American Journal of Anal yt ical Chemistry, 2011, 2, 683-688
doi:10.4236/ajac.2011.26078 Published Online October 2011 (http://www.SciRP.org/journal/ajac)
Effect of Pre-Analytica l Conditions on Salivary
Nitrite Levels
Rosana Andrea Morelatto1*, Tomás Enrique Benavidez2, Ana María Baruzzi2,
Velia Matilde Solís2, Silvia Adriana López de Blanc1
1Department of Oral Pathology, Cli ni c al St o matology I and II “B”, Faculty of Dentistry,
National University of Córdoba, Córdoba, Argenti na
2INFIQC-CONICET, Department of Physical Chemistry, Faculty of Chemical Sciences,
National University of Córdoba, Córdoba, Argenti na
Received June 29, 2011; revised July 29, 2011; accepted August 5, 2011
Abstract
The aim of the present paper was to analyze the influence of sampling and storage procedures on nitrite con-
centration values in the saliva of healthy persons. The samples were obtained and stored under varied condi-
tions, and processed using the Griess method. Results: when the salivary nitrite concentration was measured
immediately after collection a significant dependence on the collection time was observed. A mean value of
94 µmol/L (range 3 - 625) was obtained at 8:30 am. This value decreased significantly with time (p < 0.05)
reaching a value of 68 at 12:30 noon. Concerning the sample storage, a significant increase in the nitrite
concentration was observed after 2 hrs, either at 4˚C or at room temperature (p < 0.05). In spite of the high
variability between individuals the values for each individual showed a marked constancy independent of the
sampling day. According to our results, by controlling pre-analytical parameters, principally sampling and
storage procedures, reproducibility is improved.
Keywords: Salivary Nitrite, Saliva, Oral Cancer, Storage Conditions
1. Introduction
N-nitroso compounds have been shown to be potent car-
cinogens in animals [1]; in the human body they can be
formed by the interaction of nitrite and a variety of amine
precursors, developing infantile methahemoglobinemia
and gastric cancer. Nitrite has been extensively studied in
relation to carcinogenesis [2]; it is an important factor for
gastric nitrozation, and could contribute to the etiology
of lung, stomach, esophagus, nasal cavity, bladder and
oral cavity cancer, leukemia and Non Hodgkin lym-
phoma [3]. It has mutagenic effects at cellular levels and
acts on p53 gene, closely related to the head and neck
squamous cell carcinoma [4].
Several studies have confirmed that the level of salivary
nitrite is strictly dependent on salivary nitrate, and thus
dependent on the dietary nitrate intake [5-8]. Dietary
nitrate derives mainly from vegetables and drinking wa-
ter [6,7] and in the latter case it could be more hazardous
since nitrate in vegetables is counterbalanced by vitamin
C and polyphenols that inhibit nitrozation [8]. Ingested
nitrate is absorbed from the stomach or intestines and
about 25% is secreted in saliva by an anion transport
system [5]. As a result, nitrate concentrations in saliva
are approximately 10 to 20 times higher than those found
in plasma [5]. In addition, it has been estimated that 70%
of the orally ingested nitrate is reduced to nitrite by
mouth microorganisms, mainly on the posterior surface
of the tongue [9,10].
The assay of saliva is an increasing area of research
with implications in basic and clinical purposes. Re-
cently, saliva has provided a substantial tool investiga-
tion of disease processes and disorders. Although this
biological fluid is easy to collect and manipulate, special
attention must be paid to minimize variation in specimen
integrity [11].
Many authors have investigated the concentration of
nitrite in saliva considering the advantages of obtaining
this biological fluid and its possible role in the etiology
and diagnostic relevance of nitro compounds. A common
feature of in vivo studies is the large spread in results,
both between individuals and for each individual in rela-

R. A. MORELATTO ET AL. 684
tion to sampling days and time [12]. The changing com-
position of saliva, dependent on several internal and ex-
ternal factors, as well as on the complex chemistry of
nitro-compounds limits the validity of the results for
clinical purposes.
In spite of the importance of testing nitro-compounds
in saliva, the literature on appropriate sample collection
and storage is scarce. The technical variables used for
sampling storage in the literature differ substantially. Xu
et al. [13] and Bojiç et al. [14] kept the samples at –20˚C
for a maximum of one week and one month until analy-
sis, respectively. On the other hand, Mirvish et al. [15]
and Yu et al. [16], assume that the saliva should be
stored at room temperature or 4˚C for up to one day, and
at –15˚C for less than a month before analysis. Since
there are marked differences in storage conditions in the
literature, the comparison of results becomes doubtful.
