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10 NITRATE, NITRITE, AND N-NITROSO COMPOUNDS

10 NITRATE, NITRITE, AND N-NITROSO COMPOUNDS

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Table 9.14 Average Nitrate Contents of Common Foods

in the United States and Per Capita Daily Intake

Nitrate, mg/100 g

Food



Content



Ingestion



Total vegetables

Asparagus

Beet

Beans, dry

Beans, lima

Beans, snap

Broccoli

Cabbage

Carrot

Celery

Corn

Cucumber

Eggplant

Lettuce

Melon

Onion

Peas

Peppers, sweet

Pickles

Potato

Potato, sweet

Pumpkin/squash

Spinach

Sauerkraut

Tomato and tomato products

Breads

All fruits

Juices

Cured meats

Milk and milk products

Water



1.3–27.6

2.1

276.0

1.3

5.4

25.3

78.3

63.5

11.9

234.0

4.5

2.4

30.2

85.0

43.4

13.4

2.8

12.5

5.9

11.9

5.3

41.3

186.0

19.1

6.2

2.2

1.0

0.2

20.8

0.05

0.071



8609.1

2.8

546.0

10.0

6.6

258.0

127.0

548.0

104.0

1600.0

77.0

7.8

14.8

1890.0

935.0

159.0

19.8

33.5

56.0

1420.0

26.4

38.0

420.0

33.2

198.0

198.0

130.0

10.7

1554.0

25.0

71.0



other functional groups. The parent compounds of dialkylnitrosamines are secondary amines.

Nitrosamine formation can occur outside or inside

the body; the principal precursors are the various amines

and amides and nitrites (Mirvish, 1975, 1983). Thus, the

fundamental requirements are a secondary amino nitrogen

and nitrous acid. In reality, the nitrosating species is nitrous anhydride or, in the presence of thiocyanate or halides, nitrosylthiocyanate or nitrosylhalide. The precursors

to nitrosamine formation occur widely in both the environment and biological systems (Table 9.15 and 9.16). Hence,

low-level amounts of nitrosamine have been widely found

in many foods and other environments.

Nitrosamines can also be formed in vivo. In fact, the

conditions in the alimentary tract from the mouth to the



Copyright 2002 by Marcel Dekker. All Rights Reserved.



R1

R2



N N



O



Dialkylnitrosamine



R1



N N O

C NH

NH R2



Acylalkylnitrosamine



R1



N N O

C O

R2



Nitrosoguanidine

Figure 9.11 General structures of three types of nitrosamines

found in foodstuffs.



rectum are quite conducive to nitrosamine formation.

First, oral bacteria may promote the reduction of nitrate to

nitrite, so that the total nitrite load is increased (Tannenbaum et al., 1974, 1976). Second, nitrosamine formation

can also be promoted in the mouth by oral bacteria. Third,

saliva can promote the formation of cyanamides from secondary amines as well as primary amines.

Both normal and hypoacidic conditions in the stomach also favor nitrosamine formation. The normal acidity

of the stomach is ideal for nitrosation, whereas hypoacidity may allow some microorganisms to promote nitrate reduction. The dual occurrence of nitrate reduction and

nitrosamine formation in human subjects with gastric hypoacidity who are gavaged with sodium nitrate and diphenylamine was reported by Sander and Schweinsberg

(1972).

The microflora in the small intestine promotes nitrification of ammonia and organic nitrogen compounds.

Five strains of Escherichia coli from the human gut have

been demonstrated to form nitrosamines from dimethylamine, diethylamine, piperidine, pyrrolidine, and other

amines in the presence of sodium nitrite at neutral pH.

In addition, strains of Bacteroides, Bifidobacterium,

Clostridium, and Enterococcus spp., which do not reduce

nitrate, have been shown to form nitrosamines on replacement of nitrate in the reaction medium with nitrite (Hawksworth and Hill, 1971).

