Counting Nitrogen Atoms – The History of Determining Total Meat Content
Part 1: From the start of the Chemical Revolution to Boussingault
By Eben van Tonder
25 September 2018
Summary
We trace the development of the understanding of nitrogen, its prevalence, nature and its role in plant and animal nutrition. If we produce a sausage, for example, what is the percentage of fat and meat that it must contain for us to be able to call it a pork sausage? What are the threshold values for the contained fat and connective tissues? How much fillers are we allowed to add? What must we declare on the food label? Tracking the meat content, as we will see, is measuring proteins and proteins are counted by measuring the amount of nitrogen. How this came about now becomes our focus.
The manipulation of proteins is nothing new. It has been with us for millennia. The domain belonged exclusively to artisan guilds through the middle ages and back into antiquity. The weavers of silk and wool, bakers, cheese makers, tanners, and the meat curers all made their living through the manipulation of protein. As the chemical revolution started to unfold and exploding human populations placed greater demands on food production, its safe storage, and transportation for trade, new artisans emerged who plied their trade, not only based on age-old traditions but predicated on newly developed techniques and analytical methods. These men contributed to what was seen as “natural food” and a “natural analysis” of reality as opposed to the mythical approach of alchemy and the secretive methods of the ancient artisans.
Where appropriate and possible, I highlight the people who made significant contributions and attempt to frame these people within the wider context they lived in.
– Robert Boyle (1627-1691) – did important reports on the production of bone gelatin.
– Bartolomeo Beccari (1682-1766) – the preparation of gluten, the protein portion of wheat flour; he characterized the starchy material of flour that would ferment to give acid spirits indicating its “vegetable nature.” In contrast, the gluten was of “animal nature” for “within a few days it gets sour, rots and very stinkingly putrifies like a dead body.”
– Joseph Black (1728–1799) – discovered carbon dioxide. In 1756 – isolate gaseous ammonia by reacting sal ammoniac with calcined magnesia.
– Charl Wilhelm Scheele (1742 – 1786) – the first scientist to describe the characteristics of oxygen and nitrogen.
– Claude Louis Bertholett (1748 – 1822) – in 1781 became aware that something joined with hydrogen to form ammonia.
– Daniel Rutherford (1749–1819) – identified and named nitrogen, “aer malignus.”
– Joseph Priestly (1733 – 1804) – the discoverer of oxygen (1774 – 1775) and identified nitrogen (but did not name it).
– Jean Antoine Claude Chaptal (1756 – 1832) – named azote, nitrogen in 1790.
– Henry Cavendish (1731 – 1810) – in 1766, discovered hydrogen.
– Hilaire Marin Rouelle (1718 – 1779) – in 1773 identified urea in urine, the key to understanding protein metabolism.
– Torbern Bergman (1735 – 1784) – in 1782 names ammonia.
– Claude Berthollet (1748 – 1822) – in 1785 reported that the vapour from decomposing animal carcasses contained ammonia.
– Louis Proust (1754-1826) – improved the methods of gelatine manufacture.
– Antoine-Laurent de Lavoisier (1743 – 1794) – in 1790 describes experiments on the respiration of human subjects which shows that nitrogen is “absolutely useless in the act of respiration, and it appears from the lung in the same quantity and quality that it has entered it.” His may be regarded as the first metabolic experiment with nitrogen.
– Joseph Louis Gay-Lussac (1778 – 1850) – developed a new method for the identification and measurement of nitrogen.
– François Magendie (1783–1855) – in 1816 became the first to recognise that there is a major difference between the nutritional value of food containing nitrogen and those without it.
He also examined the nutritive value of gelatine and reported on it in 1842.
– Jean-Louis Prévost (1790-1850) and Jean Baptiste André Dumas (1800 – 1884) – showed in 1823 that urea was not synthesized by the kidneys and suggested the liver as the site of its formation.
– John Gorham (1783-1829) – discovered zein, the protein of corn.
– Jean Dumas (1800-1884) – improved on the method developed by Gay-Lussac to analise nitrogen.
– Jean Baptiste Boussingault (1801 – 1887) – in 1836 described that it was the nitrogen content in the soil or fertiliser which is important for plant nutrition. He was also the first to conduct a “balance” trial measuring the intake, utilization, and excretion of nitrogen by animals.
– Franz Varrentrapp (1815 – 1877) and H. Will in 1841, developed a total nitrogen method to test for and measure nitrogen.
– Johan Kjeldahl (1849-1900) – in 1883 presented a much-improved method for catalyzed digestion of nitrogenous materials in sulfuric acid which allowed for the production of ammonia quantitatively.
Introduction
A friend of mine from the bacon industry in Castlemaine, Australia recently interacted with me on the matter of total meat content in bacon. This set about a fascinating line of inquiry and provided an introduction to a missing piece on work related to nitrogen. In other articles, I looked at the discovery of nitrogen, the nitrogen cycle, the discovery of bacteria responsible for nitrification and, more important for the history of meat curing, denitrification and the subsequent application of this knowledge in identifying nitrite as the curing chemical by Dr Eduard Polenski in 1891 with its derivative of nitric oxide, identified by Haldane in 1901 when he became the first person to demonstrate nitrite is further reduced to nitric oxide (NO) in the presence of muscle myoglobin to form iron-nitrosyl-myoglobin. It is nitrosylated myoglobin that gives cured meat, including bacon and hot dogs, their distinctive red colour and protects the meat from oxidation and spoiling.
