Counting Nitrogen Atoms – The History of Determining Total Meat Content (Part 4): The Background of the History of Nutrition
By: Eben van Tonder
28/12/18

Previous Installments in Counting Nitrogen Atoms

Part 1:  From the start of the Chemical Revolution to Boussingault

Part 2:  Von Liebig and Gerard Mulder’s theory of proteins

Part 3:  Understanding of Protein Metabolism Coming of Age

Introduction

As background to the Kjeldahl method of nitrogen determination and the Jones factors, commonly used to determine lean meat content, we look at the history of the discovery of nitrogen in protein.  These methods link the relatively fixed proportion of nitrogen in protein to the calculation of protein content and are the basis of the determination of lean meat content.

The background story of nitrogen and protein is told through two lenses as described by two giants in science. The lens we concluded within our last instalment in the history of unravelling protein metabolism up to the early 1960s.  I relied mostly on the work of Hamish Nisbet Munro who was described in a biography written for the National Academy of Sciences by Robert M. Russell and Nevin S. Scrimshaw as “by far this generation’s most illustrious and productive expert on mammalian protein metabolism.”  In one of the closing paragraphs of the previous article, I quoted Hamish when he wrote, that in “1810 Gay-Lussac, pupil of Lavoisier’s colleague, Berthollet, devised a system of analysis of organic compounds which allowed the identification of the nitrogen-rich organic compounds we know as the proteins . . .  To the laboratory of Gay-Lussac came the young Liebig in 1823, to take back to Germany the new science of organic analysis and apply it to the study of biological materials. In Munich, Liebig had in 1854 as a pupil in his class in chemistry Carl Voit, who was to lay the foundations of modern studies on nitrogen balance. In Voit’s laboratory, numerous investigators from Germany and from abroad underwent a period of training—including Rubner, who especially studied the specific dynamic action of proteins; Atwater and Lusk, who continued the study of protein metabolism in America; and Cathcart, who returned to Scotland and was the teacher” of none other than Hamish Munro himself, thus endowing him with the best possible credentials to tell the story.

“In 1946, Hamish received his first grant from the British National Research Council and set up a research unit at Glasgow University to study metabolic responses to injury. He then accepted a position as assistant professor in the Physiology Department at Glasgow University and transferred to the Biochemistry Section, where he remained for 20 years, during which time he rose to the rank of professor.  Throughout this period, Hamish continued his studies on protein metabolism and also ventured into the investigation of nucleic acids and protein metabolism. Toward the end of this time, he completed his first two volumes of Mammalian Protein Metabolism, coauthored with James B. Allison.”  (Russel and Scrimshaw, 2014)  It was the last section of his first chapter of this work that we quoted in its entirety in our 3rd instalment of “Counting Nitrogen Atoms – The History of Determining Total Meat Content.”  In 1966 Hamish was recruited as professor of nutritional biochemistry and metabolism in MIT’s new Department of Nutrition and Food Science.

The second lens that we used to look at the history of nitrogen and protein and our historic understanding of the relationship between the two is through the unfolding history of nutrition.  Here we relied mostly on the work of another icon in the scientific community namely Kenneth Carpenter.

Kenneth . . . “was an eminent British nutrition scientist whose career, spanning approximately 60 years, was almost equally spent in the UK and the USA. In the UK, from 1946 to 1976, he focused on some of the key nutrition issues of the time, starting with B vitamins, including niacin, folic acid, and riboflavin, and moving on to the influence of processing and storage on protein quality. He pioneered the notion of nutrient bioavailability and the concept that a nutrient can be present in a foodstuff, as measured analytically, but is not absorbed and utilised because it is present in a non-digestible bound form. His research led to the identification of bound niacin in maize and explained why pellagra was common in most but not all maize-eating populations. He identified bound lysine in certain high-protein animal foods and explained why chickens and pigs fed rations based on ‘high-lysine’ fishmeals, meat meals or milk powder did not grow as well as would be expected. The Carpenter analytical method to measure available lysine in foods based on fluorodinitrobenzene (FDNB) is still a standard laboratory procedure. It has been used extensively in the milk industry to control lysine losses during heat processing so as to ensure the nutritional quality of infant formula and complementary foods.”  (Hurrell, R. F., 2018)  Kenneth was a historian of note even though he did not think of himself in those terms.  “In the USA from 1977 onwards, he gradually moved from nutrition research to the history of nutrition as he became more interested in the evolution of ideas in nutrition science. He is credited as making this complex history widely accessible to both serious scholars and the general public through a series of highly acclaimed monographs and papers. In his work, he saw himself as a nutrition scientist rather than historian as he did not, as does the classical historian, use archival records as a source of his information but reviewed early published literature and presented the problem as seen in the publications of the time.”  (Hurrell, R. F., 2018)

