Introduction to Bacon & the Art of Living

The story of bacon is set in the late 1800s and early 1900s when most of the important developments in bacon took place. The plotline takes place in the 2000s with each character referring to a real person and actual events. The theme is a kind of “steampunk” where modern mannerisms, speech, clothes and practices are superimposed on a historical setting.  Modern people interact with old historical figures with all the historical and cultural bias that goes with this.

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Finally - Nitrosamines
October 1990
By Eben van Tonder
(Latest review: 25 December 2022) 

Introduction

When I began this project which I called the EarthwormExpress as a kind of daily newspaper, an earthworm would write about the world he lives in, nothing glorious, but interesting. The first article I did was Concerning the direct addition of nitrite to curing brine, tipping my hand at what would be the main subject of my life’s work. The second article I did was Concerning Chemical Synthesis and Food Additives. Here I told the story of the discovery that spawned the colourants and chemical food additives industry and chemical synthesis. It is, of course, the story of Liebig, the father of agricultural Chemistry and the one responsible for formalising much of the science we know today, his student from Giessen, Hoffmann, and the young William Perkin who became the first to produce a synthetic dye when he was studying under Hoffman in London. Curing was never far from my thoughts and almost 15 years later, I am still dealing with it. As I am about to tell the story of amines that will lead me into the nitrosation of amines or the formation of nitrosamines, the subject is the same as when I first told the story.

The subject is broad, but solutions are available to eliminate nitrosamines from cured meats. At the same time, nitrate, nitrite and nitric oxide remain the key sources, intermediaries and curing reagents.

The four final chapters form a unit:

• Chapter 16.05: Finally – Nitrosamines
• Chapter 16.06: Finally – Nitrite is Physiologically Vital
• Chapter 16.07: Finally – The Human Nitrogen Cycle – Basis for Nitrite’s Physiological Value
• Chapter 16.08: Finally – Bacon, the Superfood

Hoffman and the discovery of the amines

August Wilhelm von Hoffmann studied under Liebig in Giessen. It was at this time when organic and physiological chemistry emerged from plant and animal chemistry. A pivotal moment for the career of the young Hoffmann was when the owner of a tar distillery in Offenbach, who was himself also a former student of Liebig, sent a sample of the material to Giessen for analysis. The project was assigned to Hofmann.

Hoffman found credibility in “the ‘substitution theory,’ put forward and defended by Laurent and Dumas. The substitution theory made it possible for the first time to regard a molecule as a single entity, in which individual atoms might be replaced by other elements without fundamentally changing its chemical nature.” (Meinel, 1992) This was a position held by some of Liebig’s most bitter adversaries and it came at a time when Liebig was just recovering from a crisis brought about by difficulties presented by the chemistry of nitrogen metabolism in animals.” (Meinel, 1992)

The vilification of nitrite in meat curing stems from a lack of understanding of the importance of nitrogen chemistry in human physiology and if the work of Liebig was taken to its natural conclusion, it should have been possible to predict the physiological relevance of the reactive nitrogen species of nitrate, nitrite and nitric oxide in the body of mammals. Liebig struggled for a decade with the explanation for the phenomenon of nitrogen and its relationship to nutrition and human physiology.

Prof. Christoph Meinel presents the two options that faced the trailblazing researchers in the now-formalised science of organic chemistry in his article on the life of Hoffmann which I use as the main source for this section. “If chemistry were to satisfy the demands of the neo-humanist interpretation of science then it must demonstrate its powers in the context of a theoretical interpretation of natural phenomena. Either the discipline must succeed in breaking new ground and convincingly translate its insights into practical results, thereby displaying to the world the utility it claimed to possess, or else the demand for a scientific interpretation of nature must be reduced to “hard” numbers and facts, with studious disregard for any speculative elements.”

Hoffman without apology chose the second which became a hallmark of his work. Meinel writes that “Hofmann avoided becoming involved in controversies surrounding the fundamental theories of chemistry. His papers speak instead the dispassionate language of “chemical facts.”

He was offered the directorship of the Royal College of Chemistry in London. Here, in London, “Hofmann succeeded in developing a characteristic research style. One of his earliest projects, executed in Giessen but presented before the Chemical Society in April 1845, was introduced with the remarkable assertion that a new direction had become apparent in organic chemical research. Whereas in the past one had always operated in a purely analytical mode, and rarely “with the goal of preparing particular compounds postulated through prior speculation,” now the groundwork had been laid to conduct targeted “synthetic experiments” for the artificial preparation of organic compounds. “If a chemist were to succeed in transforming naphthalene in a simple way into quinine, we would quite properly honour him as a benefactor of humanity. Such a transformation has not yet been accomplished, but that alone does not imply that it is impossible,” Hoffman wrote.

“The concept of synthesis is a key to Hofmann’s way of thinking. If the analytical phase of organic chemistry began with Liebig, in Wohler’s synthesis of urea Hofmann saw the threshold to the next higher step: an “era of synthetic chemistry.” Hofmann’s research program was eminently product-oriented. His positivistic, theory-abstaining stance corresponded to the spirit of the new age itself, the spirit of goods and the marketplace. Hofmann thought strictly in terms of classes of substances, which were to be investigated systematically and thoroughly once a pilot study had revealed some synthetic access. Analogy was his leading heuristic principle; his method was to systematically chart possible derivatives, toward which targeted syntheses were then directed.”

“There is no need to explain to chemists that Hofmann could not have selected a better starting point for such an endeavour than aniline. This particular venture led to a series of ten “Contributions to an Understanding of the Volatile Organic Bases.” The goal was to investigate the “remarkable analogy” between aniline and its derivatives, on the one hand, and ammonia, on the other. Reactions with the halogens and alkyl halides were especially interesting. It became clear that the premise of the radical theory, according to which performed ammonia must constitute one member of a “pair” in such compounds, was no longer tenable. In fact, the alkylated anilines could better be viewed as compounds in which the various hydrogen atoms of ammonia were successively replaced by organic residues. Application of the principle of homology permitted the number of possible combinations to be increased almost without limit.” “The basic structure of ammonia permits one to obtain in this way a complete range of homologous substitution products, which Hofmann later designated as primary, secondary, and tertiary amines.”

So, what is an amine? Amines are compounds and functional groups with a nitrogen atom and a lone pair. Amines are formally derivatives of ammonia (NH₃). The reader with no background in organic chemistry will be able to spot the nitrogen in the three structures below.

What is N-nitrosamines and when did it become an issue?

If you combine nitroso with amines, you get nitrosamines or as they are more formally called, N-Nitrosamines. The “n” in “n-nitrosamines” refers to the position of the nitrogen atom in the molecule. The nitrogen atom in an “n-nitrosamine” is bonded to the oxygen atom and proton on the same side of the molecule, so it is classified as an “n” nitrosated species.

Generally speaking, in organic chemistry, “s” and “n” refer to the positions of the nitrogen atom in an organic compound. “s” stands for “syndiotactic” and refers to nitrogen atoms that are positioned on alternating sides of a molecule. “n” stands for “isotactic” and refers to nitrogen atoms that are positioned on the same side of a molecule.

Nitrosated species are compounds that contain a nitrogen atom that is bonded to an oxygen atom and a proton. These compounds can be classified as either “s” or “n” based on the position of the nitrogen atom relative to the other atoms in the molecule.

For example, if a compound contains an “s” nitrosated species, the nitrogen atom would be bonded to an oxygen atom and a proton on alternating sides of the molecule. As stated above, if a compound contains an “n” nitrosated species, the nitrogen atom would be bonded to an oxygen atom and a proton on the same side of the molecule.

The distinction between “s” and “n” nitrosated species is important because it can affect the properties and reactivity of the compound. For example, “s” nitrosated species may be more stable and less reactive than “n” nitrosated species, due to the geometry of the molecule.

N-nitrosamines are known to be carcinogenic and have been identified as a potential health hazard in a variety of products, including tobacco products, rubber products, and certain foods and beverages. They are formed through a process called nitrosation, which occurs when nitrogen-containing compounds react with compounds containing an unsaturated double bond, such as fatty acids or amino acids.

Nitrosamines is then a group of organic compounds with the chemical structure R₂N−N=O, where R is usually an alkyl group. An alkyl group, very simply stated, refers to hydrogen and carbon atoms arranged in a tree structure in which all the carbon-carbon bonds are single. The nitroso group (NO⁺) binds to a deprotonated amine.