What we have not looked at before is nitrogen as a constituent of the meat protein and its nutritional value. This identification and the subsequent determination of a phenomenally stable nitrogen percentage in meat lead to a number of important applications and implications, among others, a way to determine lean meat content and total meat content in meat processing. It is interesting that Dr Polenski who first speculated that nitrite (NO2-) is “closer” to the curing reaction than nitrate (NO3-) when he compared the nitrogen content of fresh meat vs processed beef in 1891 in an analysis of the nutritional difference between the two. We will return to his article.
A good summary of the thinking early in the late 1800s and early 1900s on the subject exists in the South African Food, Drugs and Disinfectants Act No. 13 of 1929 (See note 1). As an important historical document, it sets out the determination of total meat content. It essentially remained unchanged (apart from minor updates).
The calculations of total meat content are defined in subparagraph 4 (iv) which reads as follows: “In all cases where it is necessary to calculate total meat under regulations 14 (1), (2), (3) and (4), the formula used shall be:—
Percentage Lean Meat = (Percentage Protein Nitrogen × 30 ).
Percentage Total Meat = (Percentage Lean Meat + Percentage Fat).“
The questions of interest are how did they arrive at this and how accurate an indication is it of total meat content? What is the relationship between nitrogen and nutrition? When decay takes place, what happens with the nitrogen in the protein? How does the amount of nitrogen we consume determine the total nitrogen content of our bodies or any animal or plant for that matter? What is the value of nitrogen to the body which makes it essential for nutrition? How does nitrogen move from a plant or an animal into our bodies to provide nutrition? What is the impact of processing on nutrition and the total nitrogen content? Can the standard calculation for fresh meat be applied to processed products? Lastly and equally fascinating, what are other sources of nitrogen that can increase the total nitrogen count and skew the nitrogen count in a product and its relationship and to meat content.
This short series of articles set out to deal with these fascinating issues. In this first article, we will look at the time from the start of the chemical revolution to Boussingault. Sincere thanks to my friend in Castlemaine, Australia for provoking a fascinating line of inquiry!
Early Identification of Proteins
Long before the term protein was coined, researchers referred to them by different names such as albumins or quaternary azotized substances but recognized them as set apart from the hydrates of carbon and fats by their high content of nitrogen. They found that albumins would undergo putrefaction spontaneously, in contrast to the fermentation characteristic of carbohydrates and that upon destructive heat distillation of these substances, ammonia, or “alkaline air,” was produced. Their insoluble salts with heavy metals such as mercury, silver, and lead were known. The coagulation of blood serum and egg white was recognized and in a general way, the alteration of solubility relations during denaturation had received attention. Haemoglobin was found to contain iron. Fibrin and the azotized principles of milk and cereals had been examined. The fact that these substances had something in common was clear and captured the imagination of researchers. (Sahyun, M. (Editor), 1948.)
Proteins would play such important roles in the development of the concept of nutritive value. Gelatine from bones had, for example, been prepared since the days of Robert Boyle (1627-1691) in the seventeenth century. Gelatine’s value as a food source was revived by food scarcity during the French Revolution, at which time Louis Proust (1754-1826) improved the methods of gelatine manufacture. “The famous physician and physiologist Frangois Magendie (1783-1855) served as chairman of the French commission for examining the nutritive value of gelatine in 1842. Zein, the protein of corn, was discovered at Harvard University early in the nineteenth century by John Gorham (1783-1829). Casein was well known because of its occurrence in the food trades for centuries.” (Sahyun, M. (Editor). 1948)
Discovery of gasses
In the 1770s scientists started to realise that the atmosphere is made up of various gasses. This was part of the start of the chemical revolution and in a way, the major propellant. Up to this time, gasses were not regarded as a separate chemical entity and were largely ignored in experimental work. The drawback was major and real advances became only possible as this was being resolved. One of its pioneers was Joseph Black (1728–1799). Black is credited with the discovery of carbon dioxide (fixed air). His first publication on the subject came in 1756 in an expanded form of an address given the year before to an Edinburgh literary society. (Munro and Allison, 1964)
Robison (1803) quotes Black as saying: “In the same year, however, in which my first account of these experiments was published, I had discovered that this particular kind of air, attracted by alkaline substances, is deadly to all animals that breathe it by mouth and nostrils . . . and I convinced myself that the change produced on wholesome air by breathing it consisted chiefly, if not solely, in the conversion of part of it into fixed air.” In the same lecture, he said: “Here a new and boundless field seemed open before me. We do not know how many different airs may thus be contained within our atmosphere nor what may be their separate properties.” It was the first gas to be discovered. (Munro and Allison, 1964)
The Swedish Chemist, Charl Wilhelm Scheele (1742 – 1786) prepared oxygen by heating saltpetre (KNO3) in 1770. Somewhere between 1771 and 1772, he became the first scientist to realise that “air consists of two fluids different from each other, the one that does not manifest in the least the property of attracting phlogiston while the other … is peculiarly disposed to such attraction.” (Smil, 2001: 2) Phlogiston was believed to be the substance present in all material that burns, responsible for combustion. The one substance is obviously oxygen and the other nitrogen.