A moving obituary was published in the science column of The Guardian on Thursday, 12 January 2017 by Roger Carpenter.  He noted about his style that “Kenneth wrote with both elegance and clarity.”  It is this clarity that I enjoy in his work and will return to his work many times.  His paper on the nutritive value of meat meals which was done in 1970 with Atkinson is of particular interest.  There is his work on the impact of storage on protein quality, obviously of immense interest. Another is his work on the influence of raw materials and processing on protein quality and the many articles on the effect of heat on protein that will have to be digested fully at some stage so that we can glean the full value from the master.

He did four landmark articles entitled A Short History of Nutritional Sciences, Part 1, covering 1785–1885, Part 2, covering 1885–1912, Part 3, covering 1912–1944 and Part 4, covering 1945–1985.  I already made extensive use of his work from part 1.  Part 2 – 4 are equally applicable as vital background information to our present study.  I decided to select information from these remaining 3 articles that directly speak to our subject of the history of the development our understanding of protein and in particular as it relates to the nitrogen content, in this section then, from the perspective of nutrition.  In the next instalment, I will directly move to the history of the Kjeldahl method of nitrogen determination and the Jones factors. A 6th chapter has to follow where we look critically at an evaluation of these methods of protein determination and better alternatives that emerged over the last few years.

The importance of a thorough understanding of protein in meat processing cannot be overstated since it is the essence of the subject matter. Its chemistry and physiology is the basis of every process and product. It is therefore fitting that in considering the determination of meat content, we should begin by reviewing how our current understanding of protein came about, both in terms of its metabolism and function in nutrition. 

Summary

Young, John Richardson, (1782–1804). Found that regurgitated stomach contents did not undergo acetous fermentation.

William Beaumon, (1785 – 1853). Viewed as the father of gastric physiology, he observed that gastric juice, which always contained hydrochloric acid, was secreted only in response to eating. He also saw that oily food was only slowly digested, but that it was speeded by “minuteness of division”.

Claude Bernard, (1813 – 1871). Discovered that the secretions into the small intestine from the pancreas, together with the emulsifying effect of the bile, were of the greatest importance for the digestion of fat into glycerol and free fatty acids, and its absorption.

Wilbur Atwater, (1844 – 1907). A student of Carl Voit, Atwater estimated that American workmen were generally better off and ate more than German counterparts. He also thought they worked harder and he set his standard required protein intake per day at 125 g/d.

Russell Chittenden, (1856 – 1943). A professor of Physiological Chemistry at Yale University, we review some of his work on low protein diets.

We review the discovery of amino acids and its implications to the study of nutrition.  The work of F. Gowland Hopkins (1861 – 1947) who the Nobel Prize in Physiology or Medicine in 1929, with Christiaan Eijkman, for the discovery of vitamins, and S. W. Cole who isolated tryptophan.  The work of Wilcock and  Hopkins in 1906 could show that mice lived longer on zein and tryptophan.  Tryptophan became the first amino acid to be recognized as being essential for the normal growth of young animals.

We examine the work of William Cumming Rose (1887 – 1985) and colleagues who discovered the amino acid threonine and his research determined the requirement for essential amino acids in diets.  Their work showed that mixtures of amino acids could replace protein completely in a diet.

We look at the unusual studies by Elsie Widdowson, (1906 – 2000) and dr. Robert McCance (1989 – 1993), responsible for overseeing the government-mandated addition of vitamins to food and wartime rationing in Britain during World War II.

Finally, we consider the state of the world in the 1960’s when a senior nutritionist said, “We have moved from the era of vitamin research to protein research,” and the head of the Nutrition Division of FAO (the Food and Agriculture Organization of the United Nations) wrote that, “deficiency of protein in the diet is the most serious and widespread problem in the world.”  We end by concluding that measuring nitrogen and nutritional value of meat products is not just a matter of complying with government regulations but is a deeply moral question as well, deserving the NPD departments full attention.

