Phenolic Compounds in Food and Their Effects on ... - ACS Publications

Chapter 28

Tocopherol Natural Phenolic Inhibitor of Nitrosation William J. Mergens

Downloaded by GEORGE MASON UNIV on March 8, 2016 | http://pubs.acs.org Publication Date: October 1, 1992 | doi: 10.1021/bk-1992-0507.ch028

Roche Vitamins and Fine Chemicals, Vitamins Research and Development Department, Hoffmann-La Roche, Inc., Nutley, NJ 07110

N-Nitroso compounds, once formed and present in vivo, generally do not easily or readily revert back to precursors. Instead, many of them are metabolized to, or are converted to alkylating agents as terminal or proximate carcinogens. One effective method of controlling the formation of these compounds in foods and in vivo has been to use agents that inhibit their formation from precursors (e.g., amines, amides, plus nitrosating agents). The formation of N-nitroso compounds is directly dependent on the source of nitrosating agent which recent surveys indicate are numerous, ranging from atmospheric exposure to oxides of nitrogen through bacterial reduction of nitrate in the gastrointestinal tract. The generally accepted mechanism of blocking these reactions is one of competitive kinetics between the susceptible amine (amide) and potential blocking agent for the nitrosating specie. The ultimate effectiveness of any given blocking agent then depends on being able to deliver it in sufficient concentration to the site where nitrosation takes place. α-Tocopherol and its water soluble antioxidant counterpart, ascorbic acid, have been found to be extremely efficient inhibitors of nitrosation reactions in food products as well as in vivo in man. The mechanisms which apply to these systems will be presented along with the results of many recently reported investigations.

The concern about human exposure to N-nitroso compounds relates to their carcinogenicity. It is now clearly established that the N-nitroso compounds, as a class, are highly carcinogenic and many of the tested ones have been found to produce tumors in a large variety of organs and in many animal species including nonhuman primates. While the presence of N-nitroso compounds in the diet or production through endogenous synthesis has not been positively linked with cancer in man, substantial evidence has been assembled suggesting a possible health risk from exposure to these compounds.

0097-6156/92A)507-0350$06.00/0 © 1992 American Chemical Society

Huang et al.; Phenolic Compounds in Food and Their Effects on Health II ACS Symposium Series; American Chemical Society: Washington, DC, 1992.

28.

MERGENS

Tocopherol: Natural Phenolic Inhibitor of Nitrosation

351


If iV-nitroso compounds are toxicologically significant, then it is important to prevent their formation through elimination of one or both reactants or through prevention of the reactions that lead to their formation. Many compounds have been studied in the latter role, but two in particular, atocopherol and ascorbic acid, have the unique properties of being both essential nutrients and effective blocking agents of nitrosation reactions. Before presenting the results of the many studies involving inhibition of nitrosamine formation in food and animal systems, it may be beneficial to first review some of the key aspects of N nitroso compound formation, the level of man's exposure to nitrosating agents, plus some remarks regarding the mechanism of nitrosamine inhibition by α-tocopherol, related tocopherols and ascorbic acid. Formation of JV-Nitroso Compounds Essentially all amines and amides must be considered capable of forming N-nitroso compounds to a lesser or greater degree. Excellent reviews of this subject have been published (i,2). Generally, the susceptibility of any particular compound towards the formation of a nitroso derivative reflects the mutual optimization of two conditions: (a) the presence of a potential nitrosating agent and its ultimate activation and (b) the chemical state in which the amine or amide exists in the reaction milieu. For simple basic amines ( p K > 5), the rate of nitrosation in water is maximum at p H 3.0-3.4, which is near the p K of nitrous acid, HNO2. For a given amine, the nitrosation rate decreases as the p H increases above 3.4 because the concentration of nitrous acid decreases. When the pH decreases below 3.0, the rate again decreases because the concentration of unprotonated amine decreases. At a given pH, the rate of nitrosation increases as the basicity of the amine decreases because of a higher relative concentration of unprotonated amine present In moderately acidic aqueous solutions, the nitrosating agent is essentially nitrous anhydride, N2O3, formed from 2 mois of nitrous acid (HONO, p K 3.14 at 25°C), which is in turn formed from acidification of nitrite ions. At lower pH, the more reactive nitrosating agent, Η 2 θ Ν Ο is formed. Of significant interest is the catalytic effect on nitrosation that results when certain anions combine with nitrite to form alternate nitrosating agents (3-6). Thiocyanate is the most active of such catalysts, followed by the halides. This catalyzed nitrosation of amines competes most favorably with the N2O3 mechanism under conditions of high acidity and low nitrite ion concentration. The latter occurs because the reaction becomes first order in nitrite ion concentration through the formation of an N O X nitrosating intermediate (X = SCN or halide). Secondary amines such as diethanolamine will react with a nitrosating agent according to the following reaction: a

a

a

+

R N H + HONO 2

- R NÔ + H 0 2

2

(1)

The formation of nitrosamines from secondary amines as described above is usually the most rapid of all the amine nitrosations. In fact, it should be observed that primary amines must be alkylated and tertiary amines dealkylated prior to formation of the ultimate nitroso compound through the nitrosation of the correspondingly generated secondary amine. Hence, the reaction described above is common to all the mechanisms discussed below.