Nitroso compounds refer to non-organic compounds containing the NO group. This immediately will get the readers’ attention because we know that it is NO (nitric oxide) which is responsible for the pinkish/ reddish colour in cured meat. The NO group in nitroso compounds for example directly binds to the metal via the nitrogen atom (N), giving a metal–NO fragment or moiety. A nonmetal example is the common reagent nitrosyl chloride (Cl−N=O). The most stable nitrosamines are formed from secondary amines. (Rostkowska, 1998)

They are classified as carcinogens by the International Agency for Research on Cancer (IARC), an intergovernmental agency forming part of the World Health Organization of the United Nations. and US Environmental Protection Agency (EPA).

Up to the 1950s, the meat industry and governments around the world managed any possible harmful effects of nitrite in meat curing by limiting the dosage level of nitrites. It was then assumed that any possible negative effects would be dose-dependent and related to toxicity.  

Nitrite itself had a bad reputation long before the N-Nitrosamine controversy erupted. Butler (2008) says that initially, nitrite was shown to have great potential in the medical and pharmaceutical environment during the mid and late 1800s. Law (1882) suggested very large doses (20 grains or 1.3 g) of sodium nitrite to treat epilepsy. When other physicians tried it, they found considerable consequences in large dosages of inorganic nitrite which was confirmed by Ringer and Murrell (1883) who concluded that Law had been using an impure sample of sodium nitrite that was largely sodium nitrate. Butler (2008) writes that “they attempted to establish a safe dose, but the reputation of sodium nitrite had suffered, and because of the success of GTN, nitrite disappeared from widespread use.” Despite the early promise, it was shunned by the scientific community. This was already in line with the experience of farmers and the rural world where nitrate and nitrite levels were reported in drinking water from a very early to indicate “toxicity” of the water and to prevent human disease and loss of livestock.

Notices like the one below appeared in most city newspapers in the USA and England towards the latter part of 1800.

The case in the 1940s involving methemoglobinemia did not help the case. Very high concentrations of nitrate in drinking water may cause methemoglobinemia, particularly in infants (blue baby syndrome). Nitrate is distinct from nitrite, but also close enough for it to be lumped in the general negative light. “In the 1940s, Comly first reported cases of cyanotic infants who received formula prepared with well water containing a high nitrate content. Based on the subsequent analyses of the infantile cases of methemoglobinemia, the US Environmental Protection Agency (EPA) set a Maximum Contaminant Level (MCL) for nitrate of 44 mg/L (equal to 10 mg/L nitrogen in nitrate). However, it is now thought that methemoglobinemia per se was not caused by nitrate itself, but by faecal bacteria that infected infants and produced NO in their gut. A recent report by Avery has argued that it is unlikely that nitrate causes methemoglobinemia without bacterial contamination, and also that the 40–50 mg/L limit on nitrate in drinking water is not necessary.” (Kobayashi, 2015)

By the 1950s, the public was well aware of the dangers associated with nitrite and nitrates and when the scientific community shunned further investigations into possible benefits, it sealed its fate. Well, almost. Nothing compared to what was about to come in the 1950s and 60s when the N-Nitrosamine issue emerged onto the world stage.

In 1954, Barnes and Magee found that dimethylnitrosamine (DMN) produced liver toxicity in two men in the UK who worked in a laboratory where a solvent that was introduced 10 months earlier contained DMN. One of the men passed away from bronchopneumonia and incidental reference was made to cirrhosis of his liver. A second man was investigated after developing a hernia at the place of incision for an operation and mention was made in examining this that his liver felt hard it was suspected to be cirrhotic. Liver function tests were carried out which indicated damage but three months later, after no further exposure to DMN, it was normal.

A search of the literature brought up a single report about the toxicity of DMN from 1945 which indicated an illness that occurred in an automobile factory where it was used. Experiments on dogs indicated it to be capable of causing severe liver injury. Barnes and Magee were asked by the firm to investigate the toxicity of DMN which they did. It was found to be a toxin attacking the liver in particular.

Similarly, to the case above, an outbreak of acute liver toxicity in sheep in Norway was traced to the formation of DMN in fish meals preserved by nitrite. This would become a health scare rivalled by few in the past. 1960s researchers noticed that domestic animals fed on fodder containing fish meals prepared from nitrite-preserved herring were dying from liver failure. “These events prompted the early synthetic investigations of the N-nitroso compounds, particularly by groups headed by Magee in the UK and US, and Druckrey in Germany. Their work, and that of others, rapidly showed that the N-nitroso compounds, as a group, had enormous potential for being responsible for cancer formation. This, in turn, has led to the current deep scientific interest in the analysis, formation, and occurrence of carcinogenic N-nitroso compounds in our food supply, polluted atmosphere, drinking water, beverages, cigarette smoke, cosmetics, industrial waste and by-products, and more recently to their formation in vivo.” (Mergens, 1980)

Researchers identified nitrosamines formed by a chemical reaction between the naturally occurring amines in the fish and sodium nitrite. Nitrosamines have been identified as a potent cancer-causing agent, and their potential presence in human foods became an immediate worry. An examination of a wide variety of foods treated with nitrites revealed that nitrosamines could indeed form under certain conditions. Fried bacon, especially when “done to a crisp,” consistently showed the presence of these compounds.  (Schwarcz, J) In bacon, the issue is not nitrates but the nitrites which form N-nitrosamines.

Nitrosamines that have been found in foods are (Anr reference to nitrosamines from class 2A will be marked in red, class 2B in orange and class 3 in green for the rest of the article):

• NDMA (N-nitrosodimethyamine), NDEA (N-nitrosodiethylamine) (both classified as classified as 2A, probably carcinogenic to humans); (Park, 2018)
• NDBA (N-nitrosodibutylamine), NPIP (N-nitrosopiperidine), NPYR (N-nitrosopyrrolidine), NMOR (N-nitrosomorpholine), and NSAR (N-nitrososarcosine), all classified as 2B (possibly carcinogenic to humans); (Park, 2018)
• NDPhA (Nnitrosodiphenylamine) and NPRO (N-nitrosoproline), classified as 3, not classifiable as to their carcinogenicity to humans. (Park, 2018)

This fundamentally sharpened the focus of the work of Kerr and co-workers of the 1920s in response to the general toxicity of nitrites to the specific issue of N-nitrosamine formation. Reviews from 1986 and 1991 reported that “90% of the more than 300 N-nitroso compounds that have been tested in animal species including higher primates causes cancer, but no known case of human cancer has ever been shown to result from exposure to N-nitroso compounds.”  However, despite this, there is an overwhelming body of indirect evidence that shows that a link exists and “the presence of N-nitroso compounds in food is regarded as an etiological risk factor. It has been suggested that 35% of all cancers in humans are dietary related and this fact should not surprise us. (Pegg and Shahidi, 2000)

In terms of meat curing high concentrations of nitrosamines have been reported in bacon, sausage, and ham in high rates (Cho, 1970). On the other hand, unprocessed meats showed low, if any, amounts of nitrosamines.

It is clear that the matter had to be resolved.

Where do the amines come from in the stomach?

Amines, including those that can react with nitrites to form nitrosamines, are present in the stomach as a result of the breakdown of proteins in the diet. When proteins are digested, they are broken down into smaller peptides and amino acids, and some of these amino acids can be converted into amines by bacteria in the digestive tract. The amines that are produced in the stomach can react with nitrites, which are also present in the stomach, to form nitrosamines.

Mergens (1980) states that basically, “three things are needed to form an N-nitroso compound:

a. an amine or amide substrate which is primary, secondary, or tertiary amino group or a secondary amide;

b. a nitrosating agent, such as nitrite. Nitrogen oxides (NO_x), whether derived from “smoking” processes, atmospheric pollution, direct flame combustion for heating or drying systems, all are significant sources of highly reactive nitrosating agents. The gaseous forms, N₂O₃ and NO₂ , are particularly rapid and vigorous in their chemical attack of substrates, even when in alkaline aqueous solution and extremely rapid when the gases react with substrates in lipid phase.

c. the proper chemical environment. An amine that exists in its protonated form would be much less susceptible to attack by a nitrosating agent than its free-base counterpart. For a given amine, the nitrosation rate decreases as the pH increase 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 the higher relative concentration of unprotonated amine present. The formation of nitrosamines from secondary amines is usually the most rapid of all the mechanisms. In fact, it should be observed that primary amines must be alkylated and tertiary amines dealkylated prior to the formation of the ultimate nitroso compound through the nitrosation of the correspondingly generated secondary amine. (a,b, and c by Mergens, 1980)

Early on in our discussion, we can state that the formation of nitrosamines in the stomach is not a significant source of exposure for most people. Nitrosamines are more commonly found in food products and tobacco smoke. Nitrosamines can also be formed in other parts of the body, such as the mouth and respiratory tract, when certain products, such as tobacco and some types of cured meat, are consumed.

