Saliva nitric oxide levels in relation to caries experience and oral hygiene

Journal of Advanced Research (2011) 2, 357–362

Cairo University

Journal of Advanced Research

SHORT COMMUNICATION

Saliva nitric oxide levels in relation to caries experience
and oral hygiene
Enas H. Mobarak
a
b

a,*

, Dalaal M. Abdallah

b

Restorative Dentistry Department, Faculty of Oral and Dental Medicine, Cairo University, Cairo, Egypt
Pharmacology and Toxicology Department, Faculty of Pharmacy, Cairo University, Cairo, Egypt

Received 21 January 2011; revised 3 April 2011; accepted 9 May 2011
Available online 21 June 2011

KEYWORDS
Caries experience;
Nitrate;
Nitric oxide;
Nitrite;

Salivary flow rate (SFR)

Abstract The aim of the present study was to determine the relationship between nitric oxide (NO)
concentration/rate in the unstimulated whole saliva (UWS) and stimulated whole saliva (SWS) with
the decay-missing-filled teeth (DMFT) and simplified oral hygiene (OHI-s) scores. Forty adults were
included in the study. Half of the participants (n = 20) had high DMFT-OHI-s compared to the
other half. UWS and SWS flow rates, initial and final pHs were also measured. NO concentrations
in the UWS and SWS of high and low DMFT-OHI-s groups were determined using modified Griess
reaction and NO rates were calculated. The two groups revealed no significant differences in their salivary flow rates and their initial pH. NO concentrations/rates in the UWS and SWS of high and low
DMFT-OHI-s groups were not statistically different (p > 0.05). There was no significant correlation
between NO concentration or NO rate and other tested variables (DMFT-OHI-s, initial pH and final
pH). However, a significant correlation was found between UWS NO rate and UWS flow rate
(r = 0.921, p = 0.0001) and SWS NO rate and between SWS flow rate (r = 0.921, p = 0.0001). It
could be concluded that neither NO concentration nor NO rate correlates with the dental status.
As the exposure to any salivary component (including NO) depends not only on its concentration
but also on the rate of production of such concentration, it would be of value when determining individuals’ salivary components to consider their rate values rather than their absolute concentrations.
ª 2011 Cairo University. Production and hosting by Elsevier B.V. All rights reserved.

* Corresponding author. Tel.: +20 2 37600889/20101641166; fax:
+20 233385775.
E-mail address: (E.H. Mobarak).
2090-1232 ª 2011 Cairo University. Production and hosting by
Elsevier B.V. All rights reserved.
Peer review under responsibility of Cairo University.
doi:10.1016/j.jare.2011.05.005

Production and hosting by Elsevier

Introduction
Saliva is one of the primary needs for lifelong conservation of the

dentition against dental caries. Multiple anticariogenic functions of the saliva are related to its fluid characteristics that is
mainly includes dilution and washing effects. Also, they are related to its specific components such as neutralization of acids,
maintaining supersaturated calcium/phosphate concentrations
and antibacterial defense [1]. Normally, the daily production
of saliva ranges between 0.5 and 1.0 l. It is composed of more


358
than 99% water and less than 1% solids, mostly proteins and
electrolytes. The final composition of the whole saliva in the
mouth is strongly dependant on the salivary flow rate. The concentrations of sodium, chloride and bicarbonate ions have been
reported to be increased in stimulated whole saliva (SWS) [1].
On the other hand, the unstimulated whole saliva (UWS) composition was reported to be more important for the control of
carious lesions development than that of the SWS [2]. Recent
studies showed that nitrate and nitrite in saliva play a role in
the maintenance of certain oral protective functions, in particular, the production of nitric oxide (NO) [3,4].
NO represents a free radical gas and a noxious chemical in
the atmosphere, but exists in small well-controlled concentrations in the body [5]. Actually, it is also one of the most powerful antibacterial compounds [6] acting either through
inhibition of bacterial growth or through enhancement of macrophage-mediated cytotoxicity. NO easily penetrates the cell
membrane and hence induces its microbial damage through
several mechanisms, such as inhibition of various iron containing DNA synthases [7], combination with iron sulfur centers of
mitochondrial enzymes essential for their respiration [8] and
combination with superoxide to form peroxy nitrous acid
and the highly reactive hydroxyl radical [9].
NO formation requires nitrite, a potential substrate that is
found in saliva as a product of nitrate reduction. Nitrate in saliva is thus derived from both metabolic and dietary sources so
that after its absorption in the gut it is actively transported with
the blood to the salivary glands and secreted into saliva [12].
Nitric oxide can be measured using various direct and indirect methods (e.g., gas and liquid chromatography, electron
paramagnetic resonance, mass spectrometry, spectrophotometry, electrochemistry). The short half-life and low concentrations of NO in-vivo reduce the practicality of these methods

