Fd Cosmet. Toxicol. Vol. 14, pp. 545-548. Pergamon Press 1976. Printed in Great Britain
`
`INFLUENCE OF DIETARY NITRATE ON NITRITE
`CONTENT OF HUMAN SALIVA: POSSIBLE
`RELEVANCE TO IN VIVO FORMATION
`OF N-NITROSO COMPOUNDS
`
`B. SPIEGELHALDER, G. EISENBRAND and R. PREUSSMANN
`Deutsches Kreh.eforsch1111gs:e11trwn. lnstitul fiir Toxikologie und C/1emotherapie, 69 Heidelberg I,
`B11ndesrepuh/ik Deutsch/and
`
`(Received 7 May 1976)
`
`Abstract-Vegetables and vegetable juices with concentrations of nitrate between 27 and 4031 ppm
`were ingested by 11 volunteers in 30 experiments. Variations of nitrate and nitrite concentrations
`in saliva were determined for up to 7 hr at intervals of 30 or 60 min. The increase in the amount
`of nitrate secreted by the salivary glands was found to depend directly on the amount of nitrate
`ingested. Nitrite concentration in saliva correlated directly with salivary nitrate content. This suggests
`that there is a direct relationship between the salivary nitrite concentrations and the amounts of nitrate
`ingested in the diet. The average increase of nitrite concentration in saliva was 20 ppm/100 mg nitrate
`ingested. The possible relevance of these findings to the endogenous formation of N-nitroso compounds
`in the gastro-intestinal tract is discussed.
`
`INTRODUCTION
`
`having good dental care (Kilhn, 1974). However, dif(cid:173)
`ferences in dental hygiene and care could not be the
`only cause for the large variations observed. We in(cid:173)
`vestigated, therefore, the influence of ingested nitrate
`on salivary nitrate and nitrite concentrations.
`Before completion of this study, Harada, Ishiwata,
`Nakamura, Tanimura & Ishidate (1975), Ishiwata,
`Boriboon, Harada, Tanimura & Ishidate (1975), Ish(cid:173)
`iwata, Boriboon, Nakamura, Harada, Tanimura &
`Ishidate (1975) and Ishiwata, Tanimura & Ishidate
`(1975) published results of their studies on
`the
`changes of salivary nitrate and nitrite after ingestion
`of nitrate-containing Chinese cabbage. Although no
`quantitative relationships were given, these authors
`also came to the conclusion that the nitrate content
`of the diet is the predominant factor influencing saliv(cid:173)
`ary nitrite concentration.
`A similar study, complementary to ours, is reported
`in an accompanying paper (Tannenbaum, Weisman
`& Fett, 1976).
`
`Formation of carcinogenic N-nitroso compounds
`from nitrite and nitrosatable amino compounds has
`been demonstrated in vitro under conditions similar
`to those in the human stomach, and in animal experi(cid:173)
`ments (Lijinsky, Conrad & van de Bogart, 1972; Mir(cid:173)
`vish, 1975; Sander, 1971). There is increasing evidence
`that the exogenous formation of these potent car(cid:173)
`cinogens and their presence in the environment, as
`well as their endogenous generation from various pre(cid:173)
`cursors, may represent a serious human health risk.
`The presence of nitrite in human saliva is well
`known (Varady & Szanto, 1940). It had been disre(cid:173)
`garded, however, as a possible source for the endo(cid:173)
`genous formation of N-nitroso compounds until Ste(cid:173)
`phany & Schuller (1975) undertook exploratory
`experiments which suggested a direct influence of
`orally ingested nitrate on salivary nitrite concen(cid:173)
`tration. Tannenbaum, Sinskey, Weisman & Bishop
`(1974) measured salivary nitrite in man and concluded
`that nitrite production in saliva did not seem to be
`strongly influenced by dietary composition. Varady
`& Szanto (1940) cited unpublished work by T. Ville
`Materials. All reagents used were analytical grade.
