Download Protective effect of salivary nitrate and microbial nitrate reductase

Copyright Eur J Oral Sci 2004
Eur J Oral Sci 2004; 112: 424–428
Printed in UK. All rights reserved
European Journal of
Oral Sciences
Protective effect of salivary nitrate and
microbial nitrate reductase activity
against caries
J. J. Doel1, M. P. Hector2,
C. V. Amirtham2, L. A. Al-Anzan2,
N. Benjamin3, R. P. Allaker1
1
Oral Microbiology Unit and 2Department of
Oral Growth and Development, Barts and The
London, Queen Mary's School of Medicine and
Dentistry, London, UK; 3Peninsula Medical
School, St Luke's Campus, Exeter, UK
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: 424–428. Eur J Oral Sci, 2004
To test the hypothesis that a combination of high salivary nitrate and high nitratereducing capacity are protective against dental caries, 209 children attending the
Dental Institute, Barts and The London NHS Trust were examined. Salivary nitrate
and nitrite levels, counts of Streptococcus mutans and Lactobacillus spp., and caries
experience were recorded. Compared with control subjects, a significant reduction in
caries experience was found in patients with high salivary nitrate and high nitratereducing ability. Production of nitrite from salivary nitrate by commensal nitratereducing bacteria may limit the growth of cariogenic bacteria as a result of the
production of antimicrobial oxides of nitrogen, including nitric oxide.
Dental caries is the most common disease found within the
oral cavity. Although there has been a steady decrease in
the prevalence of coronal caries in many populations in the
industrialized world over the last 20–30 yr, Ôhigh-riskÕ
groups still exist within such populations. In the UK, these
high-risk groups tend now to be limited to deprived inner
city populations, ethnic minority groups and recent
immigrants. In some developing countries an overall
increase in the prevalence of dental caries is observed
particularly where large numbers of people move from a
predominantly rural to an urban environment and, at the
same time, radically change their dietary habits (1). Dental
caries is an infective condition and is initiated by an acid
attack of tooth enamel, arising largely from the metabolism of sugars by bacteria such as Streptococcus mutans
and Lactobacillus spp. (2). Individuals with high levels of
these bacteria in saliva, i.e. > 106 colony-forming units
(CFU) ml)1 of saliva, are believed to be at high risk for
dental caries. S. mutans produces strong acids, such as
lactic acid and acetic acid, that can reduce the pH of saliva
below the critical value of 5.5 for acid demineralization (3).
Although nitrate has historically been associated with
deleterious effects in humans, such as infant methaemoglobinaemia (4), recent evidence has suggested a beneficial, antimicrobial role for inorganic nitrate in several
systems in humans, including the gastrointestinal tract,
oral cavity and skin (5–7). In relation to the oral cavity,
inorganic nitrate is present in saliva in high concentrations. Dietary nitrate, present in large quantities in foods
such as leafy green vegetables, is absorbed from the small
intestine into the bloodstream. Nitrate is then actively
concentrated by the salivary glands, so that salivary
nitrate concentrations are approximately 10-fold higher
R. Allaker, Oral Microbiology Unit, Barts and
the London, Queen Mary's School of Medicine
and Dentistry, Turner Street, London E1 2AD,
UK
Telefax: +44–207–2470657
E-mail: [email protected]
Key words: dental caries; diet; nitrate; nitric
oxide; Streptococcus mutans
Accepted for publication June 2004
than those found in plasma (8). This results in a nitrate
concentration in saliva of 1500 lm, dependent largely
upon dietary nitrate intake. Nitrate concentration varies
widely according to the quality and preparation of the
food, for example, boiling results in a loss of nitrate from
most vegetables. Some typical values of nitrate in foods
are shown in Table 1.
In mammals, nitrate is considered to be largely an inert
material, with no enzymes capable of its reduction.
However, in the oral cavity, salivary nitrate will come
into contact with bacteria that are capable of rapidly
reducing nitrate to nitrite as part of their respiration.