Digestion

The question of digestion had to be solved for is to understand how a balance of ingested nitrogen was possible.  Kenneth brilliantly reviews the impact of the work of the American, John Young at the beginning of the 1800s who had found that regurgitated stomach contents did not undergo acetous fermentation, which was contrary to the current opinion.  Then, some 20 years later there was William Beaumont who, as an army surgeon, “had the opportunity to become a pioneering physiologist. At a remote trading post a young man was accidentally shot in the stomach and the wound left a permanent fistula through which food samples could be introduced and removed. Because the victim was destitute, Beaumont took him into his house and used him as a subject intermittently for almost 10 y. He observed that gastric juice, which always contained hydrochloric acid, was secreted only in response to eating. He also saw that oily food was only slowly digested, but that it was speeded by “minuteness of division”.” (Carpenter)

“In the 1850s, “Claude Bernard discovered that the secretions into the small intestine from the pancreas, together with the emulsifying effect of the bile, were of the greatest importance for the digestion of fat into glycerol and free fatty acids, and its absorption. This and the later discoveries of the proteolytic activity in the small intestine, to be discussed later made the study of purely gastric digestion seem less important.””  (Carpenter)

Diseases Due to Nutritional Deficiencies

“In 1842 George Budd, Professor of Medicine at King’s College, London, gave a memorable lecture titled “Disorders resulting from defective nutriment,” from which these are some of his opening comments: “There is no subject of more interest to the physiologist or of more practical importance to the physician … than the disorders resulting from defective nourishment. … These disorders are, no doubt, frequently presented to us by the destitute poor in our large towns; but … from our not being acquainted with all the circumstances in which they arise, their real cause escapes us. It is only—as in ships, garrisons, prisons, and asylums—when large numbers of men … become affected with one disease, that our attention is fixed upon it, and that we can succeed in discovering its cause by considering what is peculiar in their circumstances”.  (Carpenter)

An understanding emerged that protein and the accompanying measurement of nitrogen content is not the only “wellness factor” in food. Kenneth brilliantly reviews the history of the development of the concept that nutrition is crucial to wellness at a time when the germ theory of disease was predominant.

Over the course of his life, he studied and wrote extensively on the history of the research into arctic scurvy, goitre, and cretinism, anemia among young women, beriberi, chicken polyneuritis, rickets in young children, infantile scurvy, adult scurvy, guinea pig scurvy, night blindness, and xerophthalmia and how the work on these contributed to our understanding of nutrition and its direct link to wellness.  Its importance to our goal is that it neatly introduces the subject of vitamins, minerals, and different amino acids profiles of protein.  This will especially become important in our next article when we evaluate the different ways that meat content can be measured and the total nutritional benefit of meat vs a plant-based diet.

Even looking back at the material we have covered so far, it explains some of the earlier results of animal studies that were fed only one kind of food in early “balanced trials” since the results may have been due to a vitamin deficiency and not protein deficiency.

Protein research continued

“Until this time, there had been little significant work in nutritional science in the United States, but Wilbur Atwater, born in 1844 in New England and by 1885, a professor of chemistry at Wesleyan University, was determined to change that. He had already spent several months in Munich studying the nitrogen balance procedures in use at the laboratory of Carl Voit, who had been Liebig’s protégé. Voit believed that people with sufficient income to choose the diet that they preferred would instinctively select a diet containing the amount of protein that they needed to remain healthy and productive. His estimate was that the average German workman doing moderate physical work chose to eat 118 g protein/d, and this became his standard. Atwater found that American workmen were generally better off and ate more. They also, he thought, worked harder and he set his standard at 125 g/d.” (Carpenter)

“With hindsight, it seems ironic that he should not have been more questioning concerning whether they really needed so much of this relatively expensive ingredient. Apparently, he looked to the German school of nutritionists as the authorities in a field in which he was only a newcomer. Voit accepted that vegetarians who lived on a much lower protein intake could remain in nitrogen balance, but he remained convinced that such people “exposed themselves to disadvantages”. The American group suggested that even if protein was not directly used as the fuel for muscular contraction, it provided the nervous energy required to “wish to make the effort”.” (Carpenter)

“The main thrust of Atwater’s work in this period was to analyze foods by the proximate system (nitrogen, fibre, ash, ether extract, moisture and “carbohydrate by difference”)) and to use these values to teach the poor how they could obtain their requirement for protein, the most expensive of their needs, more economically. An unfortunate effect of recommending diets only on the basis of the economic provision of protein and energy was that fruits and green vegetables became dispensable luxuries. At this period, the purchase of food typically took ∼50% of a working family’s income.”  (Carpenter)