`and W. Nestrezat suggesting that the microbiological
`reduction of nitrate in the oral cavity was responsible Standard solutions and colour reagents for the colori(cid:173)
`for the presence of nitrite in saliva. This was later metric determination of nitrate and nitrite were pre(cid:173)
`pared on the day of use. Vegetables and vegetable
`supported by the finding that ductal saliva, taken dir-
`juices were purchased in local stores during June and
`ectly from the salivary glands, contains nitrate but
`not nitrite (Goaz & Biswell, 1961; Goaz, Biswell &
`July 1975.
`Determination of nitrate and nitrite. A colorimetric
`Miller, 1964; Kilhn, 1974; Miller, Biswell & Goaz,
`1962; Tannenbaum et al. 1974).
`method, based on a recommended standard technique
`· In a previous study by our group," the nitrite con-
`(International
`Standards
`Organization,
`ISO
`tent of human saliva was found to vary widely
`3091-1975) was used with some modifications.
`Determination of nitrate in vegetables and vegetable
`between randomly selected individuals. Values ranged
`juices. A 20-g aliquot of the homogenized.sample was
`from 0·01
`to 30 ppm, with an average value of
`6'8 ppm. The majority of individuals with poor dental
`heated in 150 ml water at 95-98°C for l hr. The
`hygiene were found to have salivary nitrite concen-
`resulting suspension was transferred to a volumetric
`flask and diluted to 250 ml. The suspension was fil-
`trations higher than 5 ppm, in contrast to individuals
`545
`
`EXPERIMENT AL
`
`Human Power of N Company
`EX1057
`Page 1 of 4
`
`

`

`546
`
`B. SPtEGELHALDER, G. EISENBRAND and R. PREUSSMANN
`
`tered through a 2-g layer of Celite 545 (Serva, Heidel(cid:173)
`berg) and 20 ml of the filtrate was passed through
`a cadmium column. For analysis of vegetable juices
`with a high nitrate content, the filtrate was further
`diluted up to 250-fold with water before reduction.
`Determination of nitrate and nitrite in saliva. At spe(cid:173)
`cified intervals after ingestion of nitrate-containing
`vegetables or vegetable juices, samples of saliva
`(1 ·5 ml) were taken. The time required for saliva col(cid:173)
`lection was about 5 min. During the first 2 hr of the
`experiment, samples were collected every 30min; dur(cid:173)
`ing the following 5 hr the intervals were 60 min. The
`samples were filtered immediately through a short
`column of Celite 545. One part (0·5 g) of the filtrate
`was diluted with water to 25 ml, and 20 ml of this
`solution was reduced on a cadmium column for the
`determination of total nitrate and nitrite. Another
`part (0·5 g) of the saliva filtrate was diluted in a volu(cid:173)
`metric flask (lOOml) with water to approximately
`50 ml. After addition of the reagents required for azo(cid:173)
`dye formation, the solution was made up to 100 ml
`for determination of nitrite.
`Absorption was measured at lm•• = 538 nm against
`a reagent blank. Nitrate and nitrite contents of the
`samples were obtained from calibration graphs and
`a standard nitrate solution was measured before and
`after each experiment to confirm the reproducibility
`of the reduction technique.
`Concentrations of nitrate and nitrite in saliva were
`measured in 30 experiments with 11 adult volunteers
`after ingestion of 30-550 mg nitrate in vegetable juice
`or in spinach.
`
`results of a limited survey of various kinds of other
`vegetable foods. As can be seen, fresh root plants and
`leafy vegetables as well as some vegetable juices were
`found to contain extremely high levels of nitrate, the
`highest being 4031 ppm, found in lamb's lettuce
`( V aleriane/la /ocusta). Deep-frozen cream spinach and
`canned celery salad contained relatively low levels of
`nitrate. These results compare well with
`those
`reported by White (1975).
`
`Salivary concentrations of nitrite and nitrate after in(cid:173)
`gestion of nitrate
`Average nitrate and nitrite concentrations in saliva
`1-2 hr after a normal continental-type breakfast were
`74 ± 50 and 9 ± 5 ppm, respectively, with individual
`values of 11-232 and 3-20 ppm, respectively. These
`concentrations had started to increase 30 min after
`ingestion of nitrate in the form of vegetable juice, the
`maximum average concentrations of nitrate and
`nitrite being reached after l and 1 ·2 hr, respectively
`(Fig. la).