Different Types of Nitrosamines

Nitrosamines have been grouped into the following general categories. Don’t try and memorise them. Only be aware of them. They are Nitrisomines (NA), Volatile Nitrosamines (VNA) and Nonvolatile Nitrosamines (NVN). See Note 2 for a more complete list of examples. Park (2018) investigated the important nitrosamines of NDMA, NDEA (both classified as 2A, probably carcinogenic to humans), NMEA, NDBA, NPIP, NPYR and NMOR (all classified as 2B, possibly carcinogenic to humans).

How are they formed in Cured Meats?

Look at the three structures of amines represented above. Nitrosamines are formed by the reaction of secondary or tertiary amines with a nitrosating agent, such as nitrite, from which nitric oxide and an R-NO group formes.

“Reduction of nitrite occurs extensively in the stomach, with its very high concentration of protons from the gastric juice in combination with high levels of nitrite from the saliva. Nitrite reacts with protons to form nitrous acid   (HNO₂) with a pKa of 3.4. Nitrous acid is unstable and quickly dissociates to dinitrogen trioxide (N₂O₃) and water. Dinitrogen trioxide then forms nitric oxide and nitrogen dioxide.” (Petersson, 2008) The nitrosating agent is usually then a nitrous anhydride, formed from nitrite in an acidic, aqueous solution. Anhydrite is formed when the elements of water are removed from the substance, and N₂O₃ is an example of a nitrous anhydrate.

The resolution of the concerns related to processed meats presents itself very early in our discussion but will receive much more attention. “The reduction of nitrite to NO in the presence of protons is greatly enhanced by compounds such as vitamin C and polyphenols. In the presence of vitamin C (ascorbic acid), nitrous acid is reduced to NO without yielding nitrogen dioxide as an end product. Most vegetables, in addition to being rich in nitrate, also contain large amounts of vitamin C and polyphenols. The gastric mucosa also actively secretes vitamin C. This, taken together, ensures a very efficient reduction of nitrite to NO in the stomach after ingestion of vegetables.” (Petersson, 2008)

“N-nitrosamines are formed by reactions of organic amines and their derivatives with nitrosating compounds; however, most stable nitrosamines are formed from secondary amines. Since these precursors can, in turn, be generated from pesticides and herbicide, as well as nitrogen fertilizers, N-nitrosamines can be found as contaminants in various foods. The chemical reactions in which N-nitrosamines are formed from various sources are well reviewed by Rostkowska et al.. In foods, nitrosamines are formed by reactions of nitrogen oxide with amines. Nitrite in food, whether reduced from nitrate fertilizer or added as a preservative, is hydrogenated to hydronitrogenoxide in acidic condition.” (Park, 2018)

“The resulting hydronitrogen oxide reacts with another molecule of nitrite to form nitrogen anhydride after dehydration. Nitrogen anhydride donates nitroso group to the amines in food to produce N-nitrosamines. Secondary amine can form stable nitrosamine, while nitrosamines derived from primary amine break down quickly; it is also known that tertiary amine can hardly form nitrosamine. In spinach, cabbage, and other vegetables, nitrate has been reported to be reduced to nitrite by microorganisms. The nitrosating reaction may also occur in the stomach through the reaction of nitric oxide from nitrite or nitrate with amines in acidic conditions. The optimum pH of the reaction is 3 to 4, and synthesis of the nitrosamines in the rabbit, cat and human stomach from the precursors has been reported.” (Park, 2018)

Another culprit for nitrosamine formation is the frying of bacon. Nitrite in combination with fats (lipids) seems to be the nitrosating agent during the frying of bacon. “The formation is related to the relatively high internal temperature of bacon during frying and the relatively low moisture content of bacon as compared to other cured meat products. When bacon is cooked by other methods, particularly in a microwave oven, considerably lower amounts of nitrosamines are found.” (Scanlan, 2003) 

Bacon is not the only product of Concern

The hysteria related to bacon and the use of nitrite curing as it relates to possible nitrosamine formation is immediately put in context when we consider that cured meat is not the only source. Hotchkins states it succinctly when he writes that “originally, it was thought that the use of nitrite as a curing agent for flesh foods was the major source of these trace compounds in the diet. Subsequent research has clearly shown that other processing and packaging procedures can also introduce trace amounts of these carcinogens into foods. These procedures include drying foods in direct flame-heated air, migration from food contact surfaces and direct addition as contaminants. In addition, other reports of N-nitrosamines in foods have less well-defined routes of contamination.” See Note 1. (Hotchkiss, 1984) 

Processing and packaging techniques and methods have been identified as a major reason for nitrosamine formation. This has been known since the 70s and 80s. Hotchkiss (1984) writes that such processing and packaging procedures include “drying foods in direct flame heated air, migration from food contact surfaces and direct addition as contaminants.” Hotchkiss (1984) cautions that despite the presence of nitrosamines in food, it is actually “occupational exposures” which may be responsible for “the highest individual exposures (Fine and Rounbeh1er, 1981).

“Several groups have demonstrated that a number of foods can contain trace quantities of Volatile Nitrosamines (VNA). To date nearly all types of foods have been analyzed for VNA and, hence, some important generalizations can be made. Most importantly is that the use of nitrite as a curing agent is not solely responsible for the VNA content of foods. Several foods to which nitrite has not been intentionally added have now been shown to contain trace levels of VNAs. Equally significant is that the N-nitrosamine content of foods has decreased as a result of research in this area.

One of the major sources of nitrosamines for humans is tobacco smoke. Nitrosamines are formed when tobacco is cured or when tobacco smoke is produced, and they are present in high concentrations of cigarette smoke. Nitrosamines are also found in some types of beer. Nitrosamines can also be formed during the manufacturing process of certain rubber products and in some chemical reactions used in the production of cosmetics, pharmaceuticals, and other industrial products.

Several routes to nitrosamine formation exist, and in the debate about the use of nitrite, it is important to have a view of all of them. These includes:

1. Additives;
2. Drying processes;
3. Migration from contact surfaces;
4. Addition of preformed Nitrosamines;
5. Those for which the route is not clearly defined.

See Note 1 for a detailed discussion on routes to nitrosamine formation. The following by Hotchkiss (1984) is relevant to bacon. In order for cured meats to consistently contain more than 1 μg /kg VNA, the product must be subjected to temperatures greater than 100 C in a low moisture environment. The only cured product which meets these criteria is bacon. Other cured products only sporadically contain VNA in excess of 0.1 μg /kg (Gray and Randall, 1979). In a recent large survey, only 6 of 152 cooked sausage products had a VNA content greater than 5 μg /kg and only 4 of 91 dry sausages had similar VNA contents. In the same study, however, 11 of 12 dry-cured fried bacons contained VNA, some as high as 280 μg/kg. The fact that fried cured bacon consistently contains detectable VNA has been observed by numerous workers (Scanlan, 1975).” (Hotchkiss, 1984) Over the last couple of years the level of nitrosamines from cured meats has decreased dramatically.