352

PHENOLIC COMPOUNDS IN FOOD AND THEIR EFFECTS ON HEALTH II

Primary amines can react with a nitrosating agent leading to the formation of a nitrosamine by the following reaction mechanism (2): RNH I

2


RN-N Π

+ HONO

+

+ RNH

"

H 2

° » RN=N-OH — - — - RN=N Π

-N 2

2

R NH 2

A

HONO

+

2

+

+ H 0

• R N-NO + H 0 2

(2)

2

+

(3)

3

The initial attack of a nitrosating agent on a primary amine (I) leads to the formation of an alkyl diazonium ion (II) (Equation 2). The decomposition of this diazonium ion will normally lead to the formation of the corresponding alcohols and alkenes. These compounds are the classical deamination products. Under the proper conditions as demonstrated by Equation 3, however, this diazonium ion can react further with another mol of primary amine to form a secondary amine. This secondary amine can be nitrosated subsequently to form a nitrosamine. While the nitrosamine product is not favored for the reaction of nitrosating agent with a primary amine, the presence of certain catalysts like thiocyanate, formaldehyde, or other aldehydes can lead to at least some minor conversion of a primary amine to a secondary nitroso compound (7). Tertiary amines can be converted to secondary nitrosamines, but the reaction occurs to a significant extent only at elevated temperatures in weakly acidic media. The rate of reaction is dependent on the p K of the amine. At 25°C and p H 3.4, the nitrosation of tertiary amines is about 10,000 times slower than that of related secondary amines. The following mechanism has been proposed for nitrosative dealkylation of tertiary amines and subsequent nitrosamine formation (7-9). a

, R N - C H R + 2 HONO ^ 2

2

H I

R N + - C H R + H 0 + ONO" 2

2

NO

J-HNO H

R N—CHR 2

R N H + HONO 2

2

° » RCHO + R N H 2

• R N-NO + H 0 2

2

+ 2

(4) (1)

Quaternary amines and amine oxides also appear to react slowly with nitrite in acidic media to form nitrosamines (10). Compounds of this type are very similar to tertiary amines in their reactivity toward nitrosating agents. They also require a dealkylation step prior to further reaction to form a nitroso derivative. The chemistry of N-alkylamide, as well as the related iV-alkylurea and Nalkylcarbamate nitrosation, has not been studied as thoroughly as that of nitrosamines, mainly because suitable analytical methods have not been developed, particularly for their determination in complex systems like foods and body fluids.


28. MERGENS


353

The nitrosation of a typical amide is shown in Equation 5. In these reactions, the nitrosating agent is believed to be nitrous acid ion, H 2 0 N O (3,7,11). +

RNHCOR + H O N O 2

+

• RN—COR + H 0

+

3

(5)


NO Like amides, many ureas and carbamates occur naturally or as drugs and some readily produce nitrosoureas or nitroso-carbamates. N - A l k y l ureas and carbamates are rapidly nitrosated at pH 1 - 2. The reaction rate increases about ten times for each p H unit drop from 3 to 1 and does not show a p H maximum as has been found for the formation of nitrosamines. N-Nitroso compounds also can be rapidly obtained through the reaction of the parent compound with N2O3, or N2O4, in organic solvents (12). The reactivity of amines and amides in a lipophilic environment, such as in a cosmetic preparation, is significantly greater than in the aqueous systems described above. Once the conditions are set for a nitrosating agent and susceptible amine, for example, to enter a lipid or nonpolar phase, the reaction is generally extremely rapid. Free amines readily dissolve in aprotic solvent systems as the unprotonated base, yielding a high proportion (if not all) of the more reactive form of the molecule. The nitrosating agents N2O3, and N2O4, are gases with appreciable solubility in lipid, nonpolar solvents (13). This is in marked contrast to the poor lipid solubility of nitrite ion. Indeed, an aprotic solvent will be the first choice of an organic chemist for the nitrosation of amines by N2O3 or N2O4. It is apparent, then, that the formation of nitrosamines and nitrosamides from the corresponding parent amine or amide can be demonstrated for all classes of these compounds. Generally, however, it is still the secondary amine or amide that is the most facile reactant. The other classes require either a preliminary alkylation or dealkylation. Dietary Sources of Nitrate and Nitrite For most Americans, food is the major source of exogenous exposure to NO3" and NO2'. NO3" occurs in many foods, but estimates indicate that the majority of American dietary NO3" comes from vegetables (87-97%), with a small portion coming from cured meats (2%) (Table I). Breads, dairy products and fruit and fruit juices contribute minor amounts (11%) (14). NO2" consumption was estimated to be 0.002 and 0.004 mmol/person/day for average and high cured meat diets, respectively (Table II). Cured meats contributed from 39 to 71% of daily NO2" intake, with the balance coming from baked goods (15 to 34%), vegetables (7 to 16%), and fresh meats (3.5 to 7.7%). These estimates were based on published per capita consumption data and literature values of the NO3" and NO2" content of foods (14). Endogenous Sources of Nitrate and Nitrite Estimates of the endogenous levels of NO3' and NO2" have been derived from studies on the pharmacology and metabolism of NO3". These works have been reviewed (75-77). The human studies of Wagner et ai. (18), confirmed by Leaf etal. (19), have clearly indicated that NO3" is synthesized through a de novo pathway in


