SALIVARY MARKERS OF SYSTEMIC DISEASES

BY

Y.PRAGZNA(10T21S0113)

UNDER THE GUIDANCE OFMrs. M. SANDHYA M.PHARM

ASSOCIATE PROFESSOR

CONTENTS:

Biomarkers

Saliva physiology

Advantages & disadvantages

Saliva collection, processing & storage

Diagnostic applications

Salivary proteome & transcriptome

New technologies of measurement

BIOMARKERS:

Biomarkers are defined as cellular, biochemical and molecular characteristics by which normal/abnormal processes can be recognized and/or monitored.

SALIVARY MARKERS

Non invasive diagnosis of disease and monitoring of general health

SALIVA PHYSIOLOGY:

Saliva is the mixed product of 3 major salivary glands.

Parotid

Sub mandibular

Sub lingual

It is slightly acidic(pH 6-7).

Several physiological & pathological conditions modify saliva production quantitatively.

FUNCTIONS OF SALIVA

PROTECTIVE Lubrication Anti-microbial Mucosal integrity Lavage/cleansing Buffering Remineralization

FOOD & SPEECH RELATED

Food preparation Digestion Taste Speech Prevention of

caries

SA

LIV

A A

S A

D

IAG

NO

STIC

FLU

ID

AD

VA

NTA

GES

DIS

AD

VA

NTA

GES

It is safe, non-invasive and simple.

No needles and No cringing.

Home testing.

It has an average protein concentration of 1.5-2 mg/ml.

Cost of shipping & Storage tend to be lower.

Informative analytes are present in lower amounts in saliva than in serum.

SA

LIV

A C

OLLEC

TIO

N

WHOLE SALIVA COLLECTION

DUCTAL SECRETIONS COLLECTION

PAROTID SECRETIONS COLLECTION

SUB MANDIBULAR & SUB LINGUAL SECRETIONS COLLECTION

PROCESSING & STORAGE:

All procedures must be performed on ice:

(1)Divide the saliva samples into multiple 330μL samples placed in cryotubes at -80ºc.

Further processed for Protein analysis: Add protease inhibitors like aprotinin(0.33µL), 3.3µL PMSF.

RNA analysis: Add 1.65µL of SUPERase inhibitor.

(2)Invert gently to mix.

(3)Store all fractions at -80ºc.

Salivary/Oral Fluid Biomarkers Possibilities of Use

DNA Standard genotyping Genetic information of the hosting human

body Bacterial infection (oral microbes in the mouth)

Diagnosing carcinomas of head & neck Forensics

mRNA Information on transcription rates

RNA Viral/bacterial identification

Proteins Genetic information Translational regulation Diagnosing periodontitis Detecting dental cavities

Immunoglobulin Diagnosing viruses

Metabolites Diagnosing periodontitis

DIAGNOSTIC APPLICATIONS OF SALIVA:Drugs, hormones:

☻Intra cellular pathways Active & Passive diffusion

☻Extra cellular pathways Ultra filtration

Serum components:

☻Capillary barrier☻Interstitial spaces☻Cervicular fluid☻Membrane of acinar & ductal cells

SJOGRENS SYNDROME: There are raised concentrations of NaCl, IgA, IgG,

lactoferrin, inflammatory mediators such as PGE2, TXB2,

Interleukin-6 etc.

CYSTIC FIBROSIS: Saliva of CF patients contain increased calcium &

phosphate levels, urea & uric acid and abnormally elevated

levels of PGE2.

DIABETES MELLITUS: It effects flow rate & composition of saliva. Salivary

alterations includes higher levels of IgA, Salivary Peroxidase, glucose content, K, Salivary total protein &

amylase.

ORAL MUCOSAL DISEASES: Nitric oxide levels were increased.

ONCOLOGY:

P53 antibody can be detected in oral squamous cell carcinoma, increased levels of salivary kallikrein levels in oral malignant tumors, CA 15-3 in breast cancer.

CVS:

Determination of salivary amylase activity before 6hrs after cv surgery.

KIDNEY DYSFUNCTION:

Hypo salivation , changes in taste, ammonium smelling breath, oral mucosal pain.

EPILEPSY:

Gingival hypertrophy, IgA deficiency

VIRAL DISEASES:

Acute hepatitis A & B were diagnosed based on the presence of IgM antibodies in saliva.