`When nitrate was consumed in spinach or radish
`instead of vegetable juice, the increase of salivary
`nitrate and nitrite was much slower, the highest
`values being reached after 2-3 hr (Fig. lb).
`As can be seen from Fig. 1, the nitrate concen(cid:173)
`tration did not decrease steadily from its initial maxi(cid:173)
`mum. In most cases, a second or even. a third smaller
`peak was observed, a possible explanation for which
`is discussed later.
`For comparison of the data it is more appropriate
`to calculate the absolute amounts of secreted nitrate
`
`RESULTS AND DISCUSSION
`Nitrate content in vegetables and vegetable juices
`Table I gives the nitrate contents of vegetables and
`vegetable juices used in the experiments, together with
`
`Table I. Nitrate content of selected vegetable foods
`
`Vegetable food
`
`Juices
`Sauerkraut
`Mixed vegetables
`Carrots
`Red-beets
`Vegetables
`Celery salad (canned)
`Cream spinach
`(deep frozen)
`Red-beets salad•
`Red cabbage
`White cabbage
`Leek
`Green peppen
`Parsley root
`Fennel
`Lamb's lettuce
`Carrots
`Spinach
`Red-beet
`Radish, big
`Radish, small
`Cauliftower
`Turnip-cabbage
`Endive
`
`Nitrate content (ppm)
`
`min.
`
`max
`
`mean
`
`Number
`of
`samples
`
`152
`27
`233
`1389
`
`338
`246
`667
`2201
`
`376
`1758
`
`29
`
`75
`
`52
`
`158
`1583
`
`353
`3189
`
`115
`
`719
`
`126
`1398
`I028
`462
`1244
`
`1165
`3368
`3697
`3799
`3295
`
`247
`1894
`85
`266
`
`227
`915
`1691
`4031
`730
`1931
`2320
`2210
`2132
`60
`1010
`430
`
`2
`2
`4
`13
`
`3
`3
`I
`1
`2
`I
`1
`I
`I
`4
`7
`6
`7
`5
`I
`I
`I
`
`*A concentration of 909 ppm was reported for a sample
`grown without the use of fertilizer.
`
`~
`~
`~ 160
`i 14()
`'5
`., 120
`.5
`1! 100
`
`15 60
`
`i 80
`i 40
`
`c:
`'15
`
`Fig. 1. Concentration (ppm) of nitrate ("") and nitrite
`(--)and quotient (nitrate/nitrite,---) in saliva after in(cid:173)
`gestion of (a) 450 mg nitrate in red-beet juice (250 ml) and
`(b) 95 mg nitrate in spinach (270 g).
`
`Jf 500
`....
`~ 450
`400
`j 350
`Si 300
`.s
`~ 250
`l: 200
`c
`l5
`1!
`E!
`~
`0
`
`20
`le
`16 !
`14 ~ c
`12 ~
`10 = !:
`
`16
`
`Time ofter Ingestion, hr
`
`(b) ,--------"
`
`/
`
`/
`
`...............
`
`''-..,..../'-------------
`
`',""'
`
`....
`
`14
`
`!
`....... '...,. ... -.. ------..........---._... ........ -... -
`12 E
`·;::
`10 f
`____ .,,.
`~
`.,,.,.. e
`..:S
`... c: .,
`6
`4 ~
`"
`----~~--------...::12 0
`LO 15 2.0 2.5 3.0 3.5 4.0 45 5.0 5.5 6.0 slf'
`Time ofter Ingestion, hr
`
`Page 2 of 4
`
`

`

`Nitrate and nitrite in human saliva
`
`547
`
`and mtnte than to use concentration values. The
`absolute amounts were obtained, on the assumption
`of a constant salivary flow, by multiplication of the
`mean increase of the concentrations by the total time
`period and the salivary flow. The mean increase in
`concentration was calculated as I.CJn - C0 where Ci
`is the concentration at time i, 11 is the number of
`is the base concentration
`detenninations and C0
`before nitrate intake.