The challenge to high protein standards came finally from Russell Chittenden, Yale University’s Professor of Physiological Chemistry. He had found some relief from what may have been a rheumatic condition after he had deliberately reduced his general intake of food, and particularly that of meat, and was greatly impressed by having fully maintained both his physical and mental activity, although his intake of protein had not been >40 g/d (equivalent to 48 g for someone of the “standard” weight of 150 lb).  (Carpenter)

“Chittenden then organized three controlled trials using low protein diets. In the first, Chittenden and three scientific colleagues remained healthy and in nitrogen balance for 6 mo on daily diets containing 62 g protein on average, after adjustment to “standard” body weight. The second trial used 11 corpsmen from the U.S. army who also remained in good health and physical condition with a standardized daily intake of 61 g protein. In the final trial, a group of 7 Yale student-athletes consumed ∼64 g protein (standardized) per day, maintained their levels of athletic performance and said that they felt better for it.” (Carpenter)

“Others were reluctant to accept Chittenden’s recommendation of such diets as representing “physiological economy,” and argued that the almost universal consumption of high protein diets in prosperous countries showed an important relationship that might not become apparent in short-term trials. He replied that his critics were reversing cause and effect; people did not become rich because they ate more protein but ate meat and other more expensive high protein foods because they had already attained an income sufficient to afford them. Later studies have only confirmed Chittenden’s findings.” (Carpenter)

Protein digestion and interconversion

“Throughout the writings of Voit, Atwater, and Chittenden, there was the unstated assumption that all proteins were of equal quality. Thus, Atwater had no doubt that meat protein in the diet could safely be replaced by the same quantity of protein from beans. With hindsight, this is surprising because Mulder’s hypothesis that all proteins contained the same radical had collapsed, and even the ratio of carbon : nitrogen had been reported to differ between “legumin” extracted from beans and some animal proteins.” (Carpenter)

“For most of the 19th century, even after the breakdown of Mulder’s theory, it had been assumed by workers in nutrition that proteins ingested in foods were absorbed almost intact and then modified in some slight ways, if necessary, to convert them from “fibrin” to “albumin,” for example. However, other workers studying the physiology of digestion first showed the existence of a substance (pepsin), secreted by the stomach wall, that converted proteins into more soluble derivatives. Liebig regarded this as being no more than breaking up aggregations of molecules, allowing them to pass through the gut more easily. A few years later, the pancreas was found to secrete another substance (trypsin) that further broke down the products of treating proteins with pepsin to produce materials that were noncoagulable, diffusible through parchment and included the chemicals tyrosine and leucine. This subject has been thoroughly reviewed, with full references, by Greenstein and Winitz in an easily available volume.”  (Carpenter)

“Now, tyrosine and leucine were already known as two of the compounds, first called “amino-bodies” and then “amino acids,” that chemists had obtained by boiling proteins in strong acids. These breakdown products had not been considered of interest to nutritionists because the kind of destruction affected by strong, boiling acids had been assumed to be quite different from what happened under the mild conditions in the gut. However, the discovery of amino acids as products in a biological system was obviously highly relevant, especially because analysts had already reported that proteins appeared to differ in the relative quantities of different amino acids that they yielded on treatment with acids.”  (Carpenter)

“There always seems to be a way around unwelcome findings and in 1895 Chittenden wrote: “We may well consider the formation of these amino acids in pancreatic proteolysis as a means of quickly ridding the body of any excess of ingested protein food, with the least possible expenditure of energy on the part of the system”. Thus, he was suggesting that the proteins that the body needed were still being absorbed pretty well intact, and it was just the unwanted surplus that was being broken down before its disposal. Even in 1902, a German textbook was saying essentially the same thing: “such a profound decomposition would be a waste of chemical potential energy, and a reunion of such products is highly improbable”.” (Carpenter)

“However, other workers in Germany and Denmark were studying whether animals could use mixtures of amino acids as substitutes for dietary protein. Most found that meat proteins treated with pepsin and trypsin for long periods, and apparently free of intact protein, did serve as nutritional substitutes when fed to adult dogs, but that acid hydrolysates of protein, even after neutralization and removal of excess salts, did not.” (Carpenter)