At the same time, Daniel Rutherford (1749–1819), a pupil of Black, obtained his doctorate in Medicine in 1772 from the University of Edinburgh. In his “Dissertatio inauguralis de Aere Fixo Dicto, aut Mephitico” (Rutherford, 1772) he records the following experiment in with he placed mice in a closed in environment. He concluded that if an animal is made to respire in a closed container, it will eventually die and the air that is left is unable to support life or the combustion of a flame. He then removed the fixed or mephitic air (carbon dioxide) with a caustic potash solution (alkali). He found a residual gas still incapable of supporting respiration or fire, similar to carbon dioxide, but unlike carbon dioxide, did not precipitate lime water and was not absorbed by the alkali. He thus discovered a residue of his fixed or mephitic air. He named it “aer malignus” or noxious air.” (Munro and Allison, 1964)
Priestly, who is credited for the discovery of oxygen (1774 – 1775) presented experimental evidence similar to Rutherford’s before the Royal Society of London. He, however, did not draw conclusions regarding the possible nature of the gas (Priestley, 1772). “The question of priority in the discovery of the new gas has been discussed in considerable detail by McKie (1934), who favors the view that Rutherford has some claim to being the first investigator to recognize nitrogen as an independent substance. He was certainly the first to provide it with a name, “aer malignus.” (Munro and Allison, 1964)
The identification of nitrogen was “in the air”, so to speak and as we will see, never far removed from meat curing. Sal Ammoniac (Ammonium Chloride; NH4Cl) was used since antiquity as a curing and preserving agent of meat and was investigated by none other than Joseph Black. In 1756 he became the first to isolate gaseous ammonia by reacting sal ammoniac with calcined magnesia (Magnesium Oxide). (Black, 1893) (Maurice P. Crosland, 2004). Scientists were now widely experimenting with gasses and along with air, gasses like ammonia received a great deal of attention. It would later be discovered that nitrogen is its key constituent along with hydrogen.
Following Black, ammonia was, for example, also isolated again by Peter Woulfe in 1767 (Woulfe), by Carl Wilhelm Scheele in 1770 (kb.osu.edu) and by Joseph Priestley in 1773 and was termed by him “alkaline air”. Eleven years later in 1785, Claude Louis Berthollet ascertained its composition. (Chisholm, 1911) (Berthollet, 1785)
Priestley, in Part II of his work, Experiments and Observations, described work that he had done between the years 1773 and the beginning of 1774. In this document, he gives a reprint of an earlier publication on effluvia from putrid marshes. Here he identifies ammonia and nitrous oxide. (Schofield, RE. 2004: 98)
His discoveries on ammonia were the result of a consistent application of the English scientist, Stephen Hales’s (1677 – 1761) technique for distilling and fermenting every substance he could get his hands on or capture over mercury rather than over customary water so that the air would “release.” He heated ammonia water and collected a vapour. When it cooled down, it did not condense, proving it was air. He called it alkaline air. (Schofield, RE. 2004: 98, 103, 104)
More experiments showed him that alkaline air was heavier than common inflammable air but lighter than acid air. It dissolved easily in water, producing heat and it was slightly inflammable in the sense that a candle burned in it with an enlarged colour flame before going out. In the end, he not only described ammonia chemically, but also its mode of production, and its characteristics. (Schofield, RE. 2004: 98, 103, 104)
In 1781 the French Chemist, Claude Louis Bertholett became aware that something joined with hydrogen to form ammonia. Three years later, Claude joined Lavoisier who was responsible for unravelling the composition of saltpetre along with de Morveau and de Fourcroy, in naming the substance azote. (Smil, V. 2001: 61, 62) Lavoisier named it from ancient Greek, ἀ- (without) and zoe (life). He saw it as the part of air that can not sustain life. “The name “nitrogen” was given to it by Jean Antoine Claude Chaptal in 1790 in a French text on chemistry which was translated into English in 1791. The name he used was ‘nitrogène’ and it was intended to express “the characteristic and exclusive property of this gas, which forms the radical of the nitric acid,” and thus be chemically more specific than “azote.”” (Munro and Allison, 1964) As for ammonia, its modern name was given in 1782 by the Swedish chemist Torbern Bergman. (Myers, RL. 2007: 27) The discovery of Hydrogen is credited to Cavendish in 1766.
