92 NITROSAMINES AND RELATED N-NITROSO COMPOUNDS 

Challis, B. C.; Latif, F., J. Chem. Soc. Perkin Trans 1,1990, 1005-1009. 

White, E.H., J. Arner. Chem. Soc., 1955, 77, 6008-6010. 

Williams, D. L. H., Nitrosation, C.U.P., Cambridge, 1988. 

Kurosky, A.; Hofmann, T., Canad. J. Biochem., 1972,50, 1282-1296. 

Challis, B. C.;Fernandes, M. H. R.; Glover, B. R.; Latif, F., In The 

Relevance of N-Nitroso Conzpounds to Human Cancer; Bartsch, H.; 

O'Neill, I.; Schulte-Hermann, R. Eds.; IARC, Lyon, 1987, IARC Scient. 

Publ." NO. 84, 308-314. 

Goldstein, E.; Peck, N. F.; Parkes, N. J.; Hines, H. H.; Steffey, E. 0.; 

Tarkington, B., Amer. Rev. Respir. Diseases, 1977, 115, 403-4 12. 

Keise, M., In Methemoglobinemia- A Comprehensive Review, CRC Press, 

Cleveland, Ohio, 1974, 72-73. 

Doyle, M. R.; Hoekstra, J. W., J. Inorg. Biochem., 1981, 14, 351-358. 

Howard, A. J.; Austin, J., J. Chem. Soc., 1961, 3278-3282; ibid., J. Chem. 

SOC., 1961, 3284-3289. 

Markgraf, J. H.; Davis, H. A,, J. Chem. Ed., 1990,67, 173-176. 

Maycock, C. D.; Stoodley, R. J., J. Chem. Soc. Perkin Trans. 1,1979, 1852- 

1855. 

Pollock, J. R. A., Food Chem. Toxicol., 1985,23,701-704. 

Tricker, A. R.; Preussman, R., Carcinogenesis, 1986,7, 1523-1526. 

Outram, J. R.; Pollock, J. R. A,, In N-Nitroso Compounds: Occurrence, 

Biological Effects and Relevance to Human Cancer, O;Neill, I. K.; Von 

Borstel, R. C.; Miller, C. T.; Long, J.; Bartsch, H., Eds.; IARC, Lyon, 1984, 

IARC. Scient. Pub1.n No. 57, 71-76. 

Curtius, T.; Thompson, J., Chem. Ber., 1906,39,4140-4144. 

Dimroth, O., Ann., 1910,373, 336-346 and references cited therein. 

Lawley, P. D, In Chemical Carcinogens 2nd Edn.; Searle, C. E., Ed; ACS 

monograph 182, ACS, Washington, DC, 1984,428. 

Meier, I.; Shephard, S. E.; Lutz, W. K., Mutat. Res., 1990,237, 193-197. 

Long, F. A.; Purchase, M., J. Amer. Chem. Soc., 1950, 72, 3267-3271. 

RECEIVED July 28,1993 

Chapter 8 

Nitrosatable Secondary Amines 

Exogenous and Endogenous Exposure and Nitrosation 

In Vivo 

A. R. Tricker1, B. Pfundstein, and R. Preussmann 

Institute of Toxicology and Chemotherapy, German Cancer Research 

Center, Im Neuenheimer Feld 280, D-6900 Heidelberg, Germany 

Mean dietary exposure to N-nitrosodimethylamine (NDMA), N-nitrosopyrrolidine (NPYR) and N-nitrosopiperidhe (NPIP) is 0.20-0.3 1 

&day; exposure to the secondary amines dimethylamine (DMA), 

diethylamine (DEA), pyrrolidhe (PYR) and piperidine (PIP) is 7.4 

mg/day. These amines are also found in human saliva, gastric juice, 

blood, urine and faeces. Patient groups with chronic bacterial infections 

of the urinary tract (paraplegics, Bilharzia and bladder augmentation 

patients) and healthy controls excreted a mean of 40.5-49.7 mg 

DMAIday, 19.4-23.8 mg PYRIday and 26.1-3 1.7 mg PIPiday. In patient 

groups a mean of 0.43-1.88 pg NDMA/day, 0.13-0.39 pg NPWday 

and 0.21-0.30 pg NPIPIday was excreted in urine but not in a control 

group. The endogenous formation of amines, and volatile N-nitrosamines under some clinical conditions is considerably higher than the 

exogenous exposure. 