“It had been suspected that strong acid treatment was destroying some component of the protein because proteins, and even enzymic digests, gave a color reaction suggesting the presence of an indole derivative, but acid hydrolysates did not. Finally, in 1902, F. G. Hopkins and S. W. Cole, working in Cambridge, isolated the amino acid tryptophan, which contains an indole ring, from an enzymic digest and showed that it was destroyed by conditions of acid hydrolysis. Then in 1906, Hopkins and another colleague reported that mice receiving zein (which contains no tryptophan) as their sole protein source, lived longer if they also received a supplement of tryptophan. And in 1909, Abderhalden found that adult dogs could remain in nitrogen balance if the acid-hydrolysates of protein that they were receiving were supplemented with this amino acid. These results did not yet prove that tryptophan was utilized for protein synthesis because there was no growth, but they did show that this organic compound had some essential function.” (Carpenter)

Vitamins and Minerals

We skip over Kenneth’s review of the discovery of minerals as nutrient in the diet since it falls outside the scope of our investigation.  Save to say that it is an important constituent of ash obtained in meat analysis.  He brilliantly deals with the discovery of the value of selenium, chromium, zinc in diet and the bioavailability of minerals.

Protein

Amino acid patterns.

Precisely to our point, Kenneth returns to proteins.  He writes, “I have not tried to cover work designed to measure the quantitative requirements for individual nutrients; however, protein is not in this category. No two proteins are identical, nor is the mix of proteins from one food identical to that from another. Therefore, the question remained as to how closely the amino acid pattern of our food needed to match that of our body proteins, which in practice related to the extent to which either animal protein or synthetic amino acids were needed to balance vegetable proteins for a diet to be ideal.” (Carpenter)

“Workers hoped to overcome this problem by stating requirements in terms of individual amino acids, but in 1945 it had not been demonstrated that mixtures of amino acids could completely replace protein in the human diet. William Rose and his colleagues at the University of Illinois had been working to resolve this since 1942 and they reported their findings from 1948 to 1955.”  (Carpenter)

Rose’s findings of the amino acids needed by the growing rat and the quantities needed for nitrogen balance in young men.

Amino acids found essential for the growing rat	Daily need of human adults	Subjects tested
	g	n
Lysine	0.4–0.8	27
Tryptophan	0.15–0.25	31
Histidine	Not needed	—
Phenylalanine	0.8–1.1	22
Leucine	0.5–1.1	8
Isoleucine	0.65–0.7	8
Threonine	0.3–0.5	19
Methionine	0.8–1.1	13
Valine	0.4–0.8	23
Arginine2	Not needed	—
Total of the upper levels of essential amino acid needs	6.35

“Additional glycine and urea were added to raise the total nitrogen intake of the men to 10 g/day, equivalent to 62.5 g crude protein. In further trials with double the upper level of each essential amino acid, it was found that nitrogen balance could be maintained if urea was eliminated and the glycine reduced to 6.5 g/d, so that the total nitrogen content of the diet was only 3.85 g, equivalent to 24 g of crude protein.” (Carpenter)

“Arginine can be synthesized by the rat, but not at a sufficiently rapid rate to meet the demands for growth. Its classification, therefore, as essential or non-essential is purely a matter of definition).” (Carpenter)

“They found that young men would remain in nitrogen balance with surprisingly low levels of amino acids (equivalent to only 24 g crude protein) but only with energy intakes higher than were required with equivalent quantities of intact protein. This was disturbing because it was known that increased energy intakes had an effect on the retention of nitrogen. Even though the quantitative requirements had a question mark attached to them, Rose made the point that the list of essential amino acids required by human adults must now be complete because, in the absence of even one, nitrogen balance would not be obtained at any level of intake.” (Carpenter)

The nitrogen balance (g/d) of an experimental subject receiving 10 g nitrogen/d and either 35 or 45 kcal/d of total energy intake.

Nitrogen source	35 kcal/d1	45 kcal/d
Whole casein	+0.14 (7)1 ; +0.46 (5)	+0.63 (5)
Acid-hydrolyzed casein + tryptophan	−0.29 (8)	+0.50 (6)
Enzymically hydrolyzed casein	−0.09 (6)	—
8 essential amino acids + glycine + urea	−0.91 (6)	+0.33 (5)

Duration of test period (d).

“Elsie Widdowson and Robert McCance took advantage of the unusual situation in Germany after World War II when food rations were severely limited, so that it was ethical to compare the performance of orphanage children receiving different kinds of special supplements. One group of 47 children was provided with all the bread (85% extraction, i.e., brown but not whole wheat) that they could eat, with calcium and vitamin supplements and their small ration of milk that contributed only 8.8 g protein. Another matched group received the same with extra milk providing three times as much animal protein. These treatments continued for 6 mo under careful supervision. The children, averaging age 9–10 y, grew equally well on both diets, even though in one <12% of their energy came from protein and of this only 14% was animal protein (or “first class” according to some writers). It is unfortunate that this important study was published in a series of monographs not available in many academic libraries, although some of the results have been summarized and discussed elsewhere.” (Carpenter)

The performance of orphanage children receiving unrestricted bread and different rations of milk for 6 mo.