Park (2018), in investigating the occurrence of several important nitrosamines in food made relevant findings related to NDMA, NDEA, (both classified as 2A, probably carcinogenic to humans), NMEA, NDBA, NPIP, NPYR and NMOR (all classified as 2B, possibly carcinogenic to humans). NDMA and NDEA were most frequently detected in agricultural food products such as tofu, mung-bean jelly, acorn jelly and buckwheat jelly. NDEA was detected separately in the kimchi group. Therefore, it can be concluded that NDMA was either formed during fermentation and/or by reactions between nitrite (from nitrate) and amines. Less than 1 µg/kg was detected in the group of rice cake, flour, bread, doughnut, pickled vegetable, and croquette in all nitrosamines. NDMA ranged from ND to 1.71 µg/kg was detected in the cereal, potatoes, and beans group; other nitrosamines were detected in the quantities below 0.76 µg/kg. The maximum of 6.1 µg/kg and 4.9 µg/kg NDMA was detected in fresh vegetables and mushrooms, respectively, as well as the maximum of 6.11 µg/kg of NDBA in mushrooms. The study has demonstrated higher nitrosamine contents in vegetables than those previously reported in the literature. Since soil microorganisms are known to contribute to nitrosamine formation and given that nitrate contents in vegetables can also contribute, the fluctuation of the nitrosamine contents can be expected. It is notable that NDMA and NDEA were detected in the maximum of 2.95 and 2.22 µg/kg respectively, in snack samples. Other nitrosamines were relatively low. (Park, 2018)

“NDMA, NDBA, and NMOR were detected in fishery products, but on a very low level. Milk and milk products did not show remarkable contents of any of the tested nitrosamines. NDMA of cheese showed 0.72 µg/kg and the others, such as cake and ice cream, showed less than 0.56 µg/kg. NDMA showed the highest detection rate in meat and meat products. Dumplings which contain meat but also dozens of other ingredients, including seasoning, showed the highest amount of nitrosamines. In spite of the original concerns, NDMA ranged from 0.31 to1.54 µg/kg in processed meats, such as sausages, hams, and bacons, which were lower than the contents found in vegetables or fruits. However, NPIP and NMOR were detected in processed meats, albeit in low concentrations. Compared with the results reported by another research group, nitrosamine contents in Park (2018) study tended to be lower.” (Park, 2018)

“In oil samples, about 1 µg/kg of NDMA was detected on average. NDMA was detected in all of the samples and NMOR was detected in soybean, olive, canola, rape, and sun follower oil; however, other nitrosamines were not detected.” (Park, 2018)

“Hedler and co-authors (1979) reported 1 to 10 µg/kg of NDMA and NDEA in most edible oils. The present study showed similar results except for the fact that NDEA was not detected in the present study. Yurchenko et al. reported less than 0.71 µg/kg in all nitrosamines, but they used the method we have used in the fatless sample, which may not be sufficiently efficient to extract all nitrosamines from oils. Also, the method may not be able to remove the interference of remaining lipids for detection sensitivity. NDMA was also reported in margarine and butter, though the amount was low.” (Park, 2018)

“The highest concentration of nitrosamines was observed in seasoning samples with 13.48 µg/kg of NDMA and 6.53 µg/kg of NPIP. Since previous studies on nitrosamines in seasoning are scarce, it needs to be investigated further concerning the formation mechanism in relation to the manufacturing process. In sauce samples, 3.02 µg/kg of NDMA was detected.” (Park, 2018)

In alcoholic beverages, Park (2018) detected no nitrosamines in beer, wine, rice wine, soju, and other liquors, while trace amounts of NDMA, NPYR, and NMOR were detected in whisky. Kim (2002) found a maximum of 1.87 µg/kg NDMA in beer.

Kim (2002) used an improved method of detection and writes that “the analysis methods of nitrosamines have become more accurate and simple. From the 1960s, the nitrosamine analysis employed distillation. However, distillation could generate false results, since heat of the distillation procedure could facilitate the synthesis of nitrosamines; at the same time, low molecular weight nitrosamines could be lost during distillation due to the low vapour pressure. These days, various resins are used in the food analysis. In the present study, solid supported liquid extraction using Extrelut NT and Florisil SPE was employed. To analyze the whole spectrum of food items, the method had to be modified. For fatty foods, liquid-liquid extraction was used to extract polar organic nitrosamines from lipids. To speed up the extraction procedures, polar solvent, immiscible and lighter than oil, was searched. Acetone-water (3 : 1) mixture or acetone-acetonitrile mixture satisfied the terms. The extraction mixture was stored at −80℃ freezer to help the separation of the phases. Any remaining fat in the extract may interfere and damage analytical instrument. C-18 SPE cartridge laid with aluminium oxide powder was employed to remove the remaining fats and emulsifier. The final procedure can be used for food matrix with a large amount of fats and emulsifiers, such as dressings and creamers.

Within the consideration of nitrosamines, let’s have the discussion about cured meat, but let’s be balanced in opening it up to incorporate all food categories and let’s not make it an issue restricted to cured meat. To say that the meat industry is responsible for causing cancer is a gross oversimplification of reality to the point of being false. Having said that, the onus remains to do everything possible to remove the concern from cured meats altogether by employing the strategies available to the industry which now take centre stage in the rest of our consideration.

How can Nitrosamine Formation from Cured Meat be Prevented?

There are several mechanisms for preventing nitrosamine formation. That such mechanisms should exist stems from the existence of a nitrogen cycle in the human body where nitrate can be regarded as, among others, a reservoir of nitrogen, nitrite is an intermediary species which makes nitric oxide formation possible which is a gas that exists only for a fraction of a second. As such, nitrite is constantly present in our blood and is constantly being secreted by our saliva glands. This nitrite is ingested with and without food. Where we chew our food, even if the food itself contains no nitrite, it is ingested with the foods and in our stomach can form nitrosamines if there was no other mechanism that has evolved over time to protect us from the nitrosamine formation and the possible negative implications. When we, therefore, talk about ways that nitrosamine formation can be prevented, it is not that we are imposing on nature, something that does not exist as if there is some sort of a defect in our bodies. On the contrary! We are simply mimicking the ways that nature provided for humans that have lived healthy lives for millions of years and in this review, we can see how far we have strayed from what has been designed as something natural.

A. Blocking Mechanisms

Mergens, (1980) says that “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 molecular species in the specific system versus the nitrosation reaction. Thus, both the absolute and relative concentrations of nitrosating agent, blocking agent and amine substrate for nitrosation will determine the degree of “blocking” that will occur.”

-> In Aqueous Systems

Ascorbic acid (vitamin C) has been shown to be an excellent blocking agent against nitrosation in aqueous systems, particularly in weakly acidic conditions. The ascorbic acid competes for the nitrosating agent, forming dehydroascorbic acid and the non-nitrosating NO.

This blocking effect of ascorbic acid has been demonstrated in a large variety of systems by many investigators. Kamm has shown complete protection from liver toxicity in rats by the use of ascorbic acid fed with sodium nitrite and the readily nitrosatable aminopyrine. Greenblatt has reported similar protection against cancer induction in mice by the use of ascorbic acid in animals fed aminopyrine with sodium nitrite.

Other water-soluble blocking agents include sulfite, bisulfite, and cysteine. Phenolic compounds, such as gallic acid, tannic acid, etc., also may function as blocking agents, but often also catalyze nitrosation by facilitating transnitrosation. Therefore, phenolic compounds, with certain rare exceptions, are not considered reliable blocking agents.” (Mergens, 1980)

-> In Lipid Systems

“Alpha-tocopherol (unesterified vitamin E) is an excellent lipophilic blocking agent against nitrosation reactions. Although it is a phenolic substance, it does not catalyze nitrosation since the aromatic ring is completely substituted. It reacts very effectively in the lipid phase of food, particularly in emulsified lipid droplets or micelles, to block the extremely rapid nitrosation reactions occurring in lipid phase.

Blocking agents are not synonymous with antioxidants. For example, butylated hydroxyanisole (BRA) and butylated hydroxytoluene (BHT) have little or no activity as nitrosation-blocking agents, although they are excellent food lipid antioxidants. The reason for this is believed to be the fact that these two compounds act as free radical scavengers in their role as lipid antioxidants, whereas the elimination of a nitrosating agent required it be reduced to nitric oxide or lower. Tocopherol appears to be able to function as an antioxidant through both mechanisms. Model system studies have shown that in the process of reducing a nitrosating agent, tocopherol is converted to tocoquinone.