The purpose of this work was to evaluate the influence
of saliva collection, sampling and storage on nitrite con-
centration in order to obtain better reproducibility condi-
tions. Special attention was paid to the effects of: day
time of collection, temperature of storage, and the time
elapsed until the concentration of nitrite was determined
in the saliva samples. Intra- and inter-individual variabil-
ity was also studied.
2. Material and Methods
2.1. Subjects
This study was performed in 13 healthy (10 females and
3 males) non-smoking volunteers, from 26 to 53 years
old. Exclusion criteria used were: high alcohol intake,
consumption of food-vitamin supplements, pregnancy
and use of antibiotics or anti-inflammatories. Informed
consent of the nature of the study was signed by all the
volunteers. They were also instructed not to eat food rich
in nitrate and nitrite such as beets, carrots, green beans,
spinach, and collard greens, hot dogs, cured ham, bacon,
bologna and salami, during the previous 24 hrs and not to
eat at all at least 8 hrs before the test; although they were
allowed to brush their teeth.
2.2. General Procedures
Volumes of 3 mL of unstimulated whole saliva were
collected; individuals spat directly into a sterile plastic
tube containing 60 µL of 1 M NaOH solution in order to
stop further reduction of nitrate and to destroy vitamin C
in the incubates (20). For the deproteinization of the
samples, 3 mL of 0.15 M ZnSO4 were added to the col-
lected sample and then it was centrifuged for 15 min at
9000 g (28). The supernatant was used for the determina-
tion of nitrite concentration using the Griess method, the
one most frequently used for this metabolite analysis in
biological fluids. Proteins were removed with the pur-
pose of reducing turbidity, which might interfere with the
absorbance lecture [9].
2.2.1. Effect of Day Time of Collection
The dependence of nitrite concentration with the time of
collection was evaluated in full duplicate samples from
13 subjects at the following day times: 8:30 a.m., 10:30
a.m. and 12:30 noon. Volunteers had fasted and did not
eat anything until completion of the trial. After collection,
samples were immediately processed.
2.2.2. Effect of Storage Conditions on the Nitrite
Concentration
A sample taken from each of the 9 volunteers was di-
vided into 5 aliquots, one was processed immediately after
collection at 8:30 a.m., and four were analyzed through-
out the subsequent 4 hours under different conditions of
storage.
To analyze the influence of the time of storage, an-
other set of samples was taken from the same 9 volun-
taries, each one was divided into 4 aliquots; one was
analyzed immediately and the others were frozen and
stored at –20˚C, then defrozen and processed to deter-
mine the nitrite concentration, after 7, 14 and 21 days.
2.2.3. I ndividual Var iation over Time
To evaluate individual variation, salivary nitrite under
fasting conditions was determined in 10 subjects. Each
one was sampled once per month over a period of three
months, and the saliva was processed immediately after
collection at 8:30 a.m.
2.3. Experimental
The concentration of nitrite in saliva samples and in
standard solutions prepared daily was analyzed measur-
ing absorbance with a UV-1700 Shimadzu spectropho-
tometer at 540 nm (1 nm resolution, 0.005 absorbance
accuracy). A detection limit of 6 µmol/L and a linear
behavior up to 200 µmol/L were obtained from the cali-
bration curve. Samples with higher NO2− concentrations
were diluted. NO2− was not detectable in the blank solu-
tions that contained only NaOH and ZnSO4. The ana-
lyzed solution contained 200 µL of the sample, 400 µL
of water and 400 µL of the Griess reagent. The precision
of the Griess method is very high, not only when used
for standard solutions, but also with saliva samples.
Standard deviation in the absorbance values correspond-
ing to duplicates of each sample is ±0.005, indicating
high reproducibility of the values.

R. A. MORELATTO ET AL. 685
2.4. Statistical Methods
One-way analysis of variance with a randomized block
design was used to evaluate the effect of day time of col-
lection; the subjects were considered as blocks to reduce
the biological differences between them. The paired t test
was used to evaluate the effect of temperature and time
after collection.
Nitrite variability was assessed by means of a mixed
linear model, with REML estimations of variance com-
ponents. Best linear unbiased predictors (BLUP) of vol-
unteer effects were derived from the fitted mixed linear
model. Day of collection was regarded a fixed effect and
the volunteer effect was considered as random.
