Ana Rodrigues Costa

Euro Weight Loss-2015Frankfurt, GermanyAugust 18 – 20, 2015

How saliva can be useful in weight management programsAna Rodrigues Costa

University of Évora, Portugal

Biologic functions of saliva

Buffering

Digestion

Mineralization

Lubrication & Viscosity

Tissue Coating

Anti fungal

Anti viral and anti bacterial

Taste and food perception

Salivary Functio

ns

Bicarbonate and phosphate buffer

systems, Carbonic anhydrases, Urea, Sialin

Secretory immunoglobulin A

(IgA), lysozyme, lactoferrin, Cystatins,

Histatins, Mucins, Peroxidases

Histatins

Amylases, Cystatins, Mucins, Proline-Rich Proteins, Statherins

Mucins, Statherins

Cystatins, Histatins, Proline-Rich Proteins, Statherins

Amylases, Mucins, Lipase

Gustin, Salivary proline-rich proteins, Amylases, Lipase, Carbonic anhydrase VI

Moistening and preprocessing of food

Aiding in deglutition

Protection of the oral cavity

How is saliva produced?

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=0CB4QsARqFQoTCLO50J2eoccCFQKzFAodS8oJIQ#imgdii=BOhm7SDx2EybuM%3A%3BBOhm7SDx2EybuM%3A%3BaaH3BzbivbMU9M%3A&imgrc=BOhm7SDx2EybuM%3A

Exocrine contributions:- From the three pairs of “major” salivar

glands- From the “minor” salivar glands

Non-exocrine components:- Micro-organisms- Desquamated

epithelial cells- Leukocytes- Gingival crevicular fluid- Mucosal transudation

SALIVA – a complex body fluid

Markers derived from the circulation

Markers derived from sympathetic or parasympathetic salivar secretion stimulation

Genetic markers

Sympathetic stimulation• Secretion of a small amount of

saliva with increased protein concentration

Parasympathetic stimulation• Production of a high volume of

saliva with low protein concentration

Advantages of saliva analysis

Saliva offers distinctive advantages over blood collection - it is a readily available biofluid that meets the demands for a collection:

Non invasiveness Stress-free Inexpensive

Relatively to urine, the great advantage is:

Saliva can reflect real-time levels of biomarkers

Which saliva components can be measured?

Totalprotein

Enzymes

a-amylasecarbonic

anhydraselypase

Hormones

17a-hydroprogesteroneAldosterone

AndrostenedioneChromogranin A

CortisolDHEA

EstradiolEstriol

EstroneMelatonin

ProgesteroneTestosterone

InsulineLeptineGhreline

Inflammation

markersC-Reactive Protein

Interleukin-1 bInterleukin-6

NeopterinSecretory

Immunoglobulin ATNF-a

DNA or RNA

Metabolic

GlycoseCholesterol

LDH-cholesterolTriglycerides

Inorganic component

esNa+, K+

Cl-, Mg2-

HPO42-, HCO3-

H+

Metabolism related markers

Stress related markers

Physical exercise markers

Taste related markers

Cortisol DHEA a amylase

Glycose Cholesterol + HDL/cholesterol Triglycerides Insulin Leptin Ghrelin

Testosterone Cortisol DHEA a amylase IgA, Lysozyme

a amylase Carbonic anhydrase Lipase

In the context of weight management programs …

Saliva Proteomic analysis

Standard protocols for whole saliva collection

Passive drool

Oral Swab Method

Salivette® 

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

Unstimulated whole saliva

Stimulated whole saliva Salivary flow is stimulated by mastication for 10 min with ~1g of paraffin wax (Parafilm).- Different composition

(parotid gland has a bigger contribution)

Ideal time for saliva collection

Circadian rhythm must be taken into account

Dawes, C., J. Physiol. (1972), 220, 529-545

Collection of the saliva at the same hour of the day

Cautions during sample collection

Avoid alcohol for 12h before sample collection

Avoid eating major meals within 1h of sample collection

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).

• Brush the teeth properly without toothpaste

• Avoid food or fluid (apart from water) ingestion

• Avoid chewing gum• Avoid smoking

30 min before

Sample processing 1st centrifugation

Samples collected in tubes placed on

crushed ice

Eventual storage at -20ºC

2nd centrifugation

Eventual addition of protease inhibitors (e.g.

sodium fluoride)

2.600 g, 15 min,

4ºC

Cells and large particulate debris free

sample

18.000 g, 20 min,

4ºC

Mucins free sample(lower viscosity)

Alternative to centrifugation – use of denaturing conditions (buffers containing 4-6M guanidine hydrochloride or dithiothreitol)

Interpretation of results

- pitfalls

Salivary glands producedBlood-borne constituents

Reliable measurements assumes a constant saliva/plasma ratio (SPR)

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)

Marker of sympathetic NS

activity ?

Concentration/ function

evaluation?

How to express these results?

Chronic or acute

evaluations?

Salivary flux

1 4 7 10 13 16 19 22 25 28 31 34 37 40 43 46 49 52 55 58 61 64 67 70 73 76 79

0

0.5

1

1.5

2

2.5

1st collection - Jan 2nd collection - March 3rd collection - May

Salivary

flux (

mL/

min

)

00 01 02 030

500

1000

1500

2000

2500

3000

Salivary flux (mL/min)

[Pro

tein

] (m

g/m

L)

  FluxProt conc

Flux 1Prot conc

0.03472606 1

- 81 participants- Saliva collection

in 3 different moments (at same hour and status)

Individual salivary fluxesare diferente.

Salivary flux: 0.45 ± 0.29 mL/min(0.05 – 2.16)

Salivary flux and protein concentration are not correlated.

Measuring concentration or activity?

Enzymatic activity (U/mL)

Relative Enzymatic

activity(U/mg protein)

Concentration(Western blot)

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.

Measuring secretion? Rate of Enzymatic secretion (U/min) =

Enzymatic activity (U/mL) x salivary flow rate (mL/min)

Rate of Enzymatic secretion (U/min)

When the purpose is to interpret salivary pretein levels in terms of autonomic nervous system activity, the assessment of secretion rate can be relevant.

Final remarks

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.

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

Elsa LamyFernando Capela e SilvaCristina PinheiroAlfredo PereiraLénia Rodrigues (Student)

DEPARTAMENTO DE DESPORTO E SAÚDE

Nuno BatalhaAndré Freitas (Student)

Célia AntunesDaniela Moreira (Student)

Acknowledgements

Euro Weight Loss – 2016

Website: http://weightloss.global-summit.com/europe/

Meet the eminent gathering once again atEuro Weight Loss-2016

Vienna, AustriaSeptember 19-21, 2016