Areas of the oral cavity, notably the crypts of the tongue,
harbour bacteria in conditions of low oxygen tension (9)
that encourage the reduction of nitrate. Such ability of
the oral cavity to reduce nitrate is known to vary widely
between individuals (10). Nitrate reduction is widespread
in nature (11) with highly conserved families of enzymes
across genera. The product of nitrate respiration by
bacteria is nitrite. Bacteria respiring nitrate rapidly
extrude nitrite from their periplasm into the surrounding
milieu. Thus, saliva in the oral cavity contains 1500 lm
nitrate and 100 lm nitrite, resulting from the bacterial
respiration of salivary nitrate. This salivary production
of nitrite is reduced following the use of broad-spectrum
antibiotics (12) and is absent in germ-free rats (6), thus
supporting the role of nitrate-reducing bacteria in its
production.
Nitrite in the presence of acid will form nitrous acid
which, being inherently unstable, dismutates to form a
range of nitrogen oxides, most notably nitric oxide (NO).
NO, produced in large amounts, is recognized for its
antibacterial properties, and is part of the mechanism for
Nitrate and caries protection
Table 1
Typical values of nitrate in raw and cooked foods (24–27)
Nitrate content (mg kg)1)
Foodstuff
Raw
Cooked
Apple
Asparagus
Bacon
Banana
Beer
Beetroot
Broccoli
Brussel Sprouts
Butterhead Lettuce
Carrot
Celery
Corned beef
Cos Lettuce
French Beans
Grapes
Ham
Orange
Parsnip
Pear
Peas
Potato
Savoy cabbage
Tap water
11
13
101
402
16
1,500
400
12
3,000
170
1,200
19
870
450
46
71
13
81
14
57
154
240
10–20
ND*
0
ND
ND
ND
1,900
160
8
ND
93
1,000
ND
ND
290
ND
ND
ND
54
ND
17
67
67
ND
425
London, UK. Ethical clearance for the study was obtained
from the East London and the City Health Authority
(ELCHA). Two saliva samples were taken from each subject
using sterile cotton swabs.
Microbiological determinations
*ND, not determined.
killing used by macrophages via the action of the inducible NO synthase enzyme, upregulated by lipopolysaccharide as well as by various proinflammatory cytokines
(13). The antimicrobial effects of NO are thought to
include modifications of DNA, respiratory complexes, as
well as interactions with other reactive species; for
example, the highly reactive molecule peroxynitrite is
produced upon reaction with superoxide (14).
Accordingly, when salivary nitrite comes into contact
with the acid environment around the teeth provided by
acid-producing bacteria such as S. mutans, a bolus of
antimicrobial compounds, including nitric oxide, is
formed and results in bacteriostatic and possibly bacteriocidal effects (15). As such, in the presence of large
amounts of nitrite in the oral cavity, the growth and
possible survival of acidogenic bacteria is limited. Thus,
salivary nitrate is a reservoir from which a variety of
nitrogen oxides are formed, most notably nitric oxide.
After ingesting nitrate-rich food, increases in plasma
nitrate, salivary nitrate, salivary nitrite, and oral nitric
oxide have been noted (6). To investigate whether salivary nitrate reduction can be preventative against dental
caries, we undertook a clinical study with 209 children
presenting to the Dental Institute, Barts and The London
NHS Trust, London, UK.
One swab was placed in a glass vessel containing
20 glass beads and 1 ml of reduced transport fluid (16)
and vortexed for 30 s to disaggregate bacteria from the
swab. Of the resulting solution, 40 ll was then transferred
to selective agar using a spiral plater (Don Whitley
Instruments, Shipley, UK). Rogosa agar (Oxoid, Basingstoke, UK), selective for Lactobacillus spp., and TYCSB
agar (TYC agar, Oxoid, supplemented with 150 g l)1
sucrose and 200 U l)1 bacitracin), selective for S. mutans,
were used. Agar plates were incubated in air with 10%
CO2 (v/v) at 37C for 72 h. Identification of S. mutans
was confirmed on the basis of characteristic colony morphology and standard biochemical tests.
Nitrite and nitrate measurements
The head was cut off the second swab and placed in a
microcentrifuge tube containing 10 glass beads. The tube
was then spun at 13,250 g for 5 min, the beads discarded
and the saliva sample recovered.