A Hint of Nitrogen in Animals
The relation between nitrogen through ammonia and animal bodies was known from early on. In 1785, Claude Berthollet reported to the French Academy of Sciences that he found that the vapour that came from decomposing animal matter was ammonia. When he realised the gas, he found that it was composed of three volumes of hydrogen and one volume of nitrogen, or around 17% hydrogen and 83% nitrogen by weight. He was very accurate in his measurements and the modern values of these are given as 17.75 and 82.25% respectively. (Carpenter, 2003)
Techniques for testing for Nitrogen
Key to the identification of nitrogen in animal substances was developing the tools to test for it. “The oxidation of organic material in the presence of cupric oxide, with the collection and measurement of the resultant gases was one of the earliest. It was developed extensively by Gay-Lussac, first while he was professor at the Sorbonne, and later when he was a chemist at the Jardin des Plantes in Paris.” (Sahyun, M. (Editor). 1948)
The method of Gay-Lussac was modified by Jean Dumas (1800-1884) and used by Dumas’ contemporary, Liebig. Dumas method remained the classic method, albeit with many modifications and adaptation to micro-procedure well into the 1900s. In 1841, F. Varrentrapp and H. Will developed a total nitrogen method based on the liberation of ammonia by heating protein with alkali, followed by gravimetric estimation of the ammonia as its chloroplatinate. (Sahyun, M. (Editor). 1948)
The method was slow and tedious with fundamental inaccuracies, yet it had specific technical advantages over that of the Dumas-method when applied to metabolic observations and it was used in many early studies. It was the Danish chemist, J. Kjeldahl (1849-1900), of Carlsberg, who in 1883 presented a much-improved method for catalyzed digestion of nitrogenous materials in sulfuric acid which allowed for the production of ammonia quantitatively. (Sahyun, M. (Editor). 1948)
Nitrogen in Respiration
“The next stage is described in a correspondence which can no longer be traced but was fortunately published in 1871 by the British Association for the Advancement of Science. One of the foreign students attracted to Edinburgh by Black’s international reputation brought with him a letter dated September 19, 1789, from the French chemist Antoine Lavoisier, in which Lavoisier acknowledges the inspiration given him by Black’s researches and sends some views of his own on oxidation.”
“In a later letter to Black dated November 19, 1790, Lavoisier describes experiments on the respiration of human subjects in which he shows that oxygen is consumed and carbon dioxide evolved during this process, that the quantity of oxygen used increases by some 50% above the basal level after a meal (the modern specific dynamic action of food) and that in severe exercise, oxygen consumption can increase by as much as three-and-a-half times. The actual data are not very different from those currently accepted for oxygen consumption of man under these various conditions. Part of the letter states: “Legaz azote ne sert absolument à rien dans l’acte de la res piration et il ressort du poumon en même quantité et qualité qu’il y est entré.” (Nitrogen is absolutely useless in the act of respiration, and it appears from the lung in the same quantity and quality that it has entered it)
This experiment described a mere 18 years after the discovery of nitrogen, may be regarded as the first metabolic experiment with nitrogen, and appears (D. McKie, personal communication, 1962) to have been based on studies made by Fourcroy in the late 1780s, using gasometric methods which were published in 1791 by Séguin. The findings were negative and, although from time to time investigators have claimed that some nitrogen is lost from the body during respiration, most scientists of the present day would subscribe to Lavoisier’s view that gaseous nitrogen plays no part in the nitrogen metabolism of the mammalian organism.” (Munro and Allison, 1964) They believed that the balance of nitrogen ingested and was not recovered in stools or urine was probably lost through “insensible perspiration.” (Carpenter, 2003)
Antoine Lavoisier and Armand Seguin’s experiment of human respiration not only showed no influence of nitrogen levels but also had some positive results. It showed an increased output of carbon dioxide (carbonic acid, as they called it) during exercise. This was measured at rest and when lifting weights. By itself, it was a progression. It was believed at the time that the sole purpose of respiration was the cooling of the heart. (Carpenter, 2003)
Schematic drawing by Mme. Lavoisier of her husband measuring the carbonic acid output of his collaborator Armand Seguin, while she noted down the results. (Wellcome Institute, London) from Carpenter (2003).
Lavoisier, in collaboration with mathematician Pierre-Simon Laplace, was able to identify the slow combustion of organic compounds in animal tissue as the major source of body heat. They compared the heat produced by the guinea pig with its production of carbon dioxide and compared the results with the heat produced by a lighted candle or charcoal. An ice calorimeter was used to measure the heat production. This instrument measures the heat generated by relating it to the weight of water released from the melting of the ice surrounding the inner chamber where the animal or burning material is housed. Measurement is not very precise, but it gave consistent results allowing them to draw the conclusion of the origin of body heat. (Carpenter, 2003)
“Lavoisier had returned to further studies on respiration when he was arrested in 1793 during the Reign of Terror and kept in prison. On the day of his trial in 1794, he pleaded for a short stay of execution that would allow him to do one more experiment, but the judge is believed to have replied that the Republic had no need of “savants” (scientists), and he was guillotined the same afternoon.” (Carpenter, 2003)
“Lavoisier not only introduced order into the study of the new chemistry. He also left behind him a vigorous school of chemists, some of whom turned their attention to the study of organic compounds by procedures in which gas was either evolved or removed. In 1810 a system of organic analysis was worked out by Gay-Lussac (a pupil of Lavoisier’s collaborator, Berthollet) and Thénard; the organic material was treated with potassium chlorate and the amount of oxygen and nitrogen liberated was then measured (Partington, 1951). The Dumas procedure, still a standard gasometric method of nitrogen analysis, was devised in 1830 (Partington, 1951). The new system of organic analysis allowed the identification of nitrogen-containing substances of interest to the biologist; the first fruits of this knowledge appeared immediately with the studies made by Magendie on the importance of nitrogenous components in the diet.” (Munro and Allison, 1964)
“The presence of nitrogen in animal matter was confirmed and it was shown to be absent from sugars, starch, and fats. It was shown that wheat flour contained a component (what we call gluten) that had properties of animal matter, including the development of alkaline vapour when it was allowed to rot. Potatoes were introduced and there was a debate if it could provide an adequate substitution for wheat since it did not have anything like gluten in it. The question was asked if it was the gluten that made wheat flour such a good food.” (Munro and Allison, 1964)
The isolation of proteins from plants has a long and illustrious background which goes back to the work of Bartolomeo Baccari (1682 – 1766) who was a professor at the University of Bologna for most of his life. One of his writings appeared in 1734 entitled “de Frumento.” Here he described the preparation of gluten which was found to be the protein portion of wheat flour. The following is translated from Latin:
“This is a thing of little labor. Flour is taken of the best wheat, ground moderately lest the bran goes through the sieve, for it ought to be purified as far as possible in order that all suspicion of mixture should be removed. Then it is mixed with the purest water and agitated. What remains after this process is set free by washing, for water carries off with itself whatever it is able to dissolve. The rest remains untouched.”
