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OUTLINE Introduction

Psychosocial stress

Salivary glands

Production of saliva

Catecholamine

Autonomic nervous system

Salivary and norepinephine

Objective

Materials and Methods

Procedures for Trier social stress test (TSST)

Biochemical analyses

Psychometric measurements

Statistical analyses

Results

Discussion and conclusion

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PSYCHOSOCIAL STRESS

Psychological stress occurs when an individual perceives that

environmental demands tax or exceed his or her adaptive

capacity and it affected in sympathetic nervous system (SNS) activity

When psychosocial stress triggers a stress response, the body releases a group of

stress hormones including cortisol, epinephrine (or adrenalin) and dopamine which

lead to a burst of energy as well as other changes in the body

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PSYCHOSOCIAL STRESS

Psychosocial stress was determined by measuring

catecholamine in blood plasma or using electrophysiological

methods

- Skin conductance

- Heart rate

However, these methods are complicated and invasive methods,

therefore, using non-invasive method was interested to determine

psychosocial stress such as investigated salivary alpha-amylase

SALIVA GLANDS

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PRODUCTION OF SALIVA

Sympathetic cAMP secreted proteins

Parasympathetic Phospholipase C Ca2+ Secreted

fluid

SALIVARY ALPHA-AMYLASE

(SAA)

http://chemistry.tutorvista.com/organic-chemistry/glycosidic-linkage.html

sAA (α-1,4-α-D-glucan 4-glucanohydrolase)

It is a digestive enzyme that hydrolyses alpha-1,4 bonds of large

polysaccharides such as starch and glycogen, yielding the smaller by-

products of glucose and maltose.

It consists of two families of isoenzymes

• glycosylated

• no carbohydrate

It accounts for 40 – 50 % of total salivary gland-produced protein

CATECHOLAMINE

norepinephrine

epinephrine

dopamine

A catecholamine (CA) is a monoamine.

CA are compose of …

• norepinephrine

• epinephrine• dopamine

Source

• adrenal medulla

Effects

• physiological changes (fight-or-flight response).

• increases in heart rate, blood pressure, blood

glucose levels

• a general reaction of the sympathetic nervous

system

AUTONOMIC NERVOUS SYSTEM

WHY DO RESEARCH DETERMINE SALIVARY ALPHA-AMYLASE AND NOREPINEPHINE

http://www.hakeem-sy.com/main/node/16265

Norepinephrine from sympathetic neurons binds to both

alpha- and beta-adrenergic receptors on the acinar cell and more sensitivity with β2 receptor .

Acute response of salivary -amylase and plasma norepinephine to the laboratorystress paradism TSST in n = 12 healthy men and women

Rohleder et al., Ann. N.Y. Acad. Sci. 2004; 1032: 25-263.

In previous study

Trier social stress test (TSST)

OBJECTIVE To examine the correlation between stress-induced

sAA activity with NE and E .

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MATERIALS AND METHODS

Subject

40

18 use oral

contraceptive

(6 smoke)

22 in luteal phase

26 (9 smoke)

BMI 17-27 kg/m2

Age 18-24 years

Free of chronic stress Free of medication (Ex. Beta-block ,glucocorticoid)

free of chronic disease (Ex. Endocrine, cardiovascular)

PROCEDURES

1

• At first use salivettes to keep a saliva and keep blood sample to EDTA tube.

2• All subject do the ‘Trier social stress test (TSST)’

3• After TSST a second blood draw and saliva

sample was taken again.

4

• At 20 min and 40 min taken a blood sample and saliva sample again.