for evaluation of biological samples. Additionally, these procedures are generally unsuitable for the clinical laboratory due to
instrumentation requirements and inexpedience in processing
large number of samples. The difficulties inherent to quantification of NO can be eliminated by measuring its stable metabolites, in particular, nitrite and nitrate. Numerous techniques
for detection of these anions have been reported, including
spectrophotometric, fluorescent, chemiluminescent, and chromatographic assays. The simplest and most frequently applied
method employs colorimetric detection with (Griess reagent).
However, the conventional Griess reaction has a limitation
due to its inability to detect nitrate. Moreover, the usage of
a reducing metal such as cadmium is time consuming as it requires an extra step. Cadmium as a toxic metal needs cautious
handling and proper hazardous waste disposal. Currently,
reduction can be achieved using vanadium to overcome all
the mentioned demerits [10].
Lately, it has been reported that salivary NO might be an
important intra oral defense mechanism against caries pathogens. Additionally; its oral production rate was thought to
be dependent on the salivary flow rate [11]. Nevertheless, only
two studies concerning the relation between NO concentration
(lM/L) and the past caries experience could be traced; though
that, their results were contradicting [12,13]. In addition, none
has tested the salivary NO rate (lM/min) with regards to the
flow rate of either the UWS or SWS.
The present study was conducted to determine if there is a
relationship between NO concentrations/NO rate and subjects’
oral hygiene and past caries experience. This relation could be

E.H. Mobarak and D.M. Abdallah
of value for caries prediction and diagnosis especially that
there is a general belief that past caries experience is a good
predictor for future caries [14].
Subjects and methods
Screening and selection of subjects

All patients attending the Faculty of Oral and Dental Medicine, Cairo University, Cairo, Egypt, over one month were
screened for participation in the present study. Participants
were eligible if they had either poor oral hygiene and high
DMFT or good oral hygiene and low DMFT. This research
has been approved by the local research ethics committee
and informed consents have been taken. Participants had to
have inclusive criteria of being 20–30 years age, apparently in
good health, non smokers and not taking any local or systemic
medication in the previous two months that might affect their
saliva composition. Those who had the inclusion criteria and
accepted to sign informed consent (n = 57) were stratified
according to their gender and whether they had high OH
and DMFT scores or not. Ten participants were randomly
selected from each of these four strata.
Clinical examination
Following the European criteria [15,16], the level of dental caries status for each individual was determined by the same person using the DMFT score. In addition, the simplified oral
hygiene index (OHI-S) (debris index and calculus index) was
used to determine the oral hygiene status [17]. A patient was
considered with low DMFT-OHI-s when his DMFT score
was 62 and his OHI-S score was 61. While the participant
was considered with high DMFT-OHI-s, when the DMFT
score was P8 and OHI-s score was P4. For those with high
DMFT, the percentage of D component had to be more than
75% of the DMFT scores while those of low DMFT score had
not to have any D component in their DMFT scores. On the
day of collecting the samples, participants (n = 40) were asked
not to brush their teeth in the morning and to be fasting for at
least six hours before the sampling time.
Measuring unstimulated and stimulated SFR
The time of sampling was from 9 to 10 am. Two samples were

taken from each participant. The participant sat in an upright
position and was asked to relax with no movement or talking
for few minutes to eliminate the effect of the sympathetic tone
[18]. The UWS and SWS flow rates were obtained following
the procedures proposed by Navazesh [19] and Navazesh
and Kumar [18]. The UWS and SWS flow rates were determined immediately after collection.
Measuring the initial and calculation of final pH
The stimulated saliva sample was poured in a small beaker,
after calculating its volume, to measure its pHs (pHep, Hanna
instrument, Italy). The reading was recorded as the initial salivary pH value. One millimeter of stimulated saliva was then
mixed with 3 ml of HCl (0.005 M), after 30s, the pH was measured again and recoded as the final pH [20].