`The absolute amounts calculated thus correspond
`to the areas under the salivary concentration curves
`(Fig. 1) multiplied by the salivary flow. The relation(cid:173)
`ship between secreted nitrate and nitrite and ingested
`nitrate is demonstrated in Fig. 2. As can be seen, the
`average amounts of secreted nitrate and nitrite corres(cid:173)
`pond directly to the amounts of nitrate ingested.
`In both graphs the regression line intersects the
`x-axis at a value of 54 mg ingested nitrate, which indi(cid:173)
`cates a threshold dose below which salivary concen(cid:173)
`trations of nitrate and nitrite remain unchanged.
`Figure 3 shows that there is a direct correlation
`between the amounts of secreted nitrate and of nitrite
`produced in saliva.
`
`225
`
`(o)
`
`150
`
`200
`"' E 175
`l
`!!!
`~ 125
`~ 100
`:t: c:
`.i;; 75
`~ 50
`~
`
`25
`
`0
`
`\
`
`xx
`
`/
`
`/
`
`x
`
`x
`
`~~~XI)$( I
`
`50
`
`I
`I
`I
`J
`I
`I
`100 150 200 250 300 350 400 450 500 550
`Nitrate Ingested, mQ
`
`40
`
`36
`
`(bl
`
`"' 32
`E
`• 2
`0 >
`:; 24
`"' c:
`..,
`·- 20
`~
`" ..,
`e Q.
`
`16
`
`12
`
`GI
`+-
`
`E B
`z
`
`4
`
`0
`
`x
`
`x
`
`x
`
`x
`
`x
`x
`x
`~
`l~-.1..--1.._.1..--1..~'-'.--':-:--:=
`.,,_~y·~ x I
`50
`100 150 200. 250 300 350 400 450 500 550
`Nit rote inQested, mg
`
`Quantitative aspects of salivary nitrite
`The· average increase of nitrate and nitrite was esti(cid:173)
`mated to be 1·1 and 0·2 ppm/mg nitrate ingested, re(cid:173)
`spectively. The highest concentrations (1110 ppm
`nitrate and 155 ppm n\trite) were observed after inges(cid:173)
`tion of 470 mg nitrate in 250 ml r~d-beet juice. This
`amount of nitrate may also be ingested in, for exam(cid:173)
`ple, 100 g lamb's lettuce or 200 g radish (see Table
`1). After ingestion of these foods, average salivary.
`nitrite concentrations of about 100 ppm would be
`reached and, assuming a salivary flow of 50 ml/hr,
`about 40 mg nitrite could enter the stomach. within
`.
`5~
`
`The gastro-oral circulation of nitrate
`After ingestion, the absorption of nitrate causes an
`increased nitrate concentration in the blood which
`depends on the rate of absorption from the gastro(cid:173)
`intestinal tract. The secretion of nitrate through the
`salivary glands obviously depends on the blood level
`of nitrate. In the oral cavity, about 20% of the nitrate
`is reduced to nitrite. Nitrate and nitrite are swallowed
`and reach the stomach, where nitrite quickly disap(cid:173)
`pears by reaction with the stomach contents, by
`absorption and by other processes. Nitrate, however,
`can be reabsorbed and thus can recirculate. This is
`a very likely explanation for the observed second and
`third maxima in Fig. 1.
`
`Possible influence of salivary nitrite on the endogenous
`formation of N-nitroso compounds
`The results show that the nitrate content of the
`diet is the major factor governing the nitrite content
`of saliva. When vegetable food with a high nitrate
`content is consumed, the nitrite concentration in
`saliva can reach much higher levels than previously
`was thought possible.
`Whether nitrite in saliva can react in the oral cavity
`to fonn N-nitroso compounds has not yet been inves(cid:173)
`tigated thoroughly. The neutral pH and short resi(cid:173)
`dence time of nitrite in the mouth would be unfavour(cid:173)
`able to such a reaction, but several studies have
`shown that N-nitrosation can be strongly catalysed,
`even at neutral pH, by a variety of agents and pro(cid:173)
`cesses (Archer, Tannenbaum & Wishnok, 1976; Boy(cid:173)
`land & Walker, 1974; Keefer & Roller, 1973; Okun
`& Archer, 1976). The stomach, on the other hand,
`
`40
`
`~ 35
`
`Q.