“Afterward that which the water leaves is taken in the hands and pressed together and is gradually converted into a soft mass and beyond what I could have believed tenacious, a remarkable kind of glue and suitable for many purposes, among which it is worth mentioning that it can no longer be mixed with water. Those other parts which the water carries away with itself for some time float and render the water milky. Afterwards, they gradually settle to the bottom but do not adhere together; but like a powder return upward at the slightest agitation. Nothing is more nearly related to this than starch or better, it is indeed starch.”
The starchy material, he classified as flour. The characteristics he described is that it would ferment to give acid spirits, indicating its “vegetable nature.” The characteristic of the gluten, on the other hand, was of “animal nature” for “within a few days it gets sour and rots and very stinkingly putrifies like a dead body.”
In this time, this was the method of distinguishing proteins from carbohydrates. This view was still prevalent during the time of Mulder and Liebig’s theory of the identity of animal and plant proteins and the thought that vegetable protein consumed by herbivores becomes directly the flesh and blood of the animal. (Sahyun, M. (Editor). 1948)
“Another question raised was where the nitrogen in animal bodies came from. The largest source was, of course, the air around us and some chemists suggested that animals get the nitrogen from the air by a kind of combination must occur during an animal’s digestion of plant foods “so as to give the ingesta the characteristics that would allow them to be incorporated into the animal’s own tissues either for growth or replacement of worn-out materials.” (Carpenter, 2003)
“Several compounds were isolated that became very important later in understanding protein metabolism. One such compound was urea. In 1773 it was identified in urine by H. M. Rouelle, brother of the G. F. Rouelle under whom Lavoisier had studied chemistry. Prévost and Dumas showed in 1823 that urea accumulated in the blood when the kidneys of rabbits or cats were removed. It proved that urea was not synthesized by the kidneys, and Prévost and Dumas suggested the liver as the site of its formation.
This was also the period when the first amino acid was identified. Despite being far from our modern concept of amino acids, these observations showed the seed which would later produce the theory of the amino acid building blocks, or “bausteine,” of the protein molecule. In 1810, cystine was obtained from urinary calculi by Wollaston in England. William Prout (1785-1850) did elementary analyses of the substance but many years were to pass before sulfur would be found to be one of its component elements and would be detected as one of the products of protein disintegration. (Munro and Allison, 1964) and (Sahyun, M. (Editor). 1948)
In France, Proust, working with the flavoring matter of cheeses, in 1819 isolated from cheese a white compound which he called casein oxide. His countryman, Braconnot, director of the Horticultural Gardens in Nancy obtained leucine from sulfuric acid digests of muscle and of wool (1820). This was the first occasion on which acid hydrolysis was employed for the disintegration of proteins. Also in 1820 in the same work, Braconnot described the isolation of glycine from the acid hydrolysate of fish glue. Because of its characteristic sweet taste the product was called “sugar of gelatin.” (Munro and Allison, 1964) and (Sahyun, M. (Editor). 1948)
The discovery of tyrosine was a contribution of Liebig, whom we will look at in Part 2. He reported in a brief paper in 1846 the separation of this compound from casein after fusion with caustic potash, dissolving in water and neutralization with acetic acid. A year later he obtained the same compound from fibrin and serum albumin. The product was finally isolated by acid hydrolysis, using the earlier technique of Braconnot. (Munro and Allison, 1964) and (Sahyun, M. (Editor). 1948)
François Magendie: Nitrogen as the basis for Nutrition
A major step came from the work of Magendie (1783–1855) who linked the nitrogen of inanimate substances with that of living systems. He was the first to recognise that there is a major difference between the nutritional value of food containing nitrogen and those without it. Magendie grew up in revolutionary Paris and practised as a surgeon before changing to physiology.