Nitric Oxide levels and dental status

359

Determination of nitric oxide concentration

Statistical analysis

Saliva samples were coded before measuring the NO and decoded thereafter. Nitric oxide was determined in saliva according to the method described by Miranda et al. [10].

Data were described in terms of range, medium and mean ±
standard deviation (SD). Comparison of quantitative variables
between different groups in the present study was done using
Mann Whitney U test. Correlation between NO levels and other
variables were done using Spearman rank correlation (R). A
probability value (p-value) less than 0.05 was considered statistically significant. All statistical calculations were done using the
computer programs: Microsoft Excel version 7 (Microsoft Corporation, NY, USA) and the SPSS (Statistical Package for the

Social Science; SPSS Inc., Chicago, IL, USA) version 15 for
Microsoft Windows.

Principle
Nitric oxide is relatively unstable in the presence of molecular
oxygen, with an apparent half life of approximately 3–5 s and
is rapidly oxidized to nitrate and nitrite totally designated as
NOx. A high correlation between endogenous nitric oxide production and nitrite/nitrate (NOx) levels has been established.
Measurements of these levels provide a reliable and quantitative estimate of nitric oxide output in vivo. The assay determines the total nitrite/nitrate level based on the reduction of
any nitrate to nitrite by vanadium followed by the detection
of total nitrite (intrinsic + nitrite obtained from reduction of
nitrate) by Griess reagent. The Griess reaction entails formation of a chromophore from the diazotization of sulfanilamide
by acidic nitrite followed by coupling with bicyclic amines such
as N-(1-naphthyl) ethylenediamine. The chromophoric azo
derivative can be measured colorimetrically at 540 nm.

Results
Data of both tested groups
Table 1 shows the characteristic data of both tested groups.
Statistical analysis revealed no significant difference between
both groups regarding the UWS and SWS flow rates and the
initial pH (p > 0.5). Regarding the final pH, the difference
was statistically significant (p = 0.04).

Procedure
In the Eppendorf tube, 0.75 ml cold absolute ethanol was
added to 0.75 ml saliva then was left for 48 h in the refrigerator
to attain complete protein precipitation. The mix was then centrifugated at 4000 rpm at 12 °C for 30 min using cooling centrifuge (Heraeus, Germany). Only 250 ll of the obtained
supernatant was used to which 250 ll vanadiumyrichloride
(Aldrich, USA) was added followed by rapid addition of

125 ll sulfanilamide (2% (w/v) in 5% HCl, Sigma, USA)
and 125 ll of N-(1-Naphthyl) ethylenediamine dihydrochloride (0.1% (w/v) in distilled water, Fluka, USA). The mixture
was left at room temperature for 30 min then the absorbance
of the pink colored chromophore was measured at 540 nm
using a double beam spectrophotometer (UV-150-02, Shimadzu, Japan) against a blank treated in the same manner to
the test but using 250 ll distilled water instead of the sample.
The standard was treated exactly as the supernatant and measured against a blank reagent containing 250 ll distilled water.

NO levels of both groups are presented in Table 2. For the NO
concentration regardless the salivary flow rate, the mean value
was higher in the group of high DMFT-OHI-s (79.4 ± 21.6
for UWS and 68.9 ± 13.8 for the SWS) than in the group of
low DMFT-OHI-s (77.7 ± 16.1 for the UWS and 66.6 ± 9.8
SWS). However, these differences were not statistically significant (p = 0.86 for UWS and p = 0.50 for SWS). On the other
hand, when the NO rate was calculated with regards to the salivary flow rate (lM/L min-1), the results were reversed; where
the NO rate mean value was higher in the low DMFT-OHI-s
group (25.1 ± 15.8 for the UWS and 153.9 ± 91.1 for the
SWS) than the other group (22.8 ± 11.2 for the UWS and
133.2 ± 95.9 for the SWS). Yet, these differences were not also
statistically significant (p = 0.68 for UWS and p = 0.74) for
(SWS).