`
`xx
`
`.~ 30
`g 25
`.!::
`al zo
`g
`~ 15
`l!! 10
`·;::
`.... z 5
`Olf.~20.L__40.L__6L0--:80"::--~100':-::--~120::-~14~0-=160:--~180;""'::200:::;;;-~220
`Increose In nitrote secreted in salivo, mg
`
`Fig. 2. (a) Increase of nitrate secreted and (b) nitrite pro(cid:173)
`duced in saliva after ingestion of various amounts of
`nitrate.
`
`the increase of secreted
`Fig. 3. Relationship between
`nitrate and the nitrite produced in saliva.
`
`Page 3 of 4
`
`

`

`548
`
`B. SPIEGELHALDER, G. EISENBRAND and R: PREUSSMANN
`
`represents reaction conditions which are very favour(cid:173)
`able to N-nitrosation reactions, although the dilution
`of salivary nitrite by the stomach contents must be
`taken into account.
`Nitrosatable drugs can be excreted in the saliva.
`After oral ingestion of 500 mg aminopyrine, for in(cid:173)
`stance, we found salivary concentrations of up to
`14 ppm during the first 4 hr; subsequently levels were
`consistently between I and 4 ppm. The drug is
`excreted in the saliva as long as appreciable amounts
`of the intact drug are present in the blood (G. Eisen(cid:173)
`brand, unpublished data; Vessel, Passananti, Glen(cid:173)
`wright & Dvorchik, 1975). Experiments are under
`way to study in more detail the possible formation
`of N-nitroso compounds by nitrite in saliva.
`
`Acknowledgements-The investigations were supported by
`the Deutsche Forschungsgemeinschaft. We thank Messrs
`0. Zelesny, F. Bayer and K. Zuber for competent technical
`assistance, and Mr. P. Meinzer for preparation of software
`for the computer-based processing of experimental data.
`
`REFERENCES
`
`Archer, M. C., Tannenbaum, S. R. & Wishnok, J. S. (1976).
`Nitrosamine formation in the presence of carbonyl com(cid:173)
`pounds. In N-Nitroso Compounds. Proceedings of IVth
`International Working Conference on the Analysis and
`Formation of N-Nitroso Compounds held in Tallinn,
`1975. IARC Scient. Pub!., in press.
`Boyland, E. & Walker, S. A. (1974). Effect of thiocyanate
`on nitrosation of amines. Nature, Land. 248, 601.
`Goaz, P. W. & Biswell, H. A. (1961). Nitrate reduction
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`Goaz, P. W., Biswell, H. A. & Miller, M. C., III (1964).
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`system for the measurement of the nitrate-nitrite reduc(cid:173)
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`lshiwata, H., Boriboon, P., Nakamura, Y., Harada, M.,
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`and nitrate concentrations in human saliva after inges(cid:173)
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`
`Ishiwata, H .. Tanimura. A. & lshidate, M. (1974). Studies
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`Kiihn, H.-J. (1974). Untersuchung Uber den Nitritgehalt
`im Speichel in Abhangigkeit von Zahnzustand und
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`Lijinsky, W., Conrad, E. & van de Bogart, R. (1972). For(cid:173)
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`in Heidelberg, 1971. Edited by P. Bogovski, R. Preuss(cid:173)
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`Miller, M. C., III, Biswell, H. A. & Goaz, P. W. (1962).
`Application of the method of steepest ascent to the res(cid:173)
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`salivary sediment. J. dent. Res. 41, 549.
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`Chemistry, kinetics and in vivo occurrence. Toxic. appl.
`Pharmac. 31, 325.
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`the Analysis and Formation of N-Nitroso Compounds
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`suchstieren und ihre Bedeutung flir den Menschen. I.,
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`& 2034.
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`effect of nitrate intake on nitrite formation in human
`saliva. Fd Cosmet. Toxico/. 14, 549.
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`Nitritgehalt des Speichels, des Magensaftes und des
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`Vesell, E. S., Passananti, G. T., Glenwright, P. A. & Dvor(cid:173)
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`
`Page 4 of 4
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`