The nitrosation reaction is dependent on physicochemical factors. One of the most effective inhibitors of



Table 9.15 Nitrosamine Precursors Endogenous or Formed (Derived) in Foodstuffs

Compound

Creatine, creatinine

Trimethylamine oxide

Trimethylamine

Dimethylamine

Diethylamine

Sarcosine

Choline, lecithin

Proline, hydroxyproline

Pyrrolidine

Piperidine

Methylguanidine

Citrulline

Carnitine

Dipropylamine

Dibutylamine



Nitrosamine formeda



Food

Meats, meat products, milk, vegetables

Fish

Fish

Fish, meats, meat products, cheese

Cheese

Meats, meat products, fish

Eggs, meats, meat products, soybean, corn

Meats, meat products, other foodstuffs

Meats, meat products, paprika

Meats, meat products, cheese, black pepper

Beef, fish

Meats, meat products, vegetables

Meats, meat products

Cheese

Cheese



NSA

DMN

DMN

DMN

DEN

NSA

DMN

Npro, Npyr

Npyr

Npip

MNC

NCit

DMN

DPN

DBN



a



NSA, nitrososarcosine; DMN, dimethylnitrosamine; DEN, diethylnitrosoamine; Npro, nitrosoproline; Npyr,

nitrosopyrrolidine; Npip, nitrosopiperidine; MNC, methylnitrosocyanamide; DPN, di-N-propylnitrosoamine;

DBN, di-N-butylnitrosoamine; Ncit, nitrosocitrulline.

Source: From Concon (1988).



tions of cooking and/or processing. It must be noted, however, that the methods used in nitrosamine analysis in food

may underestimate the level of these carcinogens. The reason is that, apart from the low recoveries inherent in the

methods, determinations using separation of individual nitrosamines automatically exclude many others that may be

present. Collectively, the latter may be quite significant.

Thus, the amount of total nitrosamines in a food may be

more pertinent in assessing the hazard from these carcinogens. Therefore, the methods of analysis should also focus

on the determination of total nitrosamines rather than just

each individual compound.



nitrosamine formation is ascorbic acid, which reacts with

nitrite readily to form nitric oxide and dehydroascorbic

acid (Mirvish et al., 1972). It thus competes for any nitrite

present and hence reduces the availability of this reactant

for nitrosamine formation. Other inhibitors of the nitrosation reactions include gallic acid, sodium sulfite, cysteine,

and tannins.

Examples of nitrosamine levels in foodstuffs are

summarized in Table 9.17. Cured meats (especially fried

bacon), followed by fish and cheese, represent the major

sources of nitrosamines in the diet. The formation of these

derived toxicants in foods is also dependent on the condi-



Table 9.16 Nitrosamine Precursors That Contaminate Foodstuffs

Compound

Atrazine

Benzthiazuram

Carbaryl

Fenuron

Ferbam

Morpholine

Propoxur

Simazine

Succinic acid 2,2′-dimethyl hydrazide

Thiram

Ziram

Source: From Concon (1988).



Copyright 2002 by Marcel Dekker. All Rights Reserved.