Calculation of NO concentration (lM/L)

Correlation between the NO and other variables

The level of total nitrite/nitrate (NOx) in the saliva was expressed as lM and was calculated using the following formula:
NOx(lM) = AT/As · n · DF
where AT is the absorbance of the test sample; As is absorbance of the standard sample; n is concentration of the standard (lM) and DF is the dilution factor = 1.5/0.75 = 2.

Table 3 shows the correlation coefficient and significance
between NO levels and tested variables. There were no statistically significant correlations between the salivary NO concentrations in the UWS or SWS and any of the variables namely;
DMFT, OHI-s, initial and final pHs, and UWS and SWS flow
rates. There was also no statistically significant correlation between NO rate and DMFT, OHI-s, Initial and final pH. A significant correlation between the UWS NO rate and the UWS
flow rate was found. SWS NO rate had a statistically significant correlation with the SWS flow rate. Moreover, a statistically significant correlation between the UWS NO rate and
SWS NO rate was found.

Calculation of the NO rate (lM/min)
The exposure of the dental tissue to the NO does not only depend on its concentration in the saliva either unstimulated or
stimulated but also on the rate of such exposure. Accordingly,
the actual NO secreted in the saliva per individual with regards
to his UWS and SWS flow rate (NO rate) was calculated. This
was done according to the following equations:
NO rate in UWS (lM/min) = NOx lM/ml · UWS flow
rate (ml/min).
NO rate in SWS = NOx lM/ml · SWS flow rate (ml/min).

NO concentration and rate of both groups

Discussion
Dental caries is the most common disease in the oral cavity.
The need for its control is mandatory especially in developing
countries where an overall increase in the frequency of dental


360

E.H. Mobarak and D.M. Abdallah
Table 1

Basic characteristics of the study subjects.

DMFT
OHI-s
UWS flow rate (ml/min)
SWS flow rate (ml/min)
Initial salivary pH
Final salivary pH

First group (n = 20)
(High DMFT and OHI-s) mean ± SD

Second group (n = 20)
(Low DMFT and OHI-s) mean ± SD

p-Value

11.5 ± 5.2
2.9 ± 1.3
0.3 ± 0.1a
2.0 ± 1.4b
8.0 ± 0.1c
7.5 ± 0.2

1.5 ± 1.4
0.9 ± 0.9
0.3 ± 0.2a
2.3 ± 1.1b
8.0 ± 0.2c
7.7 ± 0.2

0.00
0.00
0.42
0.36
0.23
0.04

Same letters within rows mean no statistical significance.
DMFT = decayed missed filled per tooth score; OHI-s = simplified oral hygiene score; UWS = unstimulated whole
saliva; SWS = stimulated whole saliva.

Table 2

Nitric oxide levels of both groups.

Subjects-groups Mean ± SD
(Range, median)

NO concentration in UWS
(lM/ml)

NO concentration in SWS
(lM/ml)

NO rate in UWS
(lM/min)

NO rate in SWS
(lM/min)

High-DMFT/OHI-s

79.4 ± 21.6
(54.2–159.1, 76.5)
77.7 ± 16.1
(53.3–131.9, 76.8)
0.86

68.9 ± 13.8
(48.9–96.0, 76.5)
66.6 ± 9.8
(37.8–79.9, 65.8)
0.50

22.8 ± 11.2
(7.3–54.0, 19.8)
25.1 ± 15.8
(6.2–73.8, 22.0)
0.68

133.2 ± 95.9
(49.5–400.9, 103.5)
153.9 ± 91.1
(53.6–375.4, 120.9)
0.74

Low-DMFT/OHI-s
p-Value

DMFT = decayed missed filled per tooth score; OHI-s = simplified oral hygiene score; UWS = unstimulated whole saliva; SWS = stimulated
whole saliva.

