
André Freitas (Student)
DEPARTAMENTO DE
DESPORTO E SAÚDE
Meet the eminent gathering once again at
Euro Weight Loss-2016
Vienna, Austria
September 19-21, 2016
Euro Weight Loss – 2016
Website: http://weightloss.global-summit.com/europe/