Chemical class



Nitrosamine derivative



Secondary amine

Carbamate

Carbamate

Carbamate

Amide

Secondary amine

Carbamate

Secondary amine

Amide

Amide

Amide



N-Nitrosoatrazine

N-Nitrosobenzthiazuram

N-Nitrosocarbaryl

Dimethylnitrosamine

Dimethylnitrosamine

N-Nitrosomorpholine

N-Nitrosopropoxur

N-Nitrososimazine

Dimethylnitrosamine

Dimethylnitrosamine

Dimethylnitrosamine



Table 9.17 Nitrosamine Levels in Foodstuffs

Food

Bacon, raw

Bacon, fried



Bacon, frying fat

drippings

Luncheon meat

Salami

Danish pork chop

Sausage

Sausage, mettwurs

Chinese

Fish

Sable, raw

Salmon, raw

Shad, raw

Sable, smoked

Salmon, smoked

Salmon, sable, and

shad, smoked and

nitrate/nitrite cured

Salted, marine fish

Fish sauce

Cheese

Baby foods

Shrimp, dried

NPyr

Shrimp sauce

Squid



Canned meats

Ham and other pork

products

Beef products

Wheat flour



Nitrosaminea



Level, ppb



DMN, DEN, NPyr

NPip

NPyr

DMN, NPyr

NPyr



0

1–40

10–108

11–38

2–30

10–108



DMN, DEN

DMN, DEN

DMN, DEN

DMN

NPyr, NPip

DMN



1–4

1–4

1–4

1–3

13–105

0–15



DMN

DMN

DMN

DMN

DMN

DMN



DMN

DMN, NPyr

DMN

DMN

DMN

0–37

DMN

NPyr

DMN

NPyr

DMN

DMN

DMN

DEN



4

0

0

4–9

0–5

4–26



50–300

0–2

1–4

1–3

2–10

0–10

0–10

2–8

0–7

1–3

0–5

1–2

0–10



a



DEN, diethylnitrosamine; DMN, dimethylnitrosamine; NPyr, nitrosopyrrolidine; NPip, nitrosopiperidine.

Source: Modified from Concon (1988).



The biological activity of N-nitroso compounds has

been studied extensively. The toxicity of nitrosamines was

first recognized in 1937 by Freund, who reported two

cases of accidental poisoning from inhalation of DMN. In

1956, Magee and Barnes reported that DMN was a potent

hepatocarcinogen in rats. This report marked the beginning of worldwide interest in N-nitroso compound carcinogenesis. Since then, a large number of N-nitroso



Copyright 2002 by Marcel Dekker. All Rights Reserved.



compounds have been tested for carcinogenicity. Among

the 400-odd N-nitroso compounds assayed thus far for

carcinogenicity, over 90% have yielded positive findings.

The most widely tested N-nitroso compound, N-nitrosodiethanolamine (NDEA), has been shown to be carcinogenic in 40 species (Lijinsky, 1987). There are important

distinctions between nitrosamines and nitrosamides: the

former must be activated to carcinogens by oxidative

enzymes (e.g., cytochrome P-450), whereas the latter are

direct-acting carcinogens. Nitrosamines often produce tumors at a site(s) distant from the point of application. Selected N-nitroso compounds that may be found in foods

and their corresponding target organs are listed in Table

9.18. A number of these compounds seem to have a broad

organotropicity in a single species. The organ specificity

depends on the chemical nature of the N-nitroso compound and may depend on the dose, route of administration, and animal species. Similarly, there are large

interspecies differences in the organs affected by the same

compound.

N-Nitroso compounds have the ability to induce

transplacental carcinogenesis. In rats, kidney tumors have

been induced transplacentally by DMN; similar tumors of

the lung and liver have been induced in mice (Concon,

1988). The effect observed in the offspring depends on the

time during gestation when the N-nitroso compound is administered. Generally, embryotoxic effects are observed

when the administration is on days 1–10, teratogenic effects on days 9–16, and carcinogenic effects from day 10

to delivery (Archer, 1982).

Several factors influence the carcinogenicity of Nnitroso compounds. Potentiating factors include hormones

and their levels, other carcinogens or toxicants, viral or

bacterial infections, metals, and nutritional factors. Synergism is often observed in the presence of mycotoxins. In

contrast, reducing agents such as ascorbic acid, cysteine,

and tannins diminish the carcinogenic potential of N-nitroso compounds.