Measurement of nitrite. Nitrite concentrations were determined in 96-well plates, in duplicate, using a 50-ll saliva
sample volume. To each sample, 50 ll of sulfanilamide
solution (1% w/v sulfanilamide in 5% v/v phosphoric acid)
was added and incubated for 5 min at room temperature,
protected from light. NED solution (0.1% w/v N-1-naphthylethylenediamine dihydrochloride in distilled water;
50 ll) was then added and incubated for 5 min at room
temperature, protected from light. The development of a
characteristic purple colour was then measured in a plate
reader at 540 nm. Values of nitrite were estimated by
comparison with a standard curve of nitrite concentrations.
Measurement of nitrate. Nitrate concentrations were determined in the following manner. A 50-ll sample of saliva was
added to 950 ll of 0.5 m ammonia buffer, pH 9.0, containing
1 g l)1 EDTA. The solution was then exposed to a copperized cadmium column (Nitralyzer; WPI Instruments,
Stevenage, UK) for 5 min. This converts all the nitrate in the
solution to nitrite. Nitrite in the solution was then measured
as described above; starting concentrations of nitrate were
calculated by subtracting the values obtained pre- and
postreduction using the copperized cadmium column.
Caries assessment
Caries experience of subjects was determined as the sum of
the number of teeth that were diseased as a result of active
caries, missing as a result of having been carious and filled
owing to carious lesions.
Material and methods
Dietary nitrate estimation
Subjects
A dietary questionnaire was used to establish the approximate daily dietary nitrate intake (mg kg)1 body weight) of
an individual. This was based upon the National Diet and
Nutrition Survey (17). The dietary interview included
Consent was sought from patients attending the Dental
Institute, Barts and The London Health NHS Trust,
426
Doel et al.
questions on the type of vegetables eaten (cooked or raw),
frequency of intake and a detailed description of food and
fluid intake over the previous 24-h period.
Statistics
Student’s independent t-tests were employed to analyse
data.
Results
A total of 209 subjects (106 male and 103 female) were
recruited to the study from the London Borough of
Tower Hamlets, a deprived area with a population of low
socio-economic status. Summaries of the values for salivary nitrite and nitrate levels, S. mutans and Lactobacillus spp. counts (CFU) in saliva, caries experience and
age are shown in Table 2. To assess the ability of an
individual’s oral flora to reduce salivary nitrate to nitrite,
the ratio of nitrite in the saliva as a percentage of starting
nitrate is given (because nitrate is converted to nitrite) as
a nitrate reductase (NR) ratio as follows:
Caucasian (33%); 4 Afro-Caribbean (10%); 3 mixed race
(8%); and 2 Asian (5%). The control group consisted of
170 subjects; 88 males (52%) and 82 females (48%).
Within this group the ethnicity mix comprised: 72 Indian
subcontinent (42%); 67 white Caucasian (39%); 21 AfroCaribbean (12%); 4 mixed race (3%), 3 Asian (2%); and
3 Middle-Eastern (2%). In terms of caries experience, a
significant decrease (P < 0.05) was observed in subjects
in the high salivary nitrate/nitrate reductase group when
compared with control subjects. The former group also
revealed a lower mean value for counts of S. mutans
levels when compared to control subjects. However, this
difference was not statistically significant.
A dietary survey was also undertaken to identify
whether the intake of nitrate within the sample population was within the expected variation. Of 135 subjects
surveyed, the average intake was 3.6 mg kg)1 d)1 (range:
0.2–17.3; median 2.7). The mean value accords closely
with the current acceptable daily intake (ADI) of
3.7 mg kg)1 d)1, as published by the World Health
Organization (18). The full range of nitrate intake values
are shown in Fig. 1. It is of note that 92 (68%) fall below
the ADI, with 43 (32%) consuming more than the ADI.