In addition to the mechanical aspects of properly delivering alpha-tocopherol to the system under investigation, be it the fat portion of bacon or the lower gastrointestinal tract of man, etc., there are several other facets of “vitamin E” worth review or at least reiteration. The active form of alpha-tocopherol is the unesterified or “free alcohol” form of the vitamin, which is the form that exists in nature in plants, seeds, etc. Most of the vitamin E forms of commerce are the esters, particularly the acetate ester and, in some cases, the hemisuccinate. These esters do not naturally appear in food but were developed as a means of stabilizing isolated pure forms of the “natural” d-isomer as well as the synthetic dl material from oxidation of the phenol group during processing and storage. This is true, particularly in pharmaceutical dosages such as tablets and capsules. The esters, per se, are inactive as N-nitroso-blocking agents and, for that matter, as an antioxidant as well. Their activity, then, relies upon hydrolysis, which on oral ingestion, occurs in the intestinal lumen through the action of pancreatic lipase and the aid of bile salts as part of normal fat digestion. Overall then, in vitro applications of tocopherol as an N-nitroso compound inhibitor, for example, in bacon, cosmetic formulations, drug products, etc., would require the use of the unesterified molecule. The same is true for inhibiting nitrosation reactions which could occur in the stomach where lipase activity for hydrolysis is lacking. In the lower gastrointestinal tract, it is conceivable, although not proven, that the esterified forms of vitamin E may be active. Preliminary studies indicate that after oral administration of 400 mg vitamin E acetate to volunteers, the unabsorbed fraction of the dose (-35%) appearing in faeces is essentially all unesterified vitamin E.” (Mergens, 1980)

-> Application of Blocking Agents

“Relationship to Food Fresh natural foods appear to contain variable quantities of inherent blocking agents. Of course, vitamins C and E represent at least a part of this blocking capacity. A few experiments in our laboratories, and private comments from others, indicate additional agents could also be present, at least in some animal feeds. Presumably, these are reductones, free mercapto groups, etc. Natural foods do make use of a basic principle of utilizing blocking agents in both lipid and aqueous phases (ascorbic acid and tocopherol) to achieve more efficient prevention of N-nitrosation.” (Mergens, 1980)

“Sodium nitrite addition to meat for preservation, particularly against formation of toxin by growth of Clostridium botulinum, has been utilized for thousands of years, originating independently in several separate cultures. The nitrite can also react with amines in the meat (pyrrolidine, dimethylamine, etc.) to produce carcinogenic nitrosamines. In comminuted meat products, such as frankfurters or similar sausages, it has been shown that sodium ascorbate can prevent nitrosamine formation. A minimum of two moles ascorbate per mole of nitrite is needed. This ascorbate ratio will not interfere with the other desirable functions of nitrite in meat processing, including colour formation (nitrosomyoglobin), antibotulinal control (Perigo effect). In the U.S., the trend in such comminuted meat products is to use 120 ppm input of sodium nitrite and 550 ppm input of sodium ascorbate.” (Mergens, 1980)

“Bacon, as produced in the U.S. and prepared for eating by frying, reaches temperatures of about 170°C (for at least a few minutes). This presents a unique problem since the water content is vaporized readily, leaving the residual nitrite in the bacon, which can readily generate the lipid-soluble N2O3 nitrosating agent. The amine substrate, proline or pyrrolidine, is present in the collagen fibres buried deep in the thick adipose layer of pork belly used for bacon production. Thus, the nitrosation reaction in bacon takes place primarily in the fat phase during the later, high-temperature stages of frying as shown by Fiddler. A lipophilic nitrosamine blocking agent, suitable and safe for foods, was needed to lower the nitrosopyrrolidine content of fried bacon.” (Mergens, 1980)

“Tocopherol addition to bacon does not affect residual nitrite levels nor does it interfere with the nitrite control of botulism. In fact, it appears to act exclusively during the frying step itself to lower nitrosopyrrolidine levels in the edible portion of bacon about 2-4 fold, usually well below 5 ppb. It also lowers the substantial level of volatilized nitrosamin35 produced during bacon frying by about 4 fold (i.e., about 75%).” (Mergens, 1980)

“Fish have been preserved against microbiological contamination by treatment with nitrite for thousands of years. Nitrite, whether added directly, generated as nitrogen oxides in the smoking of fish or produced through microbial action by “salting” with selected grades of sodium chloride containing traces of nitrate all have the undesirable capacity to form nitrosamines with the plentiful amines found in fish. It has been suggested that gastric cancer may be associated with dried, salted and/or nitrite-preserved fish products. Experimental treatment of fresh fish with nitrite produced active mutagens (Ames test, without microsomal activation). This was completely prevented by ascorbic acid. The suggestion has been made that vitamin C intake on a continuous basis, with all meals, could reduce gastric cancer stemming from this type of nitrosation.” (Mergens, 1980)

-> Relationship to In Vivo Formation

“Many in vitro and in vivo studies have demonstrated their effectiveness in inhibiting nitrosamine formation and subsequent toxicity in a gastric fluid environment.” (Mergens, 1980)

“An additional physiological source of nitrite has been demonstrated as being de novo synthesis in the intestine, possibly by heterotropic nitrification. Wang et al. have found volatile nitrosamines in normal human faeces at levels which appear to outweigh that which could be ingested from exogenous sources based on estimates of total environmental exposure to these compounds. It is conceivable that nitrosamines can be synthesized in the intestine since the precursors are present. While the conditions for aqueous nitrosation reactions are not optimum at pH’s encountered in the lower gastrointestinal tract, several studies have shown that the rate of these reactions can be catalyzed. It has been suggested that the intestine might be a site for the formation of nitrosamines by bacterial action. Sander has demonstrated the formation of nitrosamines by bacterial action from precursor amines and nitrate at neutral pH, and the formation of N-nitrosodimethylamine (NDMA) upon incubation of C-dimethylamine and sodium nitrite with rat faecal contents has been reported. This production of nitrosating agent capacity is assumed to be source of faecal mutagens found in at least some individuals. These faecal mutagens, which are “apparent” N-nitroso compounds, can be reduced by the incorporation of ascorbic acid and/or alpha-tocopherol in the diet.” (Mergens, 1980)

“In careful studies of a few well-controlled individuals, ascorbic acid (4 g/day) reduced the mutagens quantitatively about 50%, 4~hile alpha-tocopherol (400 mg/day) reduced the mutagens 70-90%. Dietary fibre (50 g/day) also reduced the faecal mutagens about 50% while increased fat and protein (170 g/day) markedly increased faecal mutagen output. Some cancer researchers consider this intestinal production of mutagenic material the source of nitrosamines found in blood and urine of normal, noninfected individuals. This could, therefore, represent a major source of carcinogenic substance input to the body. If so, and this is only hypothetical so far, the use of ascorbic acid and alpha-tocopherol, perhaps with added fibre, in the daily diet may become a useful routine for chemoprophylaxis of gastrointestinal cancer.” (Mergens, 1980)

B. Dose of Nitrites

Limiting the dosage of allowed nitrite in cured meat has been an effective strategy to deal with the matter. See Chapter 15.06: Regulations of Nitrate and Nitrite post-1920’s: the problem of residual nitrite.

C. Adding bioactive Vegetable Molecules

Keuleyan (2021) found that “processed meats’ nutritional quality may be enhanced by bioactive vegetable molecules, by preventing the synthesis of nitrosamines from N-nitrosation, and harmful aldehydes from lipid oxidation, through their reformulation.” They suggest that “the precise effect of these molecules during processed meats’ digestion must be deepened to wisely select the most efficient vegetable compounds.” (Keuleyan, 2021) They found that “rutin, a plant pigment that is found in certain fruits and vegetables, chlorogenic acid, a polyphenol and the ester of caffeic acid and quinic acid that is found in coffee and black tea, and naringenin, a flavourless, colourless flavanone, a type of flavonoid, the predominant flavanone in grapefruit and found in a variety of fruits and herbs, all significantly inhibited lipid oxidation and N-nitrosation was inhibited by the presence of lipids and ascorbate.” 

Is eating cured meat on the same level as cigarette smoke in terms of nitrosamines?

We already indicated that one of the major sources of nitrosamines for humans is tobacco smoke. Even secondary smoke, also known as secondhand smoke, is a major source of nitrosamines for non-smoking individuals who are exposed to it. Nitrosamines are present in high concentrations of cigarette smoke, and they can be inhaled by people who are in close proximity to someone who is smoking.

Secondhand smoke is a mixture of the smoke that is exhaled by a smoker and the smoke that is released from the burning end of a cigarette, cigar, or pipe. It contains more than 7,000 chemicals, many of which are harmful and can cause cancer. Nitrosamines are just one class of chemicals that are present in secondhand smoke, and they are considered to be highly carcinogenic, meaning that they have the ability to cause cancer.

If one wants to compare cigarette smoke and cured meat in terms of the cancer risk, it is interesting to note that in relation to tobacco smoke there is strong evidence that tobacco use is a major cause of cancer and other serious health problems. It is estimated that tobacco use is responsible for about one-third of all cancer deaths worldwide, and it is a leading cause of preventable death and disease. The risk of developing cancer and other health problems from tobacco smoke is much higher than the risk from exposure to nitrosamines in cured meats or other sources.