F test was used for comparing variances.
Statistical analysis was done using InfoStat/Profes-
sional version 1.1 statistical software package (Faculty of
Agronomy, National University of Córdoba).
3. Results
A mean value of 94 µmol/L (range 3 - 625) was obtained
for the salivary nitrite concentration. This value corre-
sponds to fasting levels of 115 samples taken from 13
volunteers, determined immediately after collection, at
8:30 a.m.
3.1. Effect of the Day Time of Collection
In this experiment a mean concentration of 93 µmol/L at
8:30 a.m. in 13 volunteers was found. A significantly
lower value (p < 0.05) was obtained when the sample as
collected at 10:30 and 12:30 h (Figure 1). At both times
the values were not statistically different, reaching a
mean concentration 68 µmol/L at noon. These results
were analyzed using one-way ANOVA with a random-
ized block design. Although the average value is plotted
in Figure 1, the statistical analysis considers the varia-
tion between individuals.
3.2. Effect of Storage on the Nitrite
Concentration
Table 1 shows the results for saliva collected at 8:30 a.m.
from nine volunteers that were processed according to
the conditions stated previously in material and methods
section. Paired t test indicated that up to 2 hrs, both at
4˚C and at room temperature, there are no significant
differences, although a slight decrease at room tempera-
Figure 1. Effect of the specimen collection time on the Sali-
vary nitrite concentration, n = 13
Table 1. Salivary nitrite concentration (µmol/L) under different storage conditions, n = 9.
Subject Immediately
(mean ± SD)1,2 2 h - 4˚C (mean ± SD) 2 h - room T (mean ± SD) 4 h - 4˚C (mean ± SD)1 4 h - room T (mean ± SD)2
1 58 ± 33 47 ± 16 37 ± 9 41 ± 13 50 ± 27
2 93 ± 3 87 ± 5 88 ± 2 139 ± 2 98 ± 5
3 100 ± 46 60 ± 34 68 ± 42 62 ± 48 73 ± 57
4 79 ± 5 84 ± 9 86 ± 10 134 ± 8 87 ± 4
5 93 ± 5 121 ± 5 99 ± 4 194 ± 8 137 ± 14
6 444 ± 51 647 ± 28 569 ± 94 813 ± 50 554 ± 20
7 87 ± 19 98 ± 6 76 ± 1 167 ± 6 101 ± 6
8 198 ± 54 155 ± 67 144 ± 25 185 ± 110 187 ± 108
9 118 ± 57 65 ± 37 78 ± 14 51 ± 36 73 ± 39
1,2(p < 0.05). These p-values, obtained with the paired t test, are relative to the results immediately after specimen collection.

R. A. MORELATTO ET AL. 686
ture was found. Between 2 hrs and 4 hrs the values in-
creased significantly in all the samples (p < 0.05). Table 1
also shows that there were strong inter-individual differ-
ences, not only in salivary nitrite concentration, but also
in the standard deviation values (SD); for example: sub-
ject 2 has a much lower variability in his values than the
subject 3; in addition these SD are quite constant with
storage time. This assessment was confirmed performing
F test to compare the inter-individual variances with re-
spect to the variances in each sample with storage time
(). This constancy in the variability of the con-
centration in the samples, corresponding to the same sa-
liva measured after several hours at room temperature or
at 4˚C, indicates that the stability of the values is quite
dependent of the sample itself.
0.05p
To evaluate the effect of several days of storage on the
three remaining aliquots frozen at –20˚C, the analysis
was repeated after 7, 14 and 21 days and the results were
compared with the sample processed immediately. Most
of the cases showed a significant decrease in the nitrite
values during the first week (p = 0.001), while maintain-
ing similar values at the end of the second and third week
(Figure 2). These results were analyzed using one-way
ANOVA with a randomized block design.
3.3. Effect of the Day of Collection
Table 2 shows the results of applying a mixed linear
model; they indicate that the inter-individual variability
is high, inducing large difference between volunteers as
shown by the best linear unbiased predictors (BLUPs) of
volunteer effects. Even though the day of collection ef-
fect was significant, the variance within subject expressed
Figure 2. Effect of the storage at –20˚C on salivary nitrite
concentration, n = 10.
Table 2. Fitted Mixed Linear Model for salivary nitrite
concentration in ten subjects.