The potential for the endogenous gastric formation of N-nitroso compounds has 

been evaluated in kinetic models (1) using data obtained from either animal 

feeding studies (2) or calculations based on the predicted intake of precursor 

,amines from the diet (3). These studies suggest that endogenous formation and. 

exposure to dialkyl and heterocyclic volatile N-nitrosamines is not of sufficient 

magnitude to present a potential health risk. However, biological monitoring of 

specific drug nitrosation products formed in man and excreted in urine (4,5) 

provides strong evidence that endogenous nitrosation can provide a substantial 

additional exposure to N-nitroso compounds. As in the case of N-nitrosodimethylarnine (NDMA) formation from endogenous nitrosation of arninopyrine (4), animal 

studies show that sufficient endogenous nitrosation occurs for biological monitor- 

'~u~~ent address: Wissenschaftliche Abteilung, Verband der Cigarettenindustrie, 

Konigswintererstrasse 550, 53227 Bonn, Germany 

0~-6156/94/0553-0093SO&OO/O 

@ 1994 American Chemical Society 

This material may be protected by Copyright Law [Title 17 US Code]
94 NITROSAMINES AND REL4ED N-mOS0 COMPOUNDS 

ing of 7-methylguanine DNA adducts excreted in urine (6) and to induce liver 

tumors in rats (7). 

Theoretical studies suffer hm two major limitations: (i) the lack of available 

analytical data on the exposure to both nitrosatable amines in the diet &d their 

occurrence in biological fluids such as gastric juice, and (ii) the fact that 

endogenous nitrosation is not limited to chemical reactions involving dietary 

nitrate, nitrite and amines occurring under acidic conditions in the stomach. 

Extragastric nitrosation may also result from reactions involving bacteria (8), yeasts 

(9), alveolar (10) and peritoneal macrophages (11) in the oral cavity (9), urinary 

bladder (12,13), sigrnoid colon (14) and possibly the vaginal vault (IS). The 

following studies evaluate the daily dietary exposure to nitrosatable secondary 

amines and volatile N-nitrosarnines, and their endogenous occurrence in human 

saliva, gastric juice, blood, urine and faeces. 

Methods and Materials 

Exogenous exposure to secondary amines and volatile N-nitrosamines. In a 

dietary survey, 38 alcoholic drinks and 2 15 foods prepared for human comumption 

using standard culinary practices were analysed for secondary amines (16,17) and 

volatile N-nitrosamines (18). Food samples were purchased fiom supermarkets in 

Heidelberg, Germany. The choice of samples was based on the 1988 German 

Nutritional Report (19) which was used to calculate the mean daily intake of 

individual foodstuffs. A 10 day duplicate diet survey was also performed, diet 

samples were collected in plastic containers, homogenized and frozen at -20°C 

pending analysis. 

Biomonitoring studies. Both smoking and nonsmoking volunteers participating 

in the studies consumed their normal diets without additional vitamin 

supplementation. Alcohol consumption was forbidden in investigations in which 

urine samples were collected. Urine samples were collected &om spine-injured 

paraplegic patients (13), Bilharzia (12) and bladder augmentations patients (20). A 

control group was selected from candidates not taking any form of pharmaceutical 

drug treatment. Urine (24 hour and individual samples) was collected by direct 

urination into sterile plastic containers containing 10 g sodium hydroxide. Saliva 

samples were collected (3-4 hours after break€&) by expectoration into sterile 

plastic vials containing 10 M sodium hydroxide (100 pl). Gastric juice samples (5- 

10 ml) were collected by endoscopy. The pH was immediately measured using a 

glass electrode prior to stabition by addition of 10 M sodium hydroxide (200 

pl). Blood (peripheral) samples were collected using heparinized sterile tubes. 

Plasma was obtained by centrifugation at 3000 g for 15 mi. at 4°C and stabilized 

by addition of 10 M sodium hydroxide (200 pl). Faeces samples (50-100 g) were 

weighed into plastic bottles containing 0.5 M sodium hydroxide (50 rnl) and 

vigorously shaken to produce a homogenous slurry. Sodium hydroxide stabilization 

was used for all samples to prevent artefact N-nitrosamine formation and to 

prevent bacterial growth andlor bacterial reduction of nitrate to nitrite. All samples 

were stored at -20°C prior to analysis using previously reported methods for the 

analysis of nitrate, nitrite, secondary amines and volatile N-nitrosamines (21). 