	Low milk	Higher milk
Protein intake, g/d
85% extr. wheat bread	41.0	34.6
Other vegetable foods	11.5	11.5
Animal foods	8.8	26.5
Total	61.4	72.6
Total energy intake, kcal/(kg · d)	66.6	67.0
Nitrogen intake, mg/(kg · d)	322	381
Weight gain over 6 mo, kg	2.5	2.5
FAO/WHO calculations
N gain, mg/(kg · d)	13.9	13.9
Obligatory N loss, mg/(kg · d)	73	73
Theoretical N need, mg/(kg · d)	87	87
Efficiency required of dietary N, %	27	23

“These orphanage children were growing both in height and weight at a rate ∼25% above the average for their ages, perhaps as a “catch up” phenomenon. It is notable that this was possible on the low protein diet, in which mixed proteins themselves contained only ∼3.7% lysine, about half the level in our own body proteins. Human growth is extremely slow and the children were estimated to have gained on average only 14 mg N/(kg body wt·d) with an intake of 322 mg/(kg·d).” (Carpenter)

“A group at the Massachusetts Institute of Technology suggested that, although people on relatively low protein intakes were in nitrogen balance, their equilibrium might be at the expense of lower rates of protein turnover and of potential synthesis of antibodies when exposed to infection. This was an important question and by 1985 procedures were being developed for its measurement using turnover and oxidation studies with isotope-labeled amino acids. However, no definitive answer had been obtained and another worker recommended caution in justifying the need for increased protein on the basis of such measurements.” (Carpenter)

“It was also demonstrated that the requirement of growing chicks for the limiting essential amino acid, lysine, was increased from 0.85% of the diet to ∼1.1% when the total level of protein in the diet was raised from 20% to 30%. Alfred Harper and colleagues at Wisconsin then demonstrated that adding a single amino acid at a fairly high level, for example 2% l-histidine to a diet containing 12% casein (plus methionine) that supported rapid weight gains (56 g in 9 d) in young rats, could inhibit their appetite and performance (in this example to 45 g). Attempts have been made to divide effects of this general kind into toxicities, antagonisms and imbalances. However, there was no evidence that they were likely to occur in practice with humans. One possible concern was that high protein Western diets might be causing an acidosis that resulted in a compensating loss of calcium, and thus of bone.” (Carpenter)

The world protein problem.

“The period had begun therefore with studies indicating that the supply of protein, at least for diets based on cereals, was not a problem. Nevertheless, in 1960, a senior nutritionist said, “We have moved from the era of vitamin research to protein research,” and the head of the Nutrition Division of FAO (the Food and Agriculture Organization of the United Nations) wrote that, “deficiency of protein in the diet is the most serious and widespread problem in the world”.” (Carpenter)

“This idea grew from the finding that a serious disease, called “kwashiorkor” in West Africa and recognized by flaky dermatitis, hair changes, edema and apathy, was also common among 1–4-y-old children in other parts of the developing world. It was found to respond to concentrated relatively high protein nutritional supplements such as skim milk powder, and the previous idea that it was an infantile form of pellagra was abandoned because it did not respond to nicotinic acid or other B-vitamins.” (Carpenter)

“Kwashiorkor was also characterized by liver damage and, because cirrhosis of the liver was common among adults in Africa, it was initially suspected at FAO that the African diet remained protein-deficient throughout life, and that the same might be true throughout the developing world. Milk and milk powder was expensive and in short supply, and it was urged that substitutes needed to be developed.” (Carpenter)

“Much work was carried out in areas where the problem existed, for example at the Institute for Nutrition in Central America and Panama, to develop and test cheaper alternatives to milk powder based on locally available cereals and oilseed flours. These could prevent the condition from developing and also cure it, although not quite as quickly as with milk powder. Individual babies could also be deficient in electrolytes and vitamins as well as in protein and energy. Others suggested that essential fatty acids might also be deficient.” (Carpenter)