100 ½nitrite=ðnitrite þ nitrateÞ:
In order to identify those patients who had both a high
concentration of salivary nitrate and a high nitratereducing capacity, the data were further subdivided into
two groups. Those patients with levels of salivary nitrate
and a nitrite/nitrate ratio above the 50th percentile
(986.3 lm and 6.4%, respectively) were considered to
have a high nitrate level in saliva and posses a high
nitrate-reducing capacity. The two groups are shown in
Table 3. This high nitrate-reducing/high salivary nitrate
group consisted of 39 subjects: 18 males (46%) and
21 females (54%). Within this group the ethnicity mix
comprised: 17 Indian subcontinent (44%); 13 white
Table 2
Salivary nitrite and nitrate levels, nitrate reductase (NR) ratio,
Streptococcus mutans and Lactobacillus spp. counts, caries
experience and age profile of all 209 subjects recruited
Variable
Salivary nitrite (lm)
Salivary nitrate (lm)
NR* ratio (%)
Streptococcus mutans
(CFU ml)1 of saliva)
Lactobacillus spp.
(CFU ml)1 of saliva)
Caries experience
Age (yr)
Mean
91.0 ± 6.3
(range 0.0–700.9; median 70.2)
1,370.2 ± 93.0
(range 0.0–8,351.2; median 986.3)
9.3 ± 0.8
(range 0.0–100.0; median 6.3)
6.3 · 105 ± 1.5 · 105
(range 0.0–2.5 · 108; median 1.3 · 104)
3.3 · 103 ± 9.4 · 102
(range 0.0–1.4 · 105; median 0.0)
5.0 ± 0.3
(range 0.0–26.0; median 5.0)
7.2 ± 0.3
(range 0.08–16.0; median 7.0)
*The NR ratio represents an individual’s capacity to reduce
salivary nitrate.
Results are expressed as mean ± standard error (range; median). CFU, colony-forming units.
Discussion
Humans have evolved mechanisms to concentrate nitrate
in the oral cavity. As no mammalian enzymes exist to act
upon nitrate, this nitrate recycling appears to have no
clear function within the human body. However, considering the complex microflora that resides within the
specialized niches of the oral cavity, possible reasons for
this enteric nitrate recycling become apparent. The oral
flora consists of at least 500 different species identified to
date (19). Many of these bacteria, such as Veillonella
atypica are anaerobes and have an absolute requirement
for nitrate as an electron acceptor. Other bacteria, such
as Actinomyces odontolyticus, are facultative anaerobes
and will respire under conditions of low oxygen tension.
Under anaerobic conditions, as found in the deep clefts
of the tongue, such bacteria will reduce nitrate, resulting
in the production of large quantities of nitrite, which is
rapidly extruded from the bacteria.
The concentration of nitrate in the oral cavity may
thus encourage the survival of bacteria under conditions
of low oxygen tension and, in turn, enhance oral
immunity via the production of antimicrobial oxides of
nitrogen. Accordingly, individuals with a high nitrate
intake and an oral flora with the ability to reduce large
amounts of nitrate to nitrite in the oral cavity may be
protected against cariogenic acidogenic bacteria and,
thus, dental caries. Considering acidogenic bacteria,
typified by cariogenic S. mutans, the production of acid
in the presence of nitrite will result in bacteriostatic and
possibly bacteriocidal effects. It is suggested that the
antimicrobial nature of acidified nitrite is selective in that
only those bacteria in the immediate vicinity will be
affected.
To test the hypothesis that nitrate recycling and a high
nitrate-reductase activity in the oral cavity could be
Nitrate and caries protection
427
Table 3
Comparison of salivary nitrite and nitrate levels, nitrate reductase (NR) ratio, Streptococcus mutans and Lactobacillus spp. counts,
caries experience and age profile for high nitrate reducers compared with other subjects
Salivary nitrite (lm)
Salivary nitrate (lm)
NR* ratio (%)
Streptococcus mutans
(CFU ml)1 of saliva)
Lactobacillus spp.
(CFU ml)1 saliva)
Caries experience
Age (yr)
High nitrate reducers
Others
177.8 ± 20.7
(range 74.4–700.9; median 132.5)
1,486.6 ± 98.7
(range 986.3–3,655.5; median 1,294.1)
10.3 ± 0.7
(range 6.5–28.1; median 8.9)
5.2 · 105 ± 2.4 · 105
(range 0.0–9.2 · 106; median 2.3 · 104)
3.6 · 103 ± 1.8 · 103
(range 0.0–6.4 · 104; median 0.0)
4.0 ± 0.7
(range 0.0–14.0; median 3.0)
6.6 ± 0.5
(range 1.0–15.0; median 6.0)
71.1 ± 5.1
(range 0.0–327.4; median 56.2)
1,343.5 ± 112.1
(range 0.0–8,351.2; median 851.6)
9.1 ± 1.0
(range 0.0–100.0; median 4.8)
6.5 · 105 ± 1.8 · 105
(range 0.0–2.5 · 107; median 1.0 · 104)
3.2 · 103 ± 1.1 · 103
(range 0.0–1.4 · 105; median 0.0)
5.3 ± 0.3
(range 0.0–26.0; median 5.0)
7.3 ± 0.3
(range 0.08–16.0; median 7.0)
*The NR ratio represents an individual’s capacity to reduce salivary nitrate.