354


Table I. Dietary Sources of Nitrate (United States) Source


Vegetables

)imol/person/day

% of total

1050 (4200)»

86 (97)" 6

Fruits, juices

69

Water

32 (2840)

Cured meat

26

2

Baked goods, cereals

26

2

_13_

_1

1216

100

Others Total a b

b

3 (68)

b

Vegetarian diet Nitrate-rich water SOURCE: Reprinted with permission from ref. 14. Copyright 1981 National Academy of Science. Table II. Dietary Sources of Nitrite (United States) Source

|imol/persoii/day

% of total

Cured meat

6.5 (26)»

39 (71)

Baked goods, cereals

5.7

34

Vegetables

2.6 (10)

Fresh meats

1.3

8

JLZ 16.8

4 100

Others Total

b

a

15 (62)

b

a

High cured meat diet Vegetarian diet SOURCE: Reprinted with permission from ref. 14. Copyright 1981 National Academy of Science.


28.

MERGENS


355


humans in amounts of approximately 1.2 mmol/day. Thus, the quantity of endogenously formed NO3" is a significant portion of overall nitrate exposure, perhaps nearly equal in level to the estimates of ingested nitrate. Tannenbaum et ai. (17) have shown that NO3" is concentrated and secreted in human saliva where a portion is reduced by oral microflora to NO2'. The NO3" and NO2" content of human saliva peaks approximately one hour after ingestion of NO3", although there appears to be wide inter-individual variability. Assuming a 5% reduction of NO3" to salivary NO2", Hotchkiss (20) calculated that NO 2' exposure through this path would be 0.12 mmol/person/day compared to 0.02 mmol/person/day through the diet. Preventing 7V-Nitroso Compound Formation with α-Tocopherol Ascorbic Acid

and

If iV-nitroso compounds are toxicologically significant, then it is important to prevent their formation through elimination of one or both of the reactants or through prevention of the reactions leading to their formation. The formation of N-nitroso compounds can be reduced, minimized or even completely prevented by the presence of blocking agents when nitrosation potential exists. Blocking agents are essentially substances capable of rapidly reducing the nitrosating agent to the non-nitrosating nitric oxide (NO). They act as competitive substrates for the nitrosating species in the specific system (Figure 1). Both the absolute and relative concentrations of nitrosating agent, blocking agent and amine or amide substrate will determine the degree of "blocking" that will occur. Many types of compounds can function as blocking agents, but two compounds in particular, ascorbic acid and α-tocopherol, have the unique properties of being both essential nutrients and effective blocking agents for nitrosation reactions. Together, the pair can act to inhibit nitrosation in multiphase systems, a desirable characteristic for foods and for the lumen of the gastrointestinal tract. Ascorbic acid has been shown to be an excellent blocking agent against nitrosation in aqueous systems, particularly under weakly acidic conditions. The ascorbic acid competes for the nitrosating agent, forming dehydroascorbic acid and the non-nitrosating nitric oxide (NO) as indicated in Figure 2. The chemistry of atocopherol inhibition in lipid systems and possibly at the interface of micellar emulsions is quite analogous to that of ascorbic acid (Figure 2). The mechanism and kinetics of these reactions have been reported by a number of investigators (21-25). The effectiveness of α-tocopherol and ascorbic acid as inhibitors of nitrosation reactions can vary with the system under study. Simply, the interconversions that are possible among nitrogen oxide species (Figure 3) indicate that nitrosating agents can occur not only in an aqueous environment, but in the gaseous and lipid states as well. Hence, the combined use of both blocking agents is best suited for all but the simplest systems, where it can be clearly defined that nitrosations are taking place solely in the aqueous or lipophilic phase. Isomers and Derivatives of Vitamin Ε as Inhibitors. Not all forms of "vitamin E " are equally effective as inhibitors of nitrosation. Of the naturally occurring forms of vitamin E, α-tocopherol has the highest biological activity as the vitamin. There are, however, other tocopherol and tocotrienols (tocopherols with unsaturated side chains) in foods of plant origin. The structures of the α-, β-, γ- and δ- tocopherols are shown in Figure 4.