HIV:

Salivary IgA levels.

BACTERIAL INFECTIONS:

By monitoring the presence of antibodies to microorganisms found in saliva & oral cavity

AGAIN & AGAIN ABOUT TOBACCO:

Smoking causes decrease in salivary important antioxidant levels.Salivary nicotine levels are considered to be the most reliable marker

DR

UG

MO

NIT

OR

ING

ANTI-ASTHMATICS: Theophylline

ANTI EPILEPTICS: Carbamazapine, Diazepam, Ethosuximide, Lamotrigine, Topiramate, Phenytoin, Primidone, Valproic acid

ANTI ARRYTHMIC/ANTI HYPERTENSIVES: Digoxin, Metoprolol, and Procainamide.

ANTI MICROBIALS: Clarithromycin, Gentamycin, Isoniazid, Ofloxacin, Sulfanilamide

ANTIPYRETICS: Acetaminophen, Antipyrine, Paracetamol

ANTI NEOPLASTICS: Cisplatin, Methotrexate, Texol, Topotecan

ANTI VIRAL: Quinine

LITHIUM PSYCHOTROPIC: Lithium, Amitriptyline

MISCELLANEOUS: Nicotine, Ethanol, Caffeine, Tolbutamide, Quinine

ILLICIT DRUGS: Amphetamine, Barbiturates, Benzodiazepines, Opiods, Cocaine, Phencyclidine

HORMONE MONITORING:Steroids like cortisol, aldosterone, testosterone, insulin

SIALOCHEMISTRY ANALYSIS:Early monitoring of an exposure to lead, cadmium, mercury because of higher salivary levels of these elements

CLASSIFICATION OF SALIVARY BIO MARKERS:

◘PROTEOMIC

◘GENOMIC

◘MICROBIAL

◘OTHERS

DIA

GN

OS

TIC

MO

LEC

ULA

R

TAR

GETS

SALIVARY PROTEOME:

Use of ‘Divide & conquer’ strategy

Initial fractionation of proteins with separation techniques

Digestion with proteolytic enzyme, trypsin

Analysis by 1-D, 2-D LC-MS

Finally data is processed & submitted for database searching

SA

LIVA

RY P

RO

TEO

MIC

AN

ALY

SIS

SALIVARY TRANSCRIPTOME:

◘It includes mRNA molecules that cells use to convey the instructions carried by DNA for subsequent protein production.

◘At present, the main strategy for identification of salivary transcriptomic biomarkers is through microarray technology.

SA

LIVA

RY T

RA

NS

CR

IPTO

ME:

DNA MICROARRAY TECHNOLOGY

NEW TECHNOLOGIES FOR MEASURING SALIVARY BIOMARKERS:

MICROBEAD ARRAY

Micro pits within a

silicon wafer are populated with a variety of chemically sensitized bead micro reactors.

POC DEVICE

In this POC device, saliva(100-300μL) is placed into the salivary collection module, and then delivered into the Nano-biochip.

MIC

RO

BEA

D A

RR

AY T

EC

HN

IQU

E NANO-BIOCHIP

CONCLUSION:

The components of saliva act as a “Mirror of the body’s health”, widespread use and growing acceptability of saliva as a diagnostic tool is helping individuals, researchers, health care professionals and community health programs to better detect and monitor diseases and to improve the general health of the public

REFERENCES:◘Mandel ID. A contemporary view of salivary research. Crit Rev Oral Biol Med 1993; 4:599.

◘Raymond G, Schipper a, Erika Silletti a, Monique H, Vingerhoeds. Saliva as research material: biochemical, physiochemical, and practical aspects. Archives of Oral Biology 2007 ;( 52):1114-35.

◘Richard P, Jiri S, Jana V, Edgar F, Petr M, Jindrich P et al. Saliva as a diagnostic medium. Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub 2009; 153(2):103-10.

◘Eliaz Kaufman .The diagnostic applications of saliva. A review. Crit Rev Oral Biol Med 2002; 13(2):197-212.

◘www.med.kufauniv.com/ar/journals2010/res%20(1).pdf

◘www.jofs.in/files/Artical-11.pdf

◘www.linkgroup.hu/docs/pcs2/218-08CurrGen.pdf

◘www.wikipedia.com 

THANK YOU

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