ÔHigh nitrate reducersÕ comprise those subjects whose salivary nitrate and NR ratio are above the 50th percentile.
Results are expressed as mean ± standard error (range; median). CFU, colony-forming units.
Nitrate intake (mg–1 kg–1 day–1)
protective against dental caries, we examined the levels of
salivary nitrate and nitrite, S. mutans and Lactobacillus
spp. counts in saliva, and caries experience in children
presenting to the Dental Institute at Barts and The
London NHS Trust. We found that those children with
high salivary nitrate levels and a high percentage reduction of nitrate to nitrite had significantly less caries
experience, providing support to the suggestion that
nitrate recycling may be an important part of the innate
immune response in the oral cavity. To lend further
support, the mean nitrite salivary level of 0.2 mm found
in this group reaches the concentration (‡ 0.2 mm)
known to kill S. mutans in vitro under acidified conditions (15).
Further circumstantial evidence that nitrate reduction
could be important as a defence mechanism against
16
12
8
4
0
1
31
61
91
Sample number
121
Fig. 1. Distribution of estimated dietary nitrate intake for 135
subjects (mg kg)1 d)1). The unbroken line represents the
acceptable daily intake (World Health Organization guidelines)
for nitrate intake (3.7 mg kg)1 d)1).
dental caries has been demonstrated (20,21). Such studies
found that the incidence of dental caries was much
reduced in areas with a high molybdenum content in soil.
It is interesting to note in light of this fact that the nitrate
reductase enzyme requires molybdenum as a cofactor for
activity. Thus, consumption of a higher amount of
molybdenum in drinking water and vegetables could lead
to a higher level of nitrate reductase activity in such
individuals.
To conclude, we have found that patients with a high
concentration of nitrate in saliva and an oral flora with a
high capacity to reduce nitrate to nitrite, have significantly less caries history than those with low amounts of
nitrate in saliva and an oral flora with a low capacity to
reduce nitrate to nitrite. Dental caries is a particular
problem in children, and this is partly explained by a
number of salivary factors. Salivary nitrate concentrations are greatly influenced by the amount of nitrate in
the diet, which, in industrialized countries, is mainly
influenced by the intake of green vegetables (22). In
general, many children have a dislike of green vegetables
(17), and we hypothesize that increasing nitrate intake in
this group may be especially important in establishing
nitrate-reducing bacteria in the oral cavity, similar to the
effects that are seen in the gut (23). Once established, the
nitrate-reducing bacteria may suppress the growth of
acid-forming bacteria and thereby protect teeth against
caries. The major anaerobic nitrate-reducing bacteria on
the human tongue probably include V. dispar and
V. atypica. These are thought to be true commensals in
the oral cavity as a result of their ability to metabolize
lactic acid, the major causative agent of dental caries,
into weaker acids. The residency of strictly anaerobic
bacteria on the tongue is possibly aided by the presence
of secretory glands (von Ebner’s glands) at the base of
the circumvallate papillae. These glands release bicarbonate, which could protect and nourish the anaerobic
bacteria deep within the clefts. This may be part of a
symbiotic adaptation between host and bacteria, adding
428
Doel et al.
support to the hypothesis that certain commensal bacteria are encouraged to reside in the oral cavity to
enhance mucosal immunity. Therefore, probiotic therapy
at an early age to encourage colonization of the oral
cavity with micro-organisms such as V. atypica, which
contain the nitrate reductase enzyme, may be a potential
therapy against dental caries.
Acknowledgements – This work was funded by the Royal
London Hospital Special Trustees (RAC 399).
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