caries has been reported [21]. Knowing that any infectious
disease can only occur when the pathogenic organisms are sufficient in number to surmount the intra-oral defense mechanisms, and that tooth decay is an infectious disease caused
by acid attacks resulting from bacterial sugars fermentation,
would clarify the essentiality of the role of the innate-host defense mechanism against dental caries. NO might be one of the
important intra oral defense mechanisms against caries pathogens [4].
Though cross-sectional analytical studies might suffer
from the lack of blinding [22], this was not the case in the
present study as the dentist had coded the samples before
measuring NO concentrations and the decoding was done
thereafter.
DMFT score was used in present study for caries status
determination to make the results comparable with Bayindir’s
et al. earlier study [12]. However, some differences were
encountered including doubling the sample size, and measuring the salivary flow rates and initial and final pHs. Having
cut offs for DMFT scores, P8 for high DMFT group and
62 for those with low DMFT, were used to have a clear distinction between both groups. For the same reason, it was
specified that those of high DMFT score should have the main
component of D and those with low DMFT score not having
any D component. The other major difference between the two
studies was the usage of modified Griess reaction rather than
the conventional one used by the other study. These differences may explain why the outcomes of the present study contradict with Bayindir’s et al. [12]. The age group of this study
was chosen for two reasons, first to be comparable with the
Bayindir et al. [12] study. Secondly, since DMFT values are
very much age dependant, It would be expected to be more

reliable to group the study subjects according to their DMFT
at this age group rather than at younger age.

In the current study, smokers were excluded as prior investigation showed inhibition of NO production by acute or chronic
cigarette smoking [23]. Moreover, subjects were also chosen not
to be taking any local (e.g., mouthwashes) or systematic medications (such as antibiotics) that might affect their salivary components for at least two months, because it was reported by
Dougall et al. [24] that the salivary production of nitrite was reduced following the use of broad spectrum antibiotics. Additionally it was found that NO was absent in Germ-free rates [25].
The patients were also selected from those who did not take
vegetables in the last meal before fasting, for not less than 6 h,
as it was reported by Olin et al. [26] that salivary NO concentration showed significant increase for up to three hours after
ingestion of Nitrate rich food. Meanwhile, others were not able
to prove that relation [27,28].
For many years it was accepted to have samples from the
SWS to be used in caries research including analysis of its
composition, bacteriological investigation (such as counting
Streptococcus mutans and lactobacilli spp.), salivary initial
and final pH and its buffering capacity. This was because of
its expected functional role during food intake. However,
recently, Bardow et al. [29] brought the importance of the
UWS composition in caries process to light. Accordingly, in
the present study, the NO concentration was measured in the
UWS and SWS as each of them has a role in caries control
and their flow rates are quite different. Moreover, on reviewing
literatures, no study has been published that measured the NO
concentration in each of them for the same individual. Measurement of the UWS and SWS flow rates, initial and final
pH for the participants, which were found to be within the nor-


Nitric Oxide levels and dental status

361

Table 3 Correlation coefficient and significance between NO

levels and tested variables.
NO levels

Test variables

Correlation coefficient

UWS NO concentration

DMFT
OHI-s
Initial PH
Final PH
UWS
SWS

0.050
0.420
0.274
0.121
0.395
0.040

SWS NO Concentration

DMFT
OHI-s
Initial PH
Final PH
UWS

SWS

0.097
0.035
0.035
0.081
0.106
0.089

UWS NO rate

DMFT
OHI-s
Initial PH
Final PH
UWS
SWS

0.009
0.112
0.001
0.055
0.921**
0.404*

DMFT
OHI-s
Initial PH
Final PH
UWS

SWS

0.245
0.132
0.288
0.447*
0.471*
0.921**

SWS NO rate

UWS = unstimulated whole saliva, SWS = stimulated whole
saliva.
*
p < 0.05.
**
p < 0.0001.