Relationship Between Fat in Bacon and Fat and Ascorbate

Hotchkiss (1984) has also confirmed this VNA in fried bacon and has further identified the compound in the fried-out fat from bacon. NDMA and NPYR are, under most frying conditions, found in higher concentration in the fried-out fat than in the edible portion. However, Hotchkiss (1984) found NTHZ consistently occurs in higher concentrations in the edible portion regardless of the frying conditions. The mutagenicity of NTHZ has been demonstrated (Sekizawa and Shib, 1980) but the compound has not been tested in whole animals for carcinogenicity. The formation of precursors of NTHZ has also not been studied in fried bacon but thiazolidine has been identified as a browning product in a glucose-Cysteamine model system (Mihara and Shibamoto, 1980).” (Hotchkiss, 1984)

In 2007, Prof. Emilie Combet, a senior lecturer in human nutrition at the University of Glasgow, caused a major stir when she published an article, Fat transforms ascorbic acid from inhibiting to promoting acid-catalysed N-nitrosation. They found that “in absence of lipid, nitrosative stress was inhibited by ascorbic acid through conversion of nitrosating species to nitric oxide. Addition of ascorbic acid reduced the amount of N‐nitrosodimethylamine (NDMA) formed by fivefold, N‐nitrosomorpholine (NMOR) by >1000‐fold, and totally prevented the formation of N‐nitrosodiethylamine (NDEA) and N‐nitrosopiperidine (NPIP ). In contrast, when 10% lipid was present, ascorbic acid increased the amount of N‐nitrosodimethylamine (NDMA), N‐nitrosodiethylamine (NDEA) and N‐nitrosopiperidine (NPIP ) formed by approximately 8‐, 60‐ and 140‐fold, respectively, compared with absence of ascorbic acid. They suggested that “the presence of lipid converts ascorbic acid from inhibiting to promoting acid nitrosation. This may be explained by nitric oxide, formed by ascorbic acid in the aqueous phase, being able to regenerate nitrosating species by reacting with oxygen in the lipid phase.”

Keuleyan (2021), whom we referred to above, proposed the incorporation of bioactive vegetable molecules into processed meat formulations thus preventing the synthesis of nitrosamines from N-nitrosation, and harmful aldehydes from lipid oxidation. In their experimental design, they designed the reactional medium in such a way that it is supported by an oil-in-water emulsion mimicking the physicochemical environment of the gastric compartment. The model was optimized to uphold the reactions in a stable and simplified model featuring processed meat composition. They found that despite components found and herbs and vegetables, N-nitrosation was inhibited by the presence of lipids and ascorbate. Taking their emulsion design into consideration, they studied the “impacts of the structure and composition of the medium on N-nitrosation by comparing the results obtained on the emulsionized model with those on the buffer model. Results showed a significant effect on N-nitrosation modulation (p-value < 0.001). N-nitrosation was significantly reduced (by almost 30%) in the emulsified model, compared to the model containing only aqueous buffer.” They comment on it that “those results are consistent with previously shown mechanisms. Combet et al. demonstrated in 2010 that some phenolics modulated differently N-nitrosation according to the presence of lipids (under the form of bulk oil) or not. They notably showed the enhancement of N₂O₃ synthesis in a lipidic compartment (up to 400 times). This highly reactive species is able to react with both secondary amines leading to nitrosamines or with lipidic peroxyl radicals, leading to nitroso-peroxyl radicals, according to chemical competitions between the different nitrite targets. In absence of lipids, the nitroso-peroxyl radicals’ synthesis path does not exist and the balance may therefore lean towards a promoted N-nitrosation reaction compared to the measurements in the emulsified medium.”

The work of Keuleyan (2021), is, however, possibly the first time that N-nitrosation is assessed in an oil-in-water emulsion, which is the most representative medium to model in vitro digestion of food bolus. Those results not only confirm the present developed model to study chemical reactivity modulations, but also deepen a rarely explored aspect explored, i.e., the reactivity of N-nitrosation in a complex environment close to the digestive one, herein represented by the use of an oil in water emulsion.”

They observed “a slight but significant reduction in N-nitrosation was noted under the combined presence of added ascorbate to the polyphenol or vitamin in the emulsified medium (p-value < 0.05), which was around a 12% decrease. Nonetheless, the absence of a distinguished modulation by ascorbate in present systems with or without the different pure vegetable substances may be surprising as ascorbate has been demonstrated several times to inhibit N-nitrosation alone or with other phytomicronutrients. To the current knowledge, it is the first time that the chemical reactivity of N-nitrosation is assessed in an emulsified medium. Yet, the chemical reactivity of ascorbate in presence of a multiphase system may be more complex than in an aqueous system, as it was shown that N-nitrosation modulation may vary from inhibition to promotion according to the presence of bulk lipids or not. Moreover, ascorbate may also modulate the stability of other polyphenols when introduced jointly, which emphasizes the complexity of the chemical interactions between the different compounds. This effect result is interesting within the framework of processed meats reformulation, where ascorbate is very often added. The model was herein efficient to identify a modulation of N-nitrosation.” (Keuleyan, 2021)

Their work points to the limits of the Combet study and provides information to wisely reformulate processed meats to enhance their nutritional qualities. It also confirms the effectiveness of ascorbic acid as an effective strategy to combat N-nitrosamine formation from processed meats.

Is there a relationship between Exercise and Nitrosamine formation?

There is some evidence to suggest that physical activity may be associated with a reduced risk of cancer and other health problems, and it is thought that this may be due, in part, to the effects of exercise on the body’s immune system and other biological processes. However, there is limited research on the relationship between exercise and nitrosamine formation specifically.

It is known that the levels of certain hormones, such as adrenaline and cortisol, can increase during and after physical activity, and these hormones have been shown to have an effect on the metabolism of nitrates and nitrites in the body. Some studies have suggested that physical activity may increase the production of nitric oxide, a molecule that is involved in the regulation of blood pressure and other physiological processes, and that this may have an impact on the metabolism of nitrites and the formation of nitrosamines. However, more research is needed to understand the full extent of these relationships and the potential effects of exercise on nitrosamine formation.

Factors that impact nitrosamines forming cancer in humans

There are several underlying factors that can increase the risk of cancer formation from nitrosamines:

• Immune system function: Individuals with a compromised immune system, such as those with HIV/AIDS or cancer, may be more susceptible to the carcinogenic effects of nitrosamines.
• Chronic inflammation: Chronic inflammation, particularly in the gastrointestinal tract, can increase the risk of cancer from nitrosamines.
• Alcohol consumption: Heavy alcohol consumption has been linked to an increased risk of cancer from nitrosamines.
• Genetic factors: Some people may have a genetic predisposition to a higher risk of cancer from nitrosamines.
• Previous exposure to carcinogens: Individuals who have previously been exposed to other carcinogens, such as tobacco smoke or radiation, may have an increased risk of cancer from nitrosamines.
• Dosage: The amount of nitrosamines that an individual is exposed to can affect their risk of cancer. Higher doses of nitrosamines are generally associated with an increased risk of cancer.
• Duration of exposure: The longer an individual is exposed to nitrosamines, the greater their risk of cancer.
• Individual susceptibility: Some people may be more susceptible to the carcinogenic effects of nitrosamines due to genetic factors or other underlying health conditions.
• Co-exposure to other carcinogens: Exposure to other carcinogens, such as tobacco smoke or radiation, can increase the risk of cancer from nitrosamines.
• Diet: Certain dietary factors, such as the over-consumption of processed meats or grilled meats, may increase the risk of cancer from nitrosamines.
• There is some evidence to suggest that older individuals may be at increased risk for certain types of cancer, such as colorectal and stomach cancer, associated with the consumption of nitrosamines. However, it is important to note that the risk of cancer from nitrosamines is not solely determined by age, and other factors such as diet, lifestyle, and genetic predisposition can also play a role.
• Industrial emissions: Nitrosamines can be produced during the manufacturing of rubber, leather, and other chemical products. These emissions can then enter the air and be inhaled.
• Agricultural practices: Nitrosamines can also be produced during the application of fertilizers and pesticides in agriculture.
• Limit BBQ/ Braai or, rather, incorporate some of the strategies to mitigate the risk for nitrosamine formation in cured meat elucidated in this article! Barbecuing meat can potentially lead to the formation of nitrosamines. When meat is barbecued, the high heat and long cooking times can cause the amino acids and sugars in the meat to react with each other, forming compounds called heterocyclic amines (HCAs). HCAs have been shown to be carcinogenic in animal studies, and some research suggests that they may increase the risk of certain types of cancer in humans. In addition to HCAs, other compounds called polycyclic aromatic hydrocarbons (PAHs) can be formed when fat and juices from the meat drip onto the heat source and create smoke, which can then stick to the surface of the meat. Some PAHs have been shown to be carcinogenic in animal studies, and there is some evidence that they may increase the risk of cancer in humans.
• Obesity. There is some evidence to suggest that obesity may increase the risk of cancer from nitrosamines. Obesity has been linked to an increased risk of cancer in several ways, including:
  • Inflammation: Obesity is associated with chronic inflammation, which can increase the risk of cancer.
  • Hormonal changes: Obesity is associated with changes in hormone levels, including an increase in estrogen, which has been linked to an increased risk of certain types of cancer, such as breast and endometrial cancer.
  • Insulin resistance: Obesity is associated with insulin resistance, which can increase the risk of certain types of cancer, such as pancreatic and colorectal cancer.