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TRIER SOCIAL STRESS TEST

(TSST)

For TSST has divided into 2 room

Room A

Room B

BIOCHEMICAL ANALYSES

Use enzyme kinetic method was processed on Genesis RSP8/150 liquid handing system

Dilute sample at 1:625 and use 20 µl of dilute saliva and standard transferred into 96 well microplate

Standard prepared from calibrator solution with concentration 326, 163, 81.5 , 40.7, 20.38 ,10.19 and 5.01 U/L of alpha amylase

1. Salivary alpha- amylase (sAA)

PROCEDURES

Pipette 80 µl of substrate reagent to each well

Incubate at 37 ºc in water bath 90 s

Measurement at 405 nm

Incubate at 37 ºc again for 5 min

Measurement at 405 nm

water bath

BIOCHEMICAL ANALYSES

2.Norepinephrine and Epinephrine

Plasma sample was analyze for NE and E by

high-pressure liquid chromatography (HPLC) at the Laboratory for Stress Monitoring

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PSYCHOMETRIC MEASUREMENTS

Perceived stress

Depressive symptoms

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PSYCHOMETRIC MEASUREMENTS Perceived stress

Its was assessed with the 14-item version

Perceived Stress Scale (PSS) . PSS is measure

the level of stress by an individual experience

by ranging from 0 ‘never’ to 4 ‘very often’

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PSYCHOMETRIC MEASUREMENTS

Depressive symptoms

This symptom were assessed with the 20-item

of the Center of Epidemiologic Studies Depression Scale

(CES-D) that is a self-report questionnaire about the

incidence and duration of impairment experienced by individuals depressive affects.

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STATISTICAL ANALYSES Use log 10 test for normal distribution

T-test

x ²-test For the assessment of difference between group

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RESULTS

• Men had a significantly higher BMI than both groups of

women .

Smokers and non-smokers differed with a trend in terms

of sex distribution .

Results showed no association of depression scale

values with stress responses of sAA, norepinephrine, or

epinephrine .

Preliminary data analysis

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PRELIMINARY DATA ANALYSIS

Perceive stress scale overall mean indicating no

elevated perceived stress levels in our sample.

Results showed no association between PSS

values and stress responses of sAA,

norepinephrine, or epinephrine .

Results

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RESULTS

Fig. 1. Salivary alpha-amylase (sAA) in response to the acute stress induction

The sAA concentrations

over the course of the four

measurement points, we found a

significant change in the sAA

release over time during the

stress intervention and highestafter TSST .

RESULTSAfter TSST the results shown that the epinephrineresponse to acute stress was a weak predictor whereasnorepinephine was higher than epinephine.

Fig. 2. Norepinephrine (NE) and epinephrine (E) in response to the acute stress

Fig. 3. Correlation of stress-induced increases of salivary alpha-amylase (sAA) and norepinephrine (NE).

RESULTS

When correlate between

salivary alpha-amylase

and norepinephrine

response to stress

we found that p = 0.025

and its significant in psychology .

Correlation of stress-induced increases of sAA and NE

Result from previous study

The results show

that the increasing

of sAA and NE

concentration are

correlate in linear regression .

DISCUSSION

The results found significant association between stress-

induced sAA and NE responses, but not between sAA

and E responses.

The active secretion of the adrenal medulla contains

approximately 80 % E and 20 % NE ; but this proportion

is reversed in the sympathetic nerves, which contain

predominantly NE , that why NE responses to stress-induced sAA more than E responses.

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DISCUSSION

In the present study we found a significant association

between sAA and NE and the usefulness of sAA to

function as an indicator for SNS activation.

In addition, we found that stress-induced sAA and E

levels in habitual smokers, in individuals with a lower BMI, and in females using oral contraceptive.

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CONCLUSION

From this study show an association between stress

responses of sAA and plasma norepinephrine . This

relationship is stronger than the association of

norepinephrine and epinephrine responses, thus placing

the predictive power of sAA well within the expectedrange for different SNS markers.

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AUTONOMIC NERVOUS SYSTEM

The autonomic nervous system has two main divisions

Sympathetic

Promotes a "fight or flight" response, corresponds with

arousal and energy generation, and inhibits digestion

Parasympathetic

Promotes a "rest and digest" response, promotes

calming of the nerves return to regular function, and

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