No animal species tested thus far is resistant to

DMN or DEN, the two nitrosamines commonly found in

foodstuffs (Lijinsky and Taylor, 1977). Thus, the common

consensus is that humans cannot be expected to be resistant to the nitrosamines (Mirvish, 1977, 1983). These

compounds are particularly effective when exposure is

through the oral route, at small doses, and over a long period, conditions particularly relevant to humans. Furthermore, these compounds are systematically organotropic

and induce tumors in target tissues independently of the

route of administration. Biochemical studies with human

liver in vitro have produced evidence that nitrosamines are

metabolized and interact with nucleic acids (Montesano

and Magee, 1974). This finding suggests that human me-



Table 9.18 Target Tissues of Selected Nitrosamines Found in Foods

Nitrosamines



Target tissues



Dimethylnitrosamine



Liver



Diethylnitrosamine



Kidney

Lung

Nasal cavities

Liver



Di-N-propylnitrosamine



Di-N-butylnitrosamine



N-Nitrososarcosine

N-Nitrosopyrrolidine



N-Nitrosopiperidine



N-Nitrosomorpholine



N-Methyl-N-nitrosourea



Kidney

Lung

Nasal cavities

Esophagus

Forestomach

Larynx

Trachea

Bronchi

Liver

Esophagus

Tongue

Liver

Lung

Esophagus

Bladder

Forestomach

Trachea

Tongue

Esophagus

Liver

Lung

Nasal cavities

Trachea

Testes

Liver

Lung

Nasal cavities

Esophagus

Larynx

Trachea

Testis

Liver

Lung

Kidney, nasal cavities, ovaries,

esophagus

Trachea, larynx, bronchus

Central nervous system

Peripheral nervous system

Intestines

Kidney

Forestomach

Skin



Test species

Rat, mouse, European hamster, guinea pig, rabbit, rainbow trout, newt, mink, mastomys (Praomys natalensis), aquarium fish (Lebistes reticulates)

Rat, Syrian golden and European hamsters

Syrian golden hamster

Rat, rabbit

Rat, mouse, Syrian golden hamster, Chinese hamster,

guinea pig, rabbit, dog, pig, trout, grass parakeet,

monkey, Brachydanio rerio

Rat

Mouse, Syrian golden hamster

Rat, mouse, Syrian golden hamster, European hamster

Rat, mouse, Chinese hamster

Mouse, Chinese hamster

Syrian golden and European hamsters

Syrian golden and European hamsters

European hamster

Rat

Rat

Rat

Rat, mouse, guinea pig

Syrian golden and Chinese hamsters

Rat, mouse

Rat, mouse, Syrian golden and Chinese hamsters,

guinea pig

Mouse, Syrian golden hamster, guinea pig

Syrian golden hamster

Mouse

Rat

Rat

Mouse, Syrian golden hamster

Rat

Syrian golden hamster

Rat

Rat, mouse, monkey, Syrian golden hamster

Mouse, Syrian golden hamster

Rat

Rat, mouse

Rat, Syrian golden hamster

Rat, Syrian golden hamster

Mouse

Rat, mouse

Mouse

Rat

Syrian golden hamster

Rat, mouse, rabbit, dog

Rat, dog

Rat, Syrian golden hamster, rabbit

Rat, mouse

Rat, mouse

Rat, mouse, dog

(table continues)



Copyright 2002 by Marcel Dekker. All Rights Reserved.



Table 9.18 (continued)

Nitrosamines



Target tissues



Test species



N-Methyl-N-nitrosourea (continued)



N-Ethylnitrosourea



N-Methyl-N-nitrosourethane



N-Ethy-N-nitrosourethane

N-Methyl-N′-nitro-Nnitrosoguanidinea



Subcutaneous tissues

Glandular stomach, jaw, bladder,

uterus, vagina

Liver, lung, hematopoietic system

Pharynx, esophagus, trachea,

bronchus, oral cavity

Stomach, pancreas, ear duct

Central nervous system

Peripheral nervous system

Kidney

Hematopoietic system

Skin, intestines, ovary, uterus

Lung

Forestomach

Esophagus

Kidney, intestines, ovary

Pancreas, subcutaneous tissues

Forestomach, intestines

Glandular stomach



Syrian golden and European hamsters

Rat



Guinea pig

Rat, mouse

Rat, mouse

Rat, mouse

Rat, mouse

Rat

Rat, mouse

Rat, mouse, Syrian golden hamster

Rat, Syrian golden hamster

Rat

Rabbit

Rat

Rat, Syrian golden hamster



Forestomach

Stomach

Intestines

Skin

Subcutaneous tissues

Lung



Rat, mouse

Dog

Rat, mouse, Syrian golden hamster, dog

Mouse

Rat

Rabbit



Mouse

Syrian golden hamster



a



Not found in foods as such, but one of similar structures may be derived from naturally occurring guanidines, such as methylguanidines. The latter has

been detected in certain foods, e.g., meats, and is probably derived from creatinine.

