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Salivary vitamin C levels in

smokers and non- smokers

- A Comparative Study

By, Under The Able Guidance Of,

Jayaram H Nambiar, Dr. Shetty K Padma,
6th Term MBBS, Professor,
KSHEMA. Department of Pathology,
Ref. ID : 2014-03805 KSHEMA.
Tobacco use can cause reduction in
salivary antioxidant levels (1,2) and
can lead to oral cancers (3,4).
Antioxidants like uric acid, and
superoxide dismutase levels are low in
saliva, of chronic smokers(5). These
antioxidants are well known to reduce
the free radicals, reactive oxygen and
nitrogen species in inhaled cigarette
smoke, which may inhibit several
inflammatory oral pathologies and
thereby oral cancers.
Cigarette smoke contains
about 4000 toxic products, of
which about 400 are
Estimation of salivary
vitamin C is a simple and easy
way to detect the oxidative
stress induced by cigarette
unlike estimating the other
antioxidants in saliva or blood .
In chronic smoker, with at least 10 pack
years of tobacco use, there is ample chance of
significant reduction in antioxidants, including
vitamin C.
Hence 40 chronic smokers and non-smokers
will be compared with respect to their salivary
vitamin C levels. If a significant reduction in
vitamin C level is demonstrated, probably further
researches as supplementing vitamin C to chronic
smokers to prevent oral cancers may be

To estimate the levels of

Vitamin C in saliva in the two
study groups (smokers and
non smokers).
To compare the vitamin c
levels between the two groups
and find if there is any
statistically significant
Unstimulated whole Saliva sample will be
collected from 80 subjects in the age group 30-
60 years. 40 are smokers with at least 10
pack-years of tobacco use and 40 are non-
smokers of comparable age group. Saliva will
be collected between 8am and 10am to avoid
circadian rhythm variation in antioxidant
Independent sample 't' test will be used to
compare vitamin c levels from the collected
data and thus collected information will be
Summarized by descriptive statistics( mean
5ml of saliva will be collected
from the patient, centrifuged and
the supernatant obtained will be
stored at 4ºC for subsequent
Vitamin C levels will be
esimated by
DiNitro PhenylHydrazine (DNPH)
method (7)

Vitamin C or Ascorbic acid being a good

reducing agent can undergo reversible
conversion to its oxidized form
dehydro-ascorbic acid.
Vitamin C couples with 2,4-
dinitrophenylydrazine to yield an ozazone
which gives yellow colour with sulphuric acid.
Copper in the dinitrophenyl hydrazinere agent
acts as a catalyst, and the intensity of the
colour is read at 520nm .
• Sample Quantity: 100 µl
– 2,4-Dinitrophenyl hydrazine (DNPH)-
(C H N O )
6 6 4 4

– Sulphuric acid (H SO ) 2 4

– Cupric sulphate (CuSO .5H O) 4 2

– Ethyl alcohol (C H COOH) 2 5

– Thiourea (CH N S)4 2

– Trichloro acetic acid (TCA)-(CH COOCl ) 3 3

– Ascorbic acid (Vitamin C)-(C H O ) 6 8 6

• ▷ 4.5M H SO
2 4

SOLUTION A: 2, 4-dinitro phenyl
hydrazine 0.1mol/L dissolved in
500mL of 4.5M H SO .
2 4

SOLUTION B: Cupric sulphate

0.027mol/L is dissolved in 100mL
of distilled water.
SOLUTION C: - Thiourea 0.66 mol/L
is dissolved in 50% ethanol made up
to 100mL with it.
• DTC reagent is prepared by mixing
100 mL of solution A with 5mL
each of solution B and solution C

• The mixture is then heated to dissolve the

components; even then the solution will retain
some precipitate hence only the
supernatant of the reagent is utilized for
the assay.
• 5% TCA
• 5g of trichloroacetic acid is
dissolved in 100mL of distilled water.

• ⇨ 100µL of sample(Saliva) is taken in

a clean test tube and 900µL of 5%
TCA is added to it and allowed to
precipitate proteins for about ten
minutes and centrifuged.
• ⇨ 500µL of the supernatant is
separated and transferred into
another test tube.
• To this 200µL of DTC reagent is added,
the tube is plugged and the mixture is
incubated at 60˚C for 60’ in a water
• ⇨ Simultaneously a blank with 1mL of
TCA and 200µL of DTC reagent is also
incubated under similar conditions.
• ⇨ Following 60minutes of incubation,
there action mixture is cooled in an ice
bath and 1mL of 4.5M sulphuric acid
is added to it and after it reaches room
temperature the optical density is read
at 540nm against blank.