Model Term Estimate SE t-value p-value
Intercept 278.0 86.6 3.2 0.002
Day of collection 271.5 95.4 –2.8 0.006
Volunteer effect
Subject 1 –36.80
Subject 2 –10.20
Subject 3 –53.90
Subject 4 11.70
Subject 5 –37.02
Subject 6 –39.50
Subject 7 26.90
Subject 8 145.90
Subject 9 28.20
Subject 10 –35.15
Log Likelihood −346.9; R2 = 0.54.
as percentage of total variance was only 20%.
4. Discussion
In the present study we analyze the variations of salivary
nitrite in healthy people before breakfast during the morn-
ing and those related to the storage conditions, in order to
obtain reliable sampling for further clinical studies.
According to the results the values of salivary nitrite
obtained at 8:30 a.m. were higher than those in the saliva
collected two or four hours later. We therefore decided to
unify 8:30 a.m. as the hour of collection of the samples
in all the experiments to avoid variations due to the cir-
cadian rhythm.
Some authors also observed variation in salivary ni-
trate and nitrite concentrations during the day; they re-
ported enhanced values after meals with high nitrate
amounts and these levels were sustained for at least 5 hrs
post food ingestion, effect probably related with the diet
[8]. It is not possible to compare our findings with these
results considering that the conditions for collection of
the samples are completely different, and that the high
variability in the values is strongly dependent on the ni-
trite dietary intake.
Another important result was the strong influence of
the storage conditions on salivary nitrite values. In this
sense, the samples should be either processed immedi-
ately after collection, or kept at 4˚C and analyzed before
the elapsed time reaches two hours. These results are

R. A. MORELATTO ET AL.687
also not coincident with those of Tanaka [10], who af-
firms that the concentration of the anionic components
remains stable over a period of 48 hrs in the refrigerator
and for 4 hrs at room temperature.
In addition to the effects of day time of collection and
storage of the samples on the salivary nitrite concentra-
tion, we also found some interesting facts concerning the
variation between and within individuals. We obtain a
mean concentration of 94 µmol/L and a wide range of
salivary nitrite level, between 3 and 625 mol/L, results
which are in agreement with those found in the literature
[5,17-19]. In spite of the high spread of these results be-
tween individuals, it is important to remark that the in-
tra-individual variability is quite dependent on the sub-
ject (Table 1).
It should be noted, on the other hand, that the fasting
values in the monthly collected samples of the same in-
dividual immediately processed, remained quite constant,
despite the high spread of the values among different
subjects.
Measuring nitrite in saliva is of particular importance
due to its contribution to the formation of N-nitrosamines,
many of which are carcinogenic. Cancer induced by
N-nitro compounds formed in the stomach could be pre-
vented by reducing the levels of salivary nitrite accord-
ing to the studies referred to above. This purpose could
be achieved by reducing nitrate in drinking water and
diet, improving dental hygiene, and using antiseptic
mouthwashes and toothpastes [20]. Some authors point
out that improper storage of samples and bacterial con-
tamination may increase nitrite levels in saliva [10]. As
the technical variables for storage conditions found in the
literature differ significantly, comparison of results is not
possible.
Nevertheless in this paper we have found out that by
controlling some pre-analytical parameters, such as col-
lection, sampling and storage procedures, reproducibility
is improved and some valid clinical information can be
obtained.
5. Conclusions
Although the reproducibility of results is shown to be af-
fected by many parameters, variation can be greatly re-
duced if the following conditions are carefully obeyed.
1) The time of collection of the sample should be the
same in all the individuals.
2) Samples should be immediately analyzed or before
two hours after collection. Storage of samples produces
variation in the nitrite concentration even when they are
kept at 4˚C.
The saliva matrix is an upcoming area of research for
basic and clinical application purposes, with considerable
potential for growth and progress. Although the con-
ditions to stabilize nitrite ion concentrations in the sam-
ples are difficult to achieve, the relative constancy in
individual salivary nitrite levels when pre-analytical con-
ditions are kept the same, would allow more reliable co-
hort studies to evaluate their clinical relevance.
6. Acknowledgements
The authors acknowledge Consejo Nacional de Investi-
gaciones Científicas y Técnicas (CONICET), Secretaría
de Ciencia y Tecnología de la Universidad Nacional de
Córdoba (SECyT) and Agencia Nacional de Promoción
Científica y Tecnológica (ANPCyT) for financial support.
R. Morelatto thanks Secretaría de Ciencia y Tecnología
de la Universidad Nacional de Córdoba (SECyT) for the
fellowship granted.
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