Source: From Magee et al. (1976) and Concon (1988).



tabolism of nitrosamines may also produce proximate carcinogens similar to those seen in almost every animal

study. When the amounts of total nitrosamines in food,

water, and other sources are added to those formed

throughout the GI tract, the total nitrosamine load of modern populations would be considerable. Viewed from this

perspective, each carcinogen, even in trace amounts, assumes considerable significance.



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10

Toxicants and Antinutrients in Plant Foods



10.1 INTRODUCTION

Animal life on earth is primarily sustained by green plants

with photosynthetic capacity to convert carbon dioxide

and water into basic macronutrients, i.e., carbohydrates,

protein, and fat. In fact, on a global basis over 65% of food

protein and over 80% of food energy is supplied by plants,

and in terms of gross tonnage, plant products directly contribute about 82% of the total world food harvest (Deshpande, 1992). The photosynthetic process of plants,

however, is not confined to the production of basic macronutrients. It also includes the biosynthesis of a variety of

organic compounds. Traditionally, the processes generating plant compounds have been categorized as either primary or secondary metabolism. Research in plant

physiological characteristics since the 1980s, however, has

clearly shown that such a distinction between primary and

secondary metabolites is at best arbitrary. The once-popular view of a secondary metabolite as one that does not

play an indispensable role in plant life at the cellular level

is no longer valid. It is now widely recognized that plants

do not haphazardly produce a large number of chemical

compounds; rather, each metabolite is biosynthesized for a

definite purpose, and all products are interrelated according to a complex process that conserves energy and scarce

organic nutrients. Antinutritional or toxic compounds that

occur naturally in many plants can be considered secondary metabolites. Most secondary metabolites are now

known to be essential to plant life; many of them provide a

defense mechanism against bacterial, viral, and fungal attack analogous to the immune system of animals. Many

are also produced in large amounts as a direct result of

some adverse environmental condition.



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The science of nutrition is not just the science of

food and its relation to life and health. We must be concerned not only with what is required in a diet, but also

with what is actually consumed. We should be as concerned with the problems caused by an excess of a food

component as we are with the problems caused by the deficiency of an essential nutrient. In considering the physiological effects of food components, it should be noted that

these effects are always related to the level of their intake.

A useful concept is that for every food component, there

are three ranges of intake: one associated with physiological inertness, a second with physiological function or benefit, and the third with potential hazard. Although it is

arguable at what level a nutrient is physiologically inert,

there is no doubt that certain levels of intake are insufficient to maintain normal body functions. The level of nutrient requirement associated with normal health, i.e., with

physiological function and benefit, is well understood for

most nutrients. We also know with certainty that the concept of “zero risk” cannot be considered valid anymore.

One would probably not consider our food sources of energy as ever constituting a potential hazard, but there is a

consensus among nutritionists that currently the most important problem of malnutrition in the United States and in

many developed Western countries is obesity. This example clearly shows that the margin between the level of caloric intake consistent with normal physiological function

and benefit and that creating a potential hazard is narrow.

Thus, for every nutrient there is also a level of intake that

constitutes a potential hazard. The margin between the

level of function and the level of hazard varies considerably with each and must be determined in each case.