8. TRICmR &. Nitrosatable Secondary Amines 95 

Results and Discussion 

The exogenous exposure to secondary mines and volatile N-nitrosamines is 

presented in Table I. Good agreement on the mean daily intake was obtained from 

the dietary survey after correction for the daily intake according to the German 

Nutrition Report and the 10 day duplicate diet survey. However, caution must be 

excercised in the estimation of exposure to food constituents due to uncertainties 

in the average food consumption within a population. According to the German 

Nutrition Report, adult women have a total average consumption of 1.8 kg/day 

while our duplicate diet survey showed an average daily consumption of 2.4 

kglday for a 60 kg female adult. 

The available data from food surveys on volatile N-nitrosamines published over 

the last decade (reviewed in 18) indicate that: (i) the dietary intake of NDMA is 

0.5 &day or less in most countries, and (ii) the current levels are about one-third 

of those found a decade ago. The latter is true for The Netherlands, Japan and 

Germany where sufficient data is available to evaluate the dietary situation. This 

is the first dietary survey which calculates a mean daily intake for secondary 

amines. Previous estimates by Shephard et al. (3) underestimate the mean exposure 

to the most commonly encountered amines (1.7 mg DMA/day, 0.3 mg PYRIday 

and 0.06 mg PIPIday) and overestimate the exposure to other secondary amines 

(0.6 mg MBzAIday and 0.3 mg MEAlday). 

The endogenous exposure to nitrate, nitrite and amines expressed in pglrnl 

(ppm) in saliva, faeces, and blood are presented in Table 11. Only DMA, PYR and 

PIP are regularly present in these biological samples. DEA was occasionally found 

in saliva and faeces but not in blood. Individual samples were occasionally found 

to contain trace levels of EMA and DBA (data not shown). 

In the oral cavity, reduction of nitrate to nitrite in the salivary glands (22) 

produces the highest effective nitrite concentrations in the human body. The 

presence of oral bacteria, yeasts (9) and increased concentrations of the nitrosation 

catalyst thiocyanate in the saliva of smokers (23) could theoretically catalyse the 

nitrosation of secondary amines naturally present in saliva. However, the presence 

of N-nitrosamines in 'fasting' saliva has not been reported. Ingestion of food 

increases not only the salivary flow but also increases the levels of nitrite-reactive 

species in the oral cavity. The concentrations of secondary amines in the diet are 

even less than those found in saliva, thus considerably reducing the chance of 

endogenous nitrosation occurring in the oral cavity during the consumption of a 

normal diet. 

In the lower gastrointestinal tract, the biological half-life of nitrate (ca. 20 mins) 

and nitrite (ca. 5 mins) is relatively short due to both bacterial metabolism and 

absorption (24,25). Bacterial decarboxylation of amino acids produces a continuous 

source of amines within the gastrointestinal tract (26). These amines are almost 

completely absorbed (27). Our results show that only low concentrations of 

secondary amines are excreted in faeces while nitrate and nitrite were not detected. 

Contrary to an earlier investigation by Wang et al. (28) reporting both NDMA and 

NDEA to be present in normal human faeces, we did not fmd the presence of 

volatile N-nitrosamines. The only reported situation in which nitrosamine formation 

occurs in the lower intestinal tract is in the sigmoid colon of ureterosigmoidostomy 
% MTROSAMINES AND RELATED N-NlTROSO COMPOUNDS 

Table I. Exogenous Exposure to Secondary Arnines and Volatile N-Nitrosamines 

from the Diet 

Mean daily dietary exposure' 

Food survey 10 Duplicate 

Men Women diets (women) 

Secondary amines (mg/da~)~ 

DMA 

PYR 

PIP 

DEA 

DBA 

MOR 

EMA 

DPA 

MBiA 

NDMA 

NPIP 

NPYR 

'Calculated horn the 1988 German Nutritional Report (19). 

'Secondary amines abbreviated as: Dimethylamine @MA), pyrrolidine (PYR), 

piperidine (PIP), diethylamine PEA), dibutylamine @BA), morpholine (MOR), 

methylethy lamine (EMA), dipropy lamine (DPA)and methy lbenzylamine (MBzA). 

Volatile N-nitrosamines abbreviated as: N-nitrosodimethylamine (NDMA), Nnitrosopiperidine (NPIP) and N-nitrosopyrrolidine (NPYR). 

m, not detected. Limit of detection 10 pgkg for amine and 0.05 pgkg for 

volatile N-nitrosamine analysis. 