mal ranges [30,31], allowed us to focus on NO concentration
difference per se between both groups.
The higher salivary NO concentration in UWS, independently from the salivary flow rate, in the group of high
DMFT/OHI-s in the current should be interpreted with caution as the difference was not statistically significant. The other
researcher [12,13] had contradicting results. This diversity of
the results may be attributed to the difference in the methodology. For example, in Doel et al. [13] study the salivary samples
were obtained by using a swab. This method of sampling was
reported to be the least accurate one for salivary testing [18].
Additionally, no prior investigations had been done for the
NO concentration in the SWS to compare our finding with.
The reported significant correlation between the NO rate
values in UWS and the UWS flow rate itself and the NO rate

in the SWS and the SWS flow rate means that it could be speculated that the measurement or calculation of each is enough
as they are very correlated with each other. However, further
investigations are required to support this speculation.
In the present study, there was a considerable overlap between the two subject groups regarding both NO concentration and rate values. This makes it difficult to consider NO a
host defense mechanism when caries increases or oral hygiene
deteriorates as mentioned earlier by Bayindir et al. [12].
Despite the previous findings, it is worth mentioning that the
increase in intake of nitrate rich food, which is mainly in green
vegetables especially leafy ones such as lettuce and spinach,
may contribute to overall protective effect against cariogenic

pathogens affecting hard tissue [32]. Further longitudinal clinical investigation to verify such findings is still required.
Conclusion
Under the conditions of this study it could be concluded that
1. Neither NO concentration nor NO rate correlates with the
dental status.
2. As the exposure to any salivary component (including NO)
depends not only on its concentration but also on the rate of
production of such concentration, it would be of value
when determining individuals’ salivary components to
consider their rate values rather than their absolute
concentrations.

References
[1] Bardow A, Largerlf F, Nauntofte B, Tenovuo J. The role of
saliva. In: Fejerskov O, Kidd E, editors. Dental caries: The
disease and its clinical management. U.K.: Blackwell
Publishing; 2008. p. 193–4.
[2] Bardow A, Hofer E, Nyvad B, ten Cate JM, Kirkeby S, Moe D,
et al. Effect of saliva composition on experimental root caries.

Caries Res 2005;39(1):71–7.
[3] de Soet JJ, Nyvad B, Kilian M. Strain-related acid production
by oral streptococci. Caries Res 2000;34(6):486–90.
[4] Radcliffe CE, Akram NC, Hurrell F, Drucker DB. Effects of
nitrite and nitrate on the growth and acidogenicity of
Streptococcus mutans. J Dent 2002;30(7-8):325–31.
[5] Moncada S, Higgs A. The L-arginine-nitric oxide pathway. N
Engl J Med 1993;329(27):2002–12.
[6] Moncada S, Palmer RM, Higgs EA. Nitric oxide: physiology,
pathophysiology, and pharmacology. Pharmacol Rev
1991;43(2):109–42.
[7] Wink DA, Kasprzak KS, Maragos CM, Elespuru RK, Misra M,
Dunams TM, et al. DNA deaminating ability and genotoxicity
of nitric oxide and its progenitors. Science 1991;254(5034):
1001–3.
[8] Reddy D, Lancaster Jr JR, Cornforth DP. Nitrite inhibition of
Clostridium botulinum: electron spin resonance detection of
iron-nitric oxide complexes. Science 1983;221(4612):769–70.
[9] Hogg N, Darley-Usmar VM, Wilson MT, Moncada S.
Production of hydroxyl radicals from the simultaneous
generation of superoxide and nitric oxide. Biochem J
1992;281(Pt 2):419–24.
[10] Miranda KM, Espey MG, Wink DA. A rapid, simple
spectrophotometric method for simultaneous detection of
nitrate and nitrite. Nitric Oxide 2001;5(1):62–71.
[11] Allaker RP, Silva Mendez LS, Hardie JM, Benjamin N.
Antimicrobial effect of acidified nitrite on periodontal
bacteria. Oral Microbiol Immunol 2001;16(4):253–6.
[12] Bayindir YZ, Polat MF, Seven N. Nitric oxide concentrations in
saliva and dental plaque in relation to caries experience and oral

hygiene. Caries Res 2005;39(2):130–3.
[13] Doel JJ, Hector MP, Amirtham CV, Al-Anzan LA, Benjamin N,
Allaker RP. Protective effect of salivary nitrate and microbial
nitrate reductase activity against caries. Eur J Oral Sci
2004;112(5):424–8.
[14] Hausen H. Caries prediction–state of the art. Community Dent
Oral Epidemiol 1997;25(1):87–96.
[15] Debnath T. Epidemiologic indices. In: Debnath T, editor.
Ashoka’s public health and preventive dentistry. india: AITBS
Publisher and Distributer; 2002. p. 57–60.