That the basic components (i.e., carbohydrates, protein, and fat) of human diet under normal conditions do

not exert any adverse effects is taken for granted. Natural

foods in everyday diets also contain a great number of

other potentially toxic substances. However, this does not

necessarily mean that the food is hazardous to human beings. A substance that is considered toxic has a more or

less pronounced capacity to induce deleterious effects on

the organism when tested by itself in certain doses. However, this capacity is not always realized under usual dietary conditions. Humans consume a multitude of toxic

substances in their normal diet every day without showing

any signs of intoxification. This is probably because natural toxicants usually exert their effects only when they are

consumed under special conditions or when there are other

potentiating substances available. In addition, the concentration of toxicants occurring naturally in the food is often

so low that the item must be consumed in usually unrealistic amounts every day for an extended period to allow intoxification to occur. Furthermore, it should be noted that

humans can handle small amounts of various toxicants.

Similarly, most toxic effects of various chemicals that are

potentially hazardous do not have an additive effect. There

also seem to occur antagonistic reactions that make some

ingredients interfere with and reduce the toxic effects of

other components. All these facts prompt Liener (1989) to

prefer the term antinutritional to toxicants to denote such

hazardous food components, since the former is not very

restrictive and may be liberally interpreted to mean nothing more nor less than an adverse physiological response

produced in humans.

Antinutritional and toxic factors that commonly are

present in the human food chain can be classified into two

broad groups: those that occur naturally (natural or inherent) as a result of intrinsic metabolism of the animal or

plant and those that are formed (acquired) as a result of

microbial growth, accumulated from the environment, or

unintentionally introduced during handling, processing,

and storage.

In this chapter, only the naturally occurring antinutritional and toxic factors of important plant food sources

are discussed. Because of the obvious limitations of space,

coupled with the fact that several excellent reviews and

books on various aspects of naturally occurring food toxicants are available, no attempt has been made to cover all

of the natural toxic substances known to be present in

plant materials. Therefore, only certain evolutionary,

structural, biochemical, technological, nutritional, and toxicological aspects of most important antinutritional factors

that occur in the human food chain are discussed.



Copyright 2002 by Marcel Dekker. All Rights Reserved.



10.2 PROTEINASE (PROTEASE) INHIBITORS

Protein inhibitors of proteinases (or protease inhibitors)

are ubiquitous. They are present in multiple forms in numerous tissues of animals and plants as well as in microorganisms. Their gross physiological function is the

prevention of undesirable proteolysis, but detailed physiological functions have been only rarely elucidated. These

inhibitors have attracted the attention of scientists in many

disciplines. Nutritionists are concerned with their possible

adverse effects on the nutritive value of plant proteins. The

inhibitor-enzyme reactions have provided a simple model

system for protein scientists to study protein-protein interactions as well as enzyme mechanisms. Because of their

unique pharmacological properties, these inhibitors hold

considerable promise in clinical applications in the field of

medicine.

Proteinase inhibitors were initially classified on the

basis of protease inhibited, such as trypsin inhibitor. They

can also be grouped on the basis of the class of protease inhibitor (Table 10.1). More recently, classification has also

been based on similarities in the primary amino acid sequence and/or the disulfide bond location (Whitaker, 1997).

Although the inhibition of proteolytic enzymes by

extracts from animal tissues was first demonstrated in the

19th century (Fredericq, 1878), it was only in the 1930s

that their presence in plant material was recognized. Read

and Haas (1938) reported that an aqueous extract of soybean flour inhibited the ability of trypsin to liquefy gelatin.

This report was soon followed by the first isolation of a



Table 10.1 Families of Plant Proteinase Inhibitors

Serine protease inhibitors (serfins)

Bowman-Birk (trypsin/chymotrypsin)a

Kunitz (trypsin, others)a

Potato I (chymotrypsin, trypsin)a

Potato II (trypsin, chymotrypsin)a

Cucurbit (trypsin)

Cereal superfamily (amylase, trypsin)b

Ragi I-2 family (amylase, protease)b

Maize 22 kDa/thaumatin/PR (amylase, trypsin)b

Cysteine protease inhibitors (cystatins and stefins)

Cystatin superfamily

Cystatin family

Stefin family

Fitocystatin family

Metalloprotease inhibitors

Carboxypeptidase

a



Second enzyme listed binds less tightly.

Double-headed inhibitor.



b



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