“In order to appreciate the extent of Magendie’s contribution, it is necessary to go back to the views held in the previous century about the nutritional importance of different dietary components. The opinions prevailing at that time are adequately summarized by a quotation from the lectures of Albrecht Haller delivered before the students of Göttingen and published in an English edition in 1754, two years before Black announced the discovery of carbon dioxide:
“. . . The flesh of animals appears a necessary part of our nourishment, … For it appears that the flesh of animals only contains the gelatinous lymph, ready prepared for the recruit both of our fluids and solids, which, being extracted from broken vessels and fibres, is readily converted into abundance of blood. . . . None (of the vegetables) have that animal glue, which is spontaneously changeable into blood; for it is only the small portion of jelly, which is drawn from their farinaceous parts, which, after many circulations, is converted into the nature of our indigenous juices.” (Munro and Allison, 1964)
Haller’s concepts of the chemical components of tissues are expounded in more detail in his textbook “Elementa Physiologiae” published in 8 volumes between 1757 and 1765. This shows that Haller and his contemporaries regarded “fibre” as the basic structure of all organized parts of the body, and that fiber was made up of several components: “Elementa fibrae . . alia solida sunt, fluida alia. . . . Solida elementum terra est, de calcario genere, quae cum acidis fervet. . . . Altera pars fibrae humanae gluten est.” (Elements of the fibers. . other solids are fluid. . . . Concrete element is of the kind is like limestone, which ferments with acids. . . . The other part of the fibers is a human glue)
Presumably, the gelatinous lymph referred to in Haller’s lecture arose from the gluten. The reference in the lecture to the “jelly” of vegetables which can be converted into animal tissues may indicate that Haller was familiar with the experiments of Beccari (1682–1766), who had separated gluten from flour and commented that, unlike the starch of the flour which was “of vegetable nature,” the gluten was “of animal nature.” (Munro and Allison, 1964)
“From Haller’s description of nutrition, we may conclude that a substance resembling the modern protein was suspected to be a constituent of animal tissues and to a lesser extent of plants, and this dietary component was considered to be essential for the renewal of blood and tissues. The genius of Magendie was that he restated this concept in chemical terms and thus understood the profound difference in nutritive value between the nitrogenous and nonnitrogenous components of the diet.” (Munro and Allison, 1964) He became interested in the subject through his interest in the then current use of special dietary treatment for the cure and prevention of urinary calculi or kidney stones. Such diets were particularly poor in nitrogenous substances and this led him to study the effects of nitrogen-free foods upon dogs. (Sahyun, M. (Editor). 1948)
In his first work on the subject, reported to the Academy of Sciences in 1816, he addressed the question of whether animals could access atmospheric nitrogen to “animalize” ingested foods of low nitrogen content.” (Carpenter, 2003)
In his 1816 article, “Sur les propriétés nutritives des substances qui ne contiennent pas d’azote.” (On the nutritional properties of substances that do not contain nitrogen), Magendie described experiments on dogs that received only carbohydrate (sugar) or fat (olive oil) until they died, in a few week’s time. From these experiments, he concluded that a nitrogen source was an essential component of the diet. For his choice of experimental animal he was taken to task by the editor of the Journal of the Royal Institution, who felt that, since he was feeding nutrients derived from plant sources, he ought to have used herbivora. To which Magendie (1816b) replied with acidity: “il faut un peu de patience; les expériences ne se font pas comme les arti cles critiques dans les journaux.” (It takes a little patience; the experiments are not like the critical articles in the newspapers)
Although Magendie’s experiments were undoubtedly complicated by vitamin deficiencies, they were first approximations to an ideal—the long-term feeding experiment with purified foodstuffs—which has only been attained in recent years. As such, they are forerunners of the classical procedure for establishing whether a nutrient is essential to the body, namely by excluding it from the diet and then looking for symptoms attributable to its deficiency.
The distinction between nitrogenous and nonnitrogenous foods is made with even greater emphasis in Magendie’s “Elementary Compendium of Physiology for the Use of Students,” of which the first edition appeared in 1817 and the third edition was translated into English in 1829. Magendie’s compendium is the first modern textbook of physiology. Written in vigorous French prose, it breaks with the tradition of earlier books like Haller’s “Elementa Physiologiae,” (1757–65) written in turgid Latin. The contrast in outlook is even more striking; from Haller to Magendie we step out of the primaeval forests of mystery and speculation into the bright sunshine of scientific observation and deductive reasoning.
A large part of Magendie’s success in the physiology of nutrition can be attributed to the influence of Lavoisier’s vigorous school of chemistry, which had grown up in the interval. This can be seen when we compare the passage about the components of tissue fibre quoted above from Haller’s textbook with the following precise statement about the components of tissues taken from Magendie’s textbook (p. 10): . . . The proximate principles of animals are divided into azotised and non-azotised. The azotised principles of animals are albumen, fibrin, gelatin, mucus, casein, urea, uric acid, osmazome, red-colouring matter of the blood, yellow colouring principle. The non-azotised principles are: olein, stearin, fatty matter of the brain, the acetic, benzoic, lactic, formic, oxalic, rosacic, acids; sugar of milk, sugar of diabetic urine, picromel; colouring matter of bile, and of other liquids and solids, which become coloured by accident.