362
[16] World Health Organization. Oral health surveys: basic
methods., 4th Edition. Geneva: WHO, 1997.
[17] Greene JC, Vermillion JR. The Simplified Oral Hygiene Index. J
Am Dent Assoc. 1964;68:7–13.
[18] Navazesh M, Kumar SK. Measuring salivary flow: challenges
and opportunities. J Am Dent Assoc 2008;139(Suppl):35S–40S.
[19] Navazesh M. Methods for collecting saliva. Ann N Y Acad Sci
1993;694:72–7.
[20] Axelsson P. Internal modified factors involved in dental caries.
In: Axselsson P, editor. Diagnosis and risk prediction of dental
caries. Germany: Quintessence Publishing; 2000. p. 91–133.
[21] Manji F, Fejerskov O, Baelum V, Luan WM. The
epidemiological features of dental caries in African and
Chinese populations: implications of risk assessments. In:
Johnson NW, editor. Dental caries markers of high and low
risk groups and individuals. Cambridge: Cambridge University
Press; 1991. p. 62–100.

[22] Manolio TA. Design and Conduct of Observational Studies and
Clinical Trials. In: Gallin JI, editor. Principals and Practice of
Clinical Research. NY: Elsevier; 2002. p. 188–9.
[23] Carossa S, Pera P, Doglio P, Lombardo S, Colagrande P,
Brussino L, et al. Oral nitric oxide during plaque deposition.
Eur J Clin Invest 2001;31(10):876–9.
[24] Dougall HT, Smith L, Duncan C, Benjamin N. The effect of
amoxycillin on salivary nitrite concentrations: an important
mechanism of adverse reactions. Br J Clin Pharmacol
1995;39(4):460–2.

E.H. Mobarak and D.M. Abdallah
[25] Duncan C, Dougall H, Johnston P, Green S, Brogan R, Leifert
C, et al. Chemical generation of nitric oxide in the mouth from
the enterosalivary circulation of dietary nitrate. Nat Med
1995;1(6):546–51.
[26] Olin AC, Aldenbratt A, Ekman A, Ljungkvist G, Jungersten L,
Alving K, et al. Increased nitric oxide in exhaled air after intake
of a nitrate-rich meal. Respir Med 2001;95(2):153–8.
[27] Spiegelhalder B, Eisenbrand G, Preussmann R. Influence of
dietary nitrate on nitrite content of human saliva: possible
relevance to in vivo formation of N-nitroso compounds. Food
Cosmet Toxicol 1976;14(6):545–8.
[28] Tannenbaum SR, Sinskey AJ, Weisman M, Bishop W. Nitrite in
human saliva. Its possible relationship to nitrosamine
formation. J Natl Cancer Inst 1974;53(1):79–84.
[29] Bardow A, Pedersen AML, Nauntofte B. Saliva. In: Miles TS,
Nauntofte B, Sevensson P, editors. Clinical Oral
Physiology. Germany: Quintessence Publishing; 2004. p. 26–35.
[30] Mass E, Gadoth N, Harell D, Wolff A. Can salivary

composition and high flow rate explain the low caries rate in
children with familial dysautonomia. Pediatr Dent
2002;24(6):581–6.
[31] Tenovuo J. Salivary parameters of relevance for assessing caries
activity in individuals and populations. Community Dent Oral
Epidemiol 1997;25(1):82–6.
[32] Benjamin N, O’Driscoll F, Dougall H, Duncan C, Smith L,
Golden M, et al. Stomach NO synthesis. Nature
1994;368(6471):502.