356


I. Amine Nitrosation: Amine + nitrosating agent •

"Nitrosamine

Π. Principle of Inhibition: Ascorbic Acid


Amine + nitrosating agentXî2^"—" (Emulsion)

α-Tocopherol

Non-nitrosating Product (NO) Nitrosamine • Non-nitrosating Product (NO)

Figure 1. Principle of blocking nitrosation reactions using water soluble and fat soluble inhibitors. (Reproduced with permission from ref. 49. Copyright 1981 Cold Spring Harbor.)

+ NO + H 0 2

+ 2 NO Figure 2. Reactions of ascorbic acid (top) and α-tocopherol (bottom) with nitrite. The actual nitrosating agent varies with pH, reaction conditions, and/or the presence of catalysts. (Reproduced with permission from ref. 50. Copyright 1980 Plenum Publishing.)



χ Ο

Όψ** •

I

CO

Β

+1

+2

«31

+41

+5

+e"(e.g. +t ascorbic N O acid) NO

2

N 0

-o\ +e" (e.g. SOôrits . acid and salts)

+

NO

G a s state

(e.g.al+e" tocopherol) τ NO NO

r

N o n - p o l a r solvent

Figure 3. Some transformations of nitrogen oxide compounds among the gaseous, aqueous, and nonpolar (lipid) phase. The shaded area indicates potential nitrosating agents. Adapted from ref. 49. Copyright 1981 Cold Spring Harbor Laboratory.

|+e- (e.g. ascorbic NO acid)

Aqueous solution



358


From the viewpoint of blocking of nitrosation reactions, these structures are very interesting. Although all of the tocopherols can act as reactive phenols to form quinones in reducing a nitrosating agent to NO, only the α-tocopherol, being fully substituted on the aromatic ring, can completely avoid forming a C-nitroso derivative. The latter can potentially transnitrosate or even catalyze the nitrosation of a nitrosatable amine or amide (26). Kamm et al. (27) have shown that α-tocopherol is a very effective blocking agent against nitrosation reactions, whereas γ-tocopherol is somewhat less effective both in vitro and in vivo. This superiority of α-tocopherol over γ-tocopherol as a nitrosation inhibitor is in marked contrast to the superiority of γ- over α-tocopherol as an in vitro or food-type antioxidant. The difference in effectiveness of blocking nitrosation reactions, as compared with lipid oxidation, apparently is due to the lack of complete substitution on the aromatic ring in the γ isomer. In addition to the difference in nitrosation blocking ability that occurs among tocopherol isomers, it should be noted that there are some forms of vitamin Ε that are not inhibitors. These are the ester deriviatives of tocopherol (Figure 5). The esters do not appear naturally in foods, but were developed as means of stabilizing the molecule against oxidation of the phenol group during processing and storage, particularly in pharmaceutical dosage forms such as tablets and capsules. These esters per se are inactive as antioxidants or N-nitroso blocking agents. They are hydrolyzed slowly in the acidic conditions of the stomach. Lower in the gastrointestinal tract hydrolysis is much more efficient. The acetate ester may be partly absorbed unhydrolyzed and is only very slowly hydrolyzed on parenteral administration (28). With this in mind, it is always better to utilize the unesterified atocopherol, d (natural), or dl (synthetic) as the preferred nitrosation inhibitor. This may be especially true of in vivo studies seeking activity of α-tocopherol in the upper portion of the gastrointestinal tract (stomach, duodenum, etc.) where little or no free active tocopherol can be expected to be formed when tocopheryl acetate or succinate is administered. Inhibition of Nitrosation in Foods Of the various foods monitored for nitrosamine content, cured meats (primarily bacon) are probably the most important, principally because these products are the main known source of nitrite in the Western diet. The two nitrosamines usually detected in such foods are N-nitrosopyrrolidine (NPYR) and N-nitrosodimethylamine (NDMA). In cured meats other than bacon, only extremely low concentrations (

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