8. TRICKER ET AL. Nitrosatable Secondary Amines 97 

patients (14). In these patients, the bladder is surgically removed and the ureters 

implanted into the sigrnoid colon producing an enriched mixture containing urinary 

nitrate and secondary amines together with nitrate-reducing faecal bacteria. 

In blood, nitrosatable secondary amines and nitrate were detected. While we did 

not analyse for nitrosamine due to the small sample volumes collected, a previous 

study has shown the presence of NDMA in blood of patients with chronic renal 

failure (29). The presence of NDMA in blood most probably results from 

exogenous dietary nitrosamine exposure and/or formation and adsorption from the 

gastrointestinal tract prior to first-pass hepatic metabolism. However, the possibility 

that NDMA is formed in blood or tissue as a result of NO synthase activity cannot 

be neglected. 

The presence of nitrate, nitrite and secondary amines in gastric juice as a 

function of pH is shown in Figure 1. The pH of 50 gastric juice samples ranged 

between pH 1.1 and 8.1 of which 56 % were acidic (pH<4.0) and only 2 samples 

had a pHB7.0 (data not shown). Nitrate was present in all of 50 samples at a mean 

concentration of 16.0 + 22.5 pg/ml (range 0.9-72.0 pg/ml), a general decreasing 

trend in nitrate concentration was observed with increasing gastric pH. Nitrite was 

detected in 13 of 50 gastric juice samples at a mean concentration of 0.40 + 1.26 

pg/ml (range ND-8.0 pg/ml). Only 2 gastric juice samples with pHC4.0 contained 

free nitrite (0.1 and 0.6 pg/ml). At pH>4.0, 11 of 32 gastric juice samples 

contained 0.60 f 1.55 pg/ml (range ND-8.0 (~g/ml) nitrite. DMA was found in all 

50 gastric juice samples at a mean concentration of 0.82 f 0.82 pglml (range 0.20- 

4.20 pglml). Lower concentrations of 0.06 f 0.1 1 pg/ml (range ND-0.45 pglml) 

DEA were detected in 19 samples, 0.17 + 0.14 pgfrnl (range ND-0.70 pglrnl) PYR 

in 43 samples and 1.31 +- 2.44 pg/ml (range ND-15.8 pgtml) PIP in 48 samples. 

A marked decrease in amine concentration was found with increasing gastric pH. 

Isolated gastric juice samples contained EMA and DBA at concentrations of less 

than 0.06 pglml. 

In the fasting stomach, the only secondary amines of any consequence which 

occur in gastric juice are DMA, PYR and PIP. Chemical nitrosation is controlled 

by two basic physical properties: (i) the dissociation of nitrite in aqueous media for 

which the acid-nitrite equilibrium has a pKa of 3.4, and (ii) the basicity of the 

amine. Protonated amines are unreactive to the nitrite-derived nitrosating agent 

N20,. As a result, amine nitrosation rates typically show an optimum at pH 3.0-3.5 

due to complex interactions of decreasing concentrations of N203 and increasing 

concentrations of free amines with increasing pH (30). For strongly basic secondary amines such as PYR (pKa 11.3), PIP (pKa 11.2) and DMA (pKa 10.7), the 

effective concentration of the unprotonated amine which can participate in 

chemical nitrosation under acidic conditions is small. At present, the extent of 

nitrosation in the gastric compartment cannot be assessed. However, endogenous 

nitrosation of aminopyrine (8) and piperazine (9) in man and the monitoring of 

their nitrosation products in urine show that a range of amino precursors do 

undergo nitrosation in vivo resulting in considerable transient levels of Nnitrosamines after exposure to high concentrations of a single nitrosatable amine. 

The mean 24 hour levels of nitrate, nitrite, secondary amines and their 

corresponding N-nitroso derivatives in urine of three different patient groups prone 

to chronic bacterial infections of the urinary bact, and in a comparative German 
98 NITROSAMINES AND RELATED N-NITROSO COMPOUNDS 

Table 11. Nitrate, Nitrite and Secondary Amines in Saliva, Blood and Faeces 

(@ml)l 

Saliva (n=20) Blood (n=10) Faeces (n=8) - -- -- 

NO, 35.5 + 17.4 3.21 f 2.74 ND2 

(5.8-92.0) (1.4-8.1) 

NO; 8.9 k 6.3 ND N-D 

(1.6-23.0) 

DMA 0.18 + 0.05 0.91 + 0.84 0.41 + 0.46 

(ND-0.30) (0.1 5-2.40) (0.10-1.35) 

DEA ND 0.03 k 0.04 

(0.03) (ND-0.10) 

PYR 0.21 + 0.23 0.36 + 0.58 0.55 k 0.67 

(ND-0.65) (ND-2.20) (0.25-1.30) 

PIP 0.49 k 0.37 1.86 + 1.02 0.39 k 0.24 

(ND-1.30) (0.45-2.70) (0.20-0.70) 

'Mean f S.D. (range of individual results). Secondary amines abbreviated as shown 

in Table I. 