“In 1968 the United Nations published a paper entitled International Action to Avert the Impending Protein Crisis. By then, several projects had been set up, with substantial funding (some from governments and foundations), to develop processes and machinery in advanced countries for the preparation of stable, solvent-extracted high protein powders from fish [fish protein concentrate (FPC)] and other materials. This was encouraged by enthusiastic international conferences, even though the original idea had been to devise new crops or simple methods of food processing that could be adopted in underdeveloped villages. In addition, even more sophisticated processes were being planned to produce “single cell protein” (SCP) from yeasts, fungi and bacteria, grown on media ranging from molasses waste to petroleum. Doris Calloway drew attention to the poor tolerance and even toxicity of some SCP materials, and that their high content of nucleic acids was also a problem for humans, who metabolize purines only to the relatively insoluble uric acid, so that they were more suited for animal species that do not have this problem.” (Carpenter)

““Hi-tech” projects, which were supposed to be aiding relatively primitive communities, received particularly bitter criticism from the faculty at the London School of Hygiene and Tropical Medicine who referred to “a continuing process of justifying scientific enthusiasms by the drawing of facile and tenuous links between research which is intellectually exciting to the investigator and problems which are of sufficient public concern to make it politically attractive to devote funds to them”. Even in the U.S., where scientists are usually less willing to risk giving offense, the leader of the government-financed FPC project was to say later (in a book worth reading) that, “Much of the motivation for FPC development had little or nothing to do with the ostensible and well-publicized humanitarian goal”.” (Carpenter)

“This is an episode in our history that nutritional scientists would probably like to forget, but one use of history is to learn from our mistakes and to not repeat them. It was brought to an end by the realization that most kwashiorkor victims had been receiving diets that were as deficient in energy as they were in protein, and too bulky for the youngsters to take in sufficient amounts. The general need was to provide more concentrated foods and correct electrolyte deficiencies rather than concentrate just on protein. It was especially difficult to improve diets based on roots such as cassava that were very bulky as well as low in protein. The United Nations Organization, which had previously emphasized its concerns about “a world protein problem,” made no mention of it at its 1974 World Food Conference.” (Carpenter)

“The synthetic production of essential amino acids likely to be first-limiting in Third World diets was also stimulated in the 1960s. Although results with human trials were generally disappointing, these compounds have found practical uses in intensive pig and poultry feeding.” (Carpenter)

“I include the last section for an important reason.  The matter of the determination of meat content is not just an academic consideration for the purpose of complying with some arbitrary national legislative requirement.  There is an important moral consideration also.” (Carpenter)

Conclusion

With this set of articles and extracts from some of the best sources on earth, a very broad background is set for a detailed evaluation of the Kjeldahl method of nitrogen determination and the Jones factors, commonly used to determine lean meat content.  We achieved much more here.  We moved protein and its role in human nutrition to the front and central position in terms of meat processing.  Bacon and sausages, like all processed meats, are designed as delicacies for the wealthy.  There is no question about this.  It is, however, also the staple of the poor and the challenge and responsibility of the meat processor who targets these markets are to provide protein sources that are both delicious and affordable so that every person on earth can enjoy it and derive the maximum nutrition from it.  As a personal rule of thumb. Myself and my son, determined not to produce something that we are not prepared to eat ourselves or give to our family in the same quantities that clients are likely to consume.

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(c) eben van tonder

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References:

Carpenter, K. J.; A Short History of Nutritional Science: Part 1, 2, 3, 4, which appeared in The Journal of Nutrition. The links to the four articles are given below.

A Short History of Nutritional Science: Part 1 (1785–1885)

A Short History of Nutritional Science: Part 2 (1885–1912)

A Short History of Nutritional Science: Part 3 (1912–1944)

A Short History of Nutritional Science: Part 4 (1945–1985)

Hurrell, R. F..  2018.  Kenneth John Carpenter (1923 – 2016).  British Journal of Nutrition. Volume 120, Issue 5, 14 September 2018, pp. 594-596.  https://doi.org/10.1017/S0007114518001733

Russell, R. M., and Scrimshaw, N. S..  2014. Hamish N. Munro 1915–1994 Biographical Memoires.

Photo Credit:  https://www.webmd.com/diet/ss/slideshow-high-protein-diet

My Oupa Eben was a formidable man.  This is seen in his life and how he made his Boerewors.  Here are the recipe and his story.  I use Kobus’ general production method with Oupa Eben’s recipe.  Anneliese, my cousin, found it and sent it to me.