It’s important to note that the risk of cancer from nitrosamines can vary widely depending on the specific circumstances of exposure and the individual’s underlying health factors.”

Steps you can take to reduce the risk of nitrosamines in your everyday life

There are several ways that you can reduce your risk of exposure to nitrosamines and reduce the formation of nitrosamines in your body:

1. Avoid tobacco use: One of the most effective ways to reduce your risk of exposure to nitrosamines is to avoid tobacco products, including cigarettes, cigars, and chewing tobacco.
2. Eat a healthy diet: Choose a diet that is rich in fruits, vegetables, and whole grains.
3. Limit your alcohol intake: Some types of alcohol, particularly beer, can contain nitrosamines, so it is a good idea to limit your alcohol intake to reduce your exposure to these compounds.
4. Use a vitamin C supplement: Vitamin C can help to inhibit the formation of nitrosamines in the stomach by neutralizing the nitrites that are present in the stomach.
5. Avoid exposure to secondhand smoke: If you are a non-smoker, avoid being in the same room with someone who is smoking, and avoid spending time in places where smoking is allowed.
6. Exercise.

Spices Linked to Limiting N-Notrrosamine Formation

Some spices have been shown to have the potential to reduce the formation of n-nitrosamines in cured meats, although more research is needed to fully understand their effectiveness and the optimal dosages and methods of use.

Some examples of vegetables that have been studied for their potential to reduce n-nitrosamine formation in cured meats include:

1. Garlic: Garlic has been shown to have anti-carcinogenic properties and may help to inhibit the formation of n-nitrosamines in cured meats.
2. Turmeric: Turmeric contains a compound called curcumin, which has been shown to have antioxidant and anti-inflammatory properties. It may also help to inhibit the formation of n-nitrosamines in cured meats.
3. Ginger: Ginger has been shown to have antioxidant and anti-inflammatory properties and may also help to inhibit the formation of n-nitrosamines in cured meats.
4. Cumin: Cumin has been shown to have antioxidant and anti-inflammatory properties and may also help to inhibit the formation of n-nitrosamines in cured meats.
5. Basil: Basil has been shown to have antioxidant and anti-inflammatory properties and may also help to inhibit the formation of n-nitrosamines in cured meats.
6. Oregano: Oregano has been shown to have antioxidant and anti-inflammatory properties and may also help to inhibit the formation of n-nitrosamines in cured meats.

It’s important to note that the potential for n-nitrosamine formation in cured meats can be reduced by following recommended curing and storage practices and by using high-quality ingredients. It’s also important to eat a varied and balanced diet and to limit the consumption of cured meats, as they may be high in sodium, fat, and other compounds.

Conclusion

N-Notrosomines is a major issue to consider and major advances have been made since it was identified. The matter of nitrosamines in cured meat is not a major risk factor in diets due to the ever-reducing inclusion of nitrites and the legislative requirement to include ascorbate in cured meat formulations. Several additional strategies have been discussed in this work. It is suggested that a similar hurdle strategy be adopted to address the matter as is done in food safety generally. Am N-Nitrosamine management program should be part of every food safety audit.

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Note 1: Route to Nitrosamine Formation

Additives

This is the class where cured meats fall in. We are already familiar with the story as we discussed it in Chapter 15.06: Regulations of Nitrate and Nitrite post-1920’s: the problem of residual nitrite. “Since the late 1960s, a substantial research effort has resulted in a body of information concerning the occurrence and formation of VNA in cured meats. This has resulted in the knowledge that the addition of nitrite to meat is not, in most cases, sufficient to routinely cause the formation of VNA. In order for cured meats to consistently contain more than 1 μg /kg VNA, the product must be subjected to temperatures greater than 100 C in a low moisture environment. The only cured product which meets these criteria is bacon. Other cured products only sporadically contain VNA in excess of 0.1 μg /kg (Gray and Randall, 1979). In a recent large survey, only 6 of 152 cooked sausage products had a VNA content greater than 5 μg /kg and only 4 of 91 dry sausages had similar VNA contents. In the same study, however, 11 of 12 dry-cured fried bacons contained VNA, some as high as 280 μg/kg. The fact that fried cured bacon consistently contains detectable VNA has been observed by numerous workers (Scanlan, 1975).” (Hotchkiss, 1984)

“Efforts have been directed at determining the chemical mechanism and precursors to the major VNA, NPYR, found in fried bacon. While several potential precursors to NPYR have been identified, including collagen, ornithine, hydroxyproline, citrulline, putrescine and arginine, it is generally accepted that the major precursor is proline (Gray, 1976). Nakamura et al. (1976) have suggested that the mechanism is temperature dependent; at temperatures above o 175 C decarboxylation precedes nitrosation and at lower temperatures nitrosation precedes decarboxylation.” (Hotchkiss, 1984)

“In addition to NDMA and NPYR, Kimoto et a1. (1982) and Gray et a1. (1982) each has reported that fried bacon also contains NTHZ. This VNA was likely missed by many researchers due to its long retention time or its on-column decomposition. Hotchkiss (1984) have also confirmed this VNA in fried bacon and has further identified the compound in the fried-out fat from bacon. NDMA and NPYR are, under most frying conditions, found in higher concentration in the fried-out fat than in the edible portion. However, Hotchkiss (1984) found NTHZ consistently occurs in higher concentrations in the edible portion regardless of the frying conditions. The mutagenicity of NTHZ has been demonstrated (Sekizawa and Shib, 1980) but the compound has not been tested in whole animals for carcinogenicity. The formation of precursors of NTHZ has also not been studied in fried bacon but thiazolidine has been identified as a browning product in a glucose-Cysteamine model system (Mihara and Shibamoto, 1980).” (Hotchkiss, 1984)

“Nitrate may be added to certain cheeses to retard the growth of microorganisms which might cause defects. Concern has been expressed that the nitrate might be reduced to nitrite by reductase-containing microflora and that this nitrite could nitrosate amines endogenous to the product. The Danish government has published the results of a large survey of cheeses in which no correlation between the use of nitrate and concentration of VNA in the product could be made (Anon. 1980). Only very small amounts (less than 0.7 μg/kg) were found in any cheese. Sen et al. (1978), however, found 21 of 31 cheeses imported into Canada contained VNA up to 20 μg/kg. These apparent discrepancies with regard to the use of nitrate in cheese have not been resolved.” (Hotchkiss, 1984)

Drying

“In addition to the use of nitrite and nitrate as additives, a second general mechanism by which foods may become contaminated with NA is through the drying of foods in air which has been directly heated in an open flame. The highest levels of VNA resulting from this common method of food processing have been in the kilning of malted barley. Concentrations of NDMA -in the dried malt of over 100 μg/kg have been reported (Hotchkiss et al. 1980; Preussmann et al. 1981). A number of workers have shown that the NDMA in the malt survives the brewing process and can be detected in the resulting beer in concentrations expected from the dilution of the malt (Havery et al. 1981). This widespread contamination was shown to be the result of the formation of oxides of nitrogen in the air as it is heated in the flame. Oxides of nitrogen have been demonstrated to be effective nitrosating agents over a wide pH range (Challis and Kyrtopoulos, 1978).” (Hotchkiss, 1984)

“Scanlan and coworkers have extensively investigated the formation of NDMA in malt. They have demonstrated that the plant alkaloids hordenine and gramine are effective precursors of NDMA in model systems and that it is likely that NVNA may also be present in direct fired kiln dried malt (Mangino et a1. 1981).” (Hotchkiss, 1984)