The concentration of Vitamin C in

the sample is obtained by plotting the
optical densities against the standard
graph and multiplied by 10 (dilution
If a statistically significant
decrease is seen in the levels of
vitamin C in the saliva of smokers
when compared to the non smokers,
this will suggest the use of vitamin C
supplementation in smokers to
increase their salivary vitamin C
levels. This will protect them from the
oxidative stress caused by free
radicals from cigarette smoking.
So supplementation of vitaminC
in smokers can be sought after
as the remedy for the
oxidative stress caused by
cigarette smoke and thus help
reduce the role of carcinogens in
cigarette in the pathogenesis of
I understand the information given in this consent form.

I have been explained about the nature, objective , duration and expected effects of the study and about what I
have to do. I have had the time and the opportunity to enquire about the study and I have been fully satisfied
with the explanation given.
I agree to co-operate with my study doctor and voluntarily give saliva sample for analysis as required in
the study. I agree to follow or observe the specific instruction given by the attending doctor.

I know that I am at libe1ty to withdraw from the study at any time without justifying my decision to withdraw. I
also know that my decision to withdraw will have no effect on my future medical care from this hospital.

I know that the results obtained from this study will be fo1warded to suitable authority . By signing this consent
fo1m, I have not given up any of my legal rights which I am otherwise entitled to as a pa1ticipant in the study.
I am ready to participate voluntarily in this study.
I know that I will get a copy of this consent form which is signed and dated .
Name of the patient Signature of the patient

I confirm that I have explained the nature , purpose and expected effects of the study to the
patient whose name is provided above.

Signature of the person providing information

Name of the person providing

Subject Case Sheet

Study Title:
Comparison of salivary vitamin c, an
antioxidant in smoking and non-smoking adults
1)Particulars of individual:
• Name:
• Age:
• Gender:
• Date and time:
• Address:
2) Past history
• Diabetes mellitus
• Hypertension
3)Personal History
• Diet :(veg/non-veg)
 Intake of (fruits/vegetables)
• Smoking (yes/no; if yes duration)
 no. of cigarettes :
 no. of years :
 whether discontinued inbetween :
 which of the following
substances have you used
: (cigarette/beedies/beetle
nut/slaked lime)
• Alcohol (yes/no; if yes duration)
4) Drug History
5) Systemic Examination
6) Local Examination
7) Levels Vitamin C
Test Chemicals Qty Amount
in Rs

1 Trichloro acetic acid 100 gm 250/-

2 2.4 DNPH 25 gm 130/-

3 Thio urea 100 gm 175/-

4 Copper Sulphate 500 gm 650/-

5 Sulphuric Acid 500 ml 225/-

6 Ascorbic acid 100 gm 750/-

Total ………Rs 2180/-

• Reference

• 1)Blot WJ, Mclaughlin JK, Winn DM, Austin DF,

Greenberg RS, Preston-Martin S, Bern stein
L,Schoenberg JB, Stemhagen A, Fraumeni JF
Jr.Smoking and drinking inrelation to oral and
pharyngeal, cancer.Cancer Res 1988;48:3282-7.

• 2)Hayes RB, Bravo-Otero E, K leinman DV, Brown LM,

Fraum eni JF, Harty LC, Winn DM. Tobacco and alcohol
u seandoral
can cerin Puerto Rico. Cancer Causes Control

• 3)Khor GH, Siar CH, Jusoff K. Chromosome 17

aberration of or alaquamous cell carcinoma in Malaysia.
Global J Health Sci 2009;1:150-6.
• 4)Sharma SM, Mohan M, Kumari S, Sorake SH.
Evaluation of glutathione in oral squamous cell
carcinoma. J Maxillofac Oral Surg2009;8:2704

• 5)Hamid - reza Abdolsamad i, DDS;

Mohammad-taghi Goodarzi:
(Chang Gung Med J 2011;34:607-11)

• 6) Collins A, Duthie S, Ross M: Micronutrients

and oxidative stress in the aetiology of cancer.
Proc Nutr Soc, 1994, 53, 67–75.

• 7)Castelli, A., Ma r tor a n a , G. E., Frasea,A.M

. an d Meucci, E. 1981. Colorimetric detection
of plasma vitamin C comparison between 2,4-
Dinitrophenyl hydrazine and phosphotunstic
acid. Methods Acta,Vitaminol Enzymol. 3(2):
Thank you