Later (p. 470) he goes on to say: . . . Since chemical analysis has made known the nature of the different tissues of the animal economy, they have been all found to contain a considerable portion of azote. Our food being also partly composed of this simple body, the azote of our organs likewise probably comes from them; but several eminent authors think that it has its source in the respiration; others believe that it is formed by the influence of life solely. Both parties insist particularly upon the example of the herbivorous animals, which are supported exclusively upon non-azotised matter; upon the history of certain peoples that live entirely on rice and maize; upon that of negroes, who can live a long time without eating anything but sugar; lastly, upon what is related of caravans, which, in traversing the deserts, have for a long time had only gum in place of every sort of food. Were it indeed proved by these facts, that men can live a long time without azotised food, it would be necessary to acknowledge that azote has an origin different from the food; but the facts cited by no means prove this. In fact, almost all the vegetables upon which man and animals feed contain more or less azote; for example, the impure sugar that the negroes eat presents a considerable proportion of it; and with regard to the people as they say, who feed upon rice and maize, it is well known that they add milk or cheese; now casein is the most azotised of all the nutritive proximate principles. I have thought that we might acquire some more exact notions on this subject, by submitting animals, during a necessary time, to the use of food, of which the chemical composition should be known.
Thereafter follows a detailed description of the experiments taken from Magendie’s publication of 1816a, in which he fed only carbohydrate or fat to dogs. To these, he added some new studies made on dogs eating special diets and noted particularly that dogs fed exclusively on cheese or eggs, both nitrogenous foods, survived indefinitely, although they were weak. Magendie concluded from his studies that “these facts . . . make it very probable that the azote of the organs is produced by the food.”
Magendie’s inquiring mind also extended to views on how the diet was utilized by the tissues of the body. In his textbook (p. 18), he says: … The life of man and that of other organised bodies is founded upon this, that they habitually assimilate to themselves a certain quantity of matter, which we name aliment. The privation of that matter, during even a very limited period, brings with it necessarily the cessation of life. On the other side, daily observation teaches, that the organs of man, as well as those of all living beings, lose, at each instant, a certain quantity of that matter which composes them; nay, it is on the necessity of repairing these habitual losses that the want of aliment is founded. From these two data, and from others which we shall make known afterwards, we justly conclude, that living bodies are by no means always composed of the same matter at every period of their existence. . . . It is extremely probable that all parts of the body of man experience an intestine movement, which has the double effect of expelling the molecules that can or ought no longer to compose the organs, and replacing them by new molecules. This internal, intimate motion, constitutes nutrition. And again (p. 468), … Nutrition is more or less rapid according to the tissues. The glands, the muscles, skin, etc. change their volume, colour, consistence, with great quickness; the tendons, fibrous membranes, the bones, the cartilages, appear to have a much slower nutrition, for their physical properties change but slowly by the effect of age and disease.
This prophetic insight into the continual renewal of body constituents, differing in rate in different tissues, succumbed to the theories of Liebig, Voit, Folin and others, and was not regained until more than a century later when Schoenheimer’s publication in 1942 of “The Dynamic State of Body Constituents” demonstrated the instability of tissue components by isotopic means.” (Munro and Allison, 1964)
“An important, unmentioned assumption behind Magendie’s work was that an animal species could be used as a model for humans; in other words, that our bodies were essentially of the same general character as those of animals. This may have arisen, at least in part, as a result of an interest in France for studies in comparative anatomy.” (Carpenter, 2003)
Jean Baptiste Boussingault
Another active investigator in France in the 1830s, with a quite different background from that of Magendie, was also studying the source of an animal’s nitrogen-rich tissues. This was Jean Baptiste Boussingault, the great “farmer of Bechelbrom,” who had learned his chemistry in a school for mining engineers. After a period of adventurous geological exploration in South America, he returned, married a farm owner’s daughter and put his mind to agricultural science. He obtained a position at the Sorbonne in Paris, where he collaborated with J. B. Dumas, one of the leading French chemists, and divided his year between Paris and the farm.” (Carpenter, 2003)
It was Boussingault who realised in 1836, over sixty centuries after it was noted and recorded that manure and legumes were beneficial to crop production, that it was the nitrogen content in the soil or fertiliser which is important for plant nutrition. In 1838, he performed a number of experiments where he grew legumes in sand with no nitrogen in it. The legumes continued to grow and the only conclusion he could come to was that they took their nitrogen from the air. How they did it, he still had no idea. (, J. N, et al., 2013) He was able to show that this was not possible for cereal grain.
“He then turned to cows and horses, whose common feeds had the reputation of being exceptionally low in nitrogen. His approach was first to find the level of feeding that kept his animals at constant weight, and then for 3 days to record the animal’s feed, excreta and, in the case of the cow, its milk, and also to analyze all these for their nitrogen content. With the horse, receiving altogether some 8.5kg hay and oats per 24 hours, the daily nitrogen intake was 139g, and the nitrogen recovered in urine and dung came to only 116g. The cow, fed on hay and potatoes, had a daily intake of 201g nitrogen and the recovered output, including 46g from milk, was only 175g. He concluded that the animals’ feed provided sufficient nitrogen to meet their needs and that there was no need to hypothesize that they had to obtain nitrogen from the atmosphere.