“The first reports of NDMA in beer indicated average concentrations in the range of 2 to 6 μg/kg (Spiegelhalder et a1. 1981). While these levels seem, at first, low it is possible to consume 1 to 2 kg of beer in a single serving. This represents more NDMA exposure than from any other food source. Spiegelhalder et a1. (1980) have estimated that 64% of a West German’s dietary NDMA came from beer. There is recent evidence, however, that the VNA content of beer has decreased sharply (Mangino et a1. 1981). This decrease is due to the widespread use of sulfur dioxide or indirect heating of the drying gases in the malting industry. The application of sulfur dioxide during the early part of the kilning process may be either by direct injection of gaseous sulfur dioxide or by burning elemental sulfur in the drying air. The inhibition by sulfur dioxide is most likely due to the formation of bisulfite which may react with the nitro sating agent in a redox reaction.” (Hotchkiss, 1984)

“Other foods which are dried in direct flame heated air have also been shown to contain trace amounts of VNA, albeit at lower levels than malt. Most notable is the finding that nonfat dried milk may contain traces of NDMA. Several reports have shown NDMA levels of 0.1 to approximately 5 μg/kg (Libbey et al. 1980; Lakritz and Pensabene, 1981). In a recent nationwide survey of 57 nonfat dried milks conducted by the US Food and Drug Administration, an average NDMA level of 0.6 μg/kg was found with 48 samples being positive (Havery et al. 1981). Apparent NPYR and NPIP were also detected in sub μg/kg concentrations. Because nonfat dry milk is diluted lOx before consumption some have considered it not to be a significant problem while others have been concerned because of the widespread use of this product by the young.” (Hotchkiss, 1984)

“Other dried foods have been shown to sporadically contain detectable VNA. Sen and Seaman (1981) analyzed nonfat dry milk, dried soups, and instant coffee and found VNA in all dried milks, 3 of 20 dried soups and 5 of 10 instant coffees, most at levels of less than 1 μg/kg. Perhaps more importantly, 3 of 8 dried infant formulas contained detectable VNA. Fazio and Havery (1981) have observed VNA in soy isolates and concentrates and dried cheeses.” (Hotchkiss, 1984)

Migration

“In addition to formation from direct additives or from the direct flame drying process, recent evidence indicates VNA may enter foods through migration from food contact surfaces. In 1981 Spiegelhalder and Preussmann (1981) reported that a number of rubber products including nursing nipples contained substantial levels of VNA and that these compounds could migrate to water and milk. Later Havery and Fazio (1982) investigated one brand of nipple available in the US. They confirmed the presence of VNA in this product and demonstrated that when inverted nipples were sterilized in milk or formul, migration occurred.” (Hotchkiss, 1984)

“We have investigated the VNA content of 8 types of rubber nipples available in the US from several domestic and foreign manufacturers (Babish et al. 1982). One or more VNA were detected in all nipples tested and when each nipple was boiled for 3 minutes in 150 ml water or incubated 3 hours at 37^oC, 6 to 44% migration occurred. Total VNA contents ranged from 42 to 617 μg/nipple (nipple weight is approximately 5 gm) and most nipples contained more than one VNA.” (Hotchkiss, 1984)

“Direct food contact paper and paperboard packaging may also be a source of VNA and nitrosatable amines in foods. Analyses of 34 food packages by GC-TEA revealed 9 to be contaminated with NMOR. Perhaps more importantly, all packaging materials examined had levels of the parent amine morpholine ranging from 98 to 842 μg/kg (Hotchkiss and Vecchio, 1982). Morpholine is easily nitrosated and there is evidence that it may be nitrosated in the stomach to produce the carcinogenic N-nitroso derivative (Mirvish, 1975). Two experiments indicated that both the NMOR and morpholine may migrate to dry foods. First, when a food package was found to contain NMOR and morpholine, the food closest in the package often also contained NMOR and morpho line (Hoffmann et ale 1982). Secondly, when paperboards which contained NMOR and morpho line were incubated at elevated temperatures in closed vessels with dry foods migration could be demonstrated. Further research is needed to determine the extent of the contamination and degree of migration under normal conditions.” (Hotchkiss, 1984)

Direct Addition

“When agricultural chemicals or food additives contain preformed VNA, it is conceivable that a portion of the VNA contaminate could be added to food. For example, meat curing premixes which contained salt, sugar, spices and nitrite and were designed to facilitate the mixing of curing brines were shown to contain relatively high levels of VNA including NPIP (Sen et ale 1974). This VNA resulted from the nitrosation of piperidine ring-containing compounds in the spices. The NPIP was then added along with the cure solution to the meat and could be detected in the product.” (Hotchkiss, 1984)

“Certain agricultural chemicals were shown at one time, to contain mg/kg quantities of VNA (Ross et al. 1977). Although current levels have been greatly reduced (Oliver, 1981) it has been demonstrated under laboratory conditions that when these mixtures are applied to food crops, absorption of the VNA either directly through the plant or indirectly through the soil is possible (Khan, 1981). For example, Dean-Raymond and Alexander (1976) have shown that radiolabeled NDMA incorporated into soil could be taken up by edible plants. A recent survey of dried waste sludge also indicates most sludges contain small amounts of VNA (Mumma et al. 1982). If sludge is incorporated into soil uptake may be possible. It should be noted that no confirmed report of VNA in foods as a result of the use of pesticides or sludge in actual field use has appeared. On the contrary, Ross et al. (1978) analyzed soil, run off water and edible plant tissue after the application of a commercial herbicide containing NDPA and failed to detect the VNA in any sample. As pointed out by Oliver, (1981) it is difficult to draw conclusions about the VNA contamination of foods based on laboratory experiments.” (Hotchkiss, 1984)

“Another potential source of direct addition of NA to food maybe through the use of processing water which has been deionized by anion exchangers. Kimoto et al. (1980) have shown that NDMA and NDEA at levels of less than 1 μg/kg can be detected in water which has been passed through an anion exchange column. This is a common treatment process in food plants.” (Hotchkiss, 1984)

Miscellaneous

“In addition to the above four mechanisms by which food may become contaminated with small amounts of VNA, other less well-defined or uncorroborated contamination processes have been reported. For example, one group of Japanese workers reported that broiling fish under a gas flame may result in substantial increases in the VNA content of the food (Matsui et al. 1980). The average NDMA content of 20 fish and seafood products increased 3 fold after broiling under a gas flame and one dried squid sample increased in NDMA from 84 to 313 μg /kg. When broiled under an electric element or covered with aluminium foil smaller increases in NDMA content were seen. Presumably, VNA is being formed by a mechanism similar to that occurring in dried foods such as malt and nonfat dried milk. Further work is needed to evaluate this source of dietary NA. Smoking fish has also been reported to result in the nitrosation of the amines associated with fish (Kann et aL 1980).” “Another potential source of direct addition of NA to food may be through the use of processing water which has been deionized by anion exchangers. Kimoto et al. (1980) have shown that NDMA and NDEA at levels of less than: 1 μg/kg can be detected in water which has been passed through an anion exchange column. This is a common treatment process in food plants.” (Hotchkiss, 1984)

Note 2: Examples of Nitrosamines

The Non-Volatile Nitrosamines are again broken down into various classes and types.

-> the N-nitrosamino acids, e.g. N-nitrosohydroxyproline (NHPRO) 

-> N-nitrosoproline (NPRO)

-> N-nitrososarcosine (NSAR)

-> N-nitroso-thiazolidine-4-carboxylic acid (NTCA)

-> N-nitroso-2-methyl-thiazolidine-4-carboxylic acid (NMTCA)

Volatile nitrosamines are similar to non-volatile nitrosamines in that they are formed when nitrogen-containing compounds react with amines or amides under certain conditions. They are, however, volatile, meaning that they are more likely to evaporate or become airborne. Formally, “volatile” refers to those compounds amenable to gas chromatography without derivatization. It includes sub-classes such as:

-> N-nitrosodimethylamine (NDMA) 

-> N-nitrosopyrrolidine (NPYR) 

-> N-nitrosopiperidine (NPIP) 

-> N-nitrosodiethylamine (NDEA)

NDEA has been evaluated as the most potent carcinogen among the known meat-related VNAs (Peto et al., 1984).” (Herrmann, 2015)

---

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