'ND, not detected. Limit of detection 1.0 pglml for nitrate and nitrite, 0.01 pglml 

for secondary amine analysis. 

Arnine concentration (~g/rnl) 

5 

Gastric juice pH 1-1.9 2-2.9 3-3.9 4-4.9 5-5.9 6-6.9 

Nitrate (pprn) 28.9 10.7 14.5 19.5 12.3 17.1 

Nitrite (pprn) 0 0 0.09 0.15 0.34 0.48 

Figure 1. Concentrations of Nitrate, Nitrite and Secondary Arnines in Gastric Juice 

as a Function of pH. 

8. TRICKEX ET AL. Nitrosatable Secondary Amines 99 

control group of healthy subjects, are given in Table 111. Urine contains the highest 

concentrations of both secondary amines and nitrate within the human body. Under 

normal conditions, in the absence of bacterial infections, nitrosation within the 

urinary tract does not occur. In the thee patient groups with bacterial colonisation 

of the urinary tract, excretion of NDMA, NPIP and NPYR most probably occur as 

a result of bacterial mediated nitrosation or as an indirect result of the infection 

which stimulates NO production. Aerobic bacterial cultures showed a complex 

Table 111. Daily Excretion of Nitrate, Nitrite, Secondary Amiies and Volatile NNitrosarnines in Urinei 

Patient groups with bacterial infections of the 

urinary tract 

Control Paraplegic Bladder Bilharzia 

group patients augmentations patients 

(n=40) (n=30) (n= 1 2) (n=50) 

(0-5) 

23.8k15.3 

(7.4-45.0) 

31.7k17.4 

6.6-98.1) 

0.43k0.38 

(ND- 1.40) 

ND 

158.1k166.7 

(1 6.0-855) 

3.36k4.02 

(ND-45.0) 

37.8k17.2 

(9.8-80.7) 

0.69k1.75 

(ND-9.04) 

2 1.9323.2 

(1.4-78.8) 

24.2K29.7 

(0.6-1 17.1) 

1.88k4.63 

(ND-32.1) 

0.10k0.18 

(ND- 1.90) 

0.16k0.25 

(ND-0.90) 

0.21+0.18 

(ND-0.60) 

'Mean f S.D. (range of individual results). Secondary amines and volatile Nnitrosamines abbreviated as shown in Table I. 

'ND, not detected. Limit of detection 1.0 pdml for nitrate and nitrite, 0.01 pg/d 

for secondary amine, and 0.05 pg/l for volatile N-nitrosamine analysis. 
100 NITROSAMINES AND RELATED N-NITROSO COMPOUNDS 

bacterial flora including nitrate-reducing bacteria in urine samples from both 

paraplegic patients and patients with bladder augment-ations. Bacterial cultures 

were not obtained from Bilharzial patients. However, other studies confii that 

chronic bacterial infections are usually present in such patients (31). 

In summary, our results show the concentrations of nitrosatable secondary 

amine precursors to volatile N-nitrosamines in the diet and in different 

compartments of the human body in which endogenous nitrosation may occur. The 

endogenous formation of these mines is far greater than the exogenous exposure 

from the diet. The principal site of amine formation is in the lower gastrointestinal 

tract from which amines are almost totally absorbed and circulated in blood. From 

blood, amines may diffuse into acidic gastric juice where on protonation there is 

little net transport from the gastric lumen back into blood. As a result, most amines 

enter an enterogastric cycle prior to renal excretion. 

Acknowledgements 

The authors are grateful to Dr. D. Stickler, School of Pure and Applied Biology, 

UWIST, Cardiff, Wales, Prof. M. H. Mostaffa, Institute of Graduate Studies and 

Research, University of Alexandria, Alexandria, Egypt, and Dr. T. Kalble, 

Urological Department, University of Heidelberg, Germany for collection of 

biological fluids used in these studies. 

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RECEIVED July 19,1993