These seem to have been the first of the many thousands of “balance” trials that would continue to be carried out until the present day. Unfortunately, the only method of analysis for nitrogen that had been developed at that time required him first to dry his samples, which could be expected to result in loss of ammonia when he was drying urine and dung. This could explain the apparent “positive” balance in these animals that were assumed to be in a steady state.” (Carpenter, 2003)
Why the focus on nitrogen?
“Even before carrying out his balance experiments with herbivores, Boussingault had proposed that the relative nutritional values of plant foods could be assessed from their contents of nitrogen. His justification for this went roughly as follows: “Magendie has shown that foods that do not contain nitrogen cannot continue to support life, therefore the nutritional value of a vegetable substance resides principally in the gluten and vegetable albumin that it contains.” Investigators at this time certainly knew that animal bodies also contained minerals that they must have obtained from their food. Even earlier, two workers had written that: “Beans are so nourishing because they contain starch, an animal matter, phosphate, lime, magnesia, potash, and iron. They yield at once the aliments and the materials proper to form and color the blood and to nourish the bones”. Perhaps in response to such criticism, Boussingault explained, “I am far from regarding nitrogenous materials alone as sufficient for the nutrition of animals; but it is a fact that where nitrogenous materials are present at high levels in vegetables they are generally accompanied by the other organic and inorganic substances which are also needed for nutrition”. It is clear from the context that the “organic substances” to which he is referring are starches and not any hypothetical trace nutrients.
Was there any reason at this period for investigators to suspect that other nutrients might also be needed to constitute a complete diet? One might think that the problem of scurvy appearing among sailors and the evidence for the value of fruits and green foods in the prevention of the disease, would have suggested it. However, even James Lind, famous for his controlled clinical trial of different potential antiscorbutics, believed that they were active in countering the bad effects of sea air, and were not required by people living on land any more than quinine would be of any value for people not living in a malarious area. Also, it was clear that dogs, the animals being used by the French workers, thrived without such supplementary food items.” (Carpenter, 2003)
Synthesis by plants
Dumas, a colleague of Boussingault’s concluded in the early 1800s that the plant kingdom alone was capable of synthesizing the kinds of nitrogenous compounds abundant in animal tissues. Then, from the observation that the overall reactions of animals were characterized by oxidation, he made the further generalization that the animal kingdom was only capable of oxidizing the materials that are obtained from its plant food. (Carpenter, 2003)
Conclusion
It is to the work of François Magendie in his 1816 publications that we credit the concept of nitrogen as the basis for nutrition. Its presence in all animal matter was at this time firmly established and sets animals apart from plants. This is the cornerstone of the practice in recent years to link the determination of total meat content to nitrogen.
Of course, it is not the full story yet. Not by a mile! Next, we will pick up the development thread by starting to look at the work of Justus von Liebig and look at the emergence of the concept of the protein.
(c) eben van tonder
Note 1
The calculation of total meat content is set out in par. 14 of the act and it revolved around the amount of nitrogen present. Processed meats are described in 14 (3)as “simple or mixed” and “shall be meat which has been subjected to cooking, curing, drying, smoking and any combination of such processes. It may contain common salt, saltpetre, sodium or potassium nitrite, sugar, vinegar, spices and/ or permitted colouring matter, but no other foreign substances. The minimum total meat content shall be 95 percent and the amount of nitrite calculated as sodium nitrite, shall not exceed 200 p.p.m. in the finished article. If packed in any container, fat agar-agar and/or gelatin may be used as a packing medium.”
For our purposes, par (4) (i) is important, dealing with manufactured meat products and being described as “meat products which have undergone one or more of the processes enumerated in 14 (3) in addition to mincing and/or grinding, and include polonies, saveloys, meat pastes, brawn, meatloaves or rolls and similar articles containing meat, but exclude food products of the nature of sausage rolls and meat pies.”
Par 4 (ii) says that “manufactured meat products shall be made from meat as defined in regulation 14 (1) ( a) with spices and flavouring with or without milk, eggs, agar-agar, gelatin and wholesome farinaceous (containing starch) or other vegetable substances. They may contain added phosphates, not exceeding 0,5 percent of the final product, added ascorbic acid, permitted preservatives and colouring matter, saltpeter, and potassium or sodium nitrite: provided that the finished article shall not contain more than 200 p.p.m. of nitrite calculated as sodium nitrite. The total meat content shall not be less than 75 percent. If packed in any container, brine, fat, agar-agar and/or gelatin may be used as a packing medium.”
Par 4 (iii) deals with canned meat and the calculations of total meat content are defined in subparagraph 4 (iv). It reads as follows: “In all cases where it is necessary to calculate total meat under regulations 14 (1), (2), (3) and (4), the formula used shall be:—
Percentage Lean Meat = (Percentage Protein Nitrogen × 30 ).
Percentage Total Meat = (Percentage Lean Meat + Percentage Fat).“
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