Meat:  20kg meat in proportion: 8, 8, 4 fat (spek).  8 pork en 8 beef.  800mL grape vinegar. Not spiced vinegar.  Run the mix through the kidney plate or 13 mm. Meat and vinegar.

Then add spices.

Spice mix:
8 tablespoons salt
6 heaped tablespoons coriander
6 teaspoons pepper
1/2 cup vinegar
2 tablespoons brown sugar
1 teaspoon cloves
(2 tablespoons thyme) – optional
(2 teaspoons nutmeg) – optional
(100g MSG) – optional

Sprinkle evenly over meat pieces. Mince and mix immediately without kneeing meat. Do NOT form into balls. The casings must be soaked in luke warm water overnight. Run water through each casing to ensure it is wet on the inside.

After mixing it all in by hand, run through 4.5 plate and stuff.

Use sheep casings.

For commercial sales, add sodium sulfite, 1g per kg FP.

My Uncle, Oom Jan Kok, sent me the following on his dad, and my grandfather, Oupa Eben:

Ebenhaezer Kok: Seun, Eggenoot, Pa, Oupa, Boer, Kerkmens en gemeenskapsmens.

Eben Kok
Pa, Oupa en Oupagrootjie.

Enenhaezer Kok is gebore op 18 Junie 1911 in Heilbron in die Vrystaat.

Agtergrondgeskiedenis en Familieherkoms

Hy was die tweede seun van Johannes Wilem Kok (Gebore 04 April 1880 te Winburg in die Vrystaat) en Maria Margaretha Klingbiel (Gebore op 17 Oktober 1879).Oupa Jan neem as 19 jarige deel aan die Anglo Boereoorlog en word as krygsgevangene na Dyatalawakamp op Ceylon gestuur waar hy op 1 Augustus 1901 in hut 54 intrek. Hier op die eiland, as krygsgevangene, kry hy die roeping om hom as Sendeling te laat oplei en na die vredesluiting in 1902 skryf hy in as student aan die Sendinginstituut te Wellington. Hy voltooi sy studies en begin sy bediening in Heilbron se Sendinggemeente op 7 April 1906. Hy werk aanvanklik vir ‘n jaar op proefbasis en word daarna permanent in die gemeente bevestig. Dit was ‘n groot, uitgestrekte gemeente wat die dorpe Petrus Steyn, Edenville, Koppies, Oranjeville, Viljoensdrif en Heilbron ingesluit het.

Oupa Jan is op 08 Julie 1907 getroud met met Maria Margaretha Klingbiel (gebore op 17 Oktober 1879 en oorlede op 27 Augustus 1942. Hy trou weer op 4 Julie 1944 met ouma Hannie wat eintlik maar die enigste Ouma was wat ek geken het. Dit was haar derde huwelik.

Aanvanklik het hy sy werk in hierdie uitgestrekte gebied met ‘n trapfiets gedoen. Na die trapfiets kon hy darem ‘n perd aanskaf en het later ‘n perdekar gehad en kon uiteindelik in 1933 sy eerste motor koop.

Na dertig jaar in die bedienig (1936), word ‘n gesamentlike diens van die blanke gemeente en die Sendinggemeente gehou om die geleentheid feestelik gevier. As blyk van waardering word aan hom ‘n toga geskenk. Hierdie diens is gehou op 29 Maaart 1936. Op 26 April 1936 word ‘n soortgelyke Feesdiens in die Sendinggemeente gehou en by die geleentheid het Oupa Jan se broer, Hendrik, wat ook ‘n sendeling was op Marquard woon die geleentheid by. Eerwaarde Hendrik Kok preek op hierdie besondere dag uit Josua 4: 5, 6 – “ . . .en Josua het vir hulle gesê: Trek uit voor die ark van die Here julle God tot binne in die Jordaan , en tel vir julle klippe op, elke man een op sy skouer volgens die getal van die stamme van die kinders van Israel sodat dit ‘n teken onder julle kan wees. As julle kinders later vra en sê: Wat beteken hierdie klippe vir julle? Die tema van sy preek handel oor die gedenkklippe op ons lewenspaaie wat ons herinner aan die groot dinge wat die Here in ons lewens laat gebeur.

(Die inligting in hierdie berig kom uit Die Kerkbode van 27 Mei 1936)
Oupa Jan tree af op 27 April 1947 en is oorlede op Heilbron op 26 Junie 1950.
Eben was die tweede oudste van 5 Kinders. Hulle was:

1. Johannes Willem Kok (Johan, gebore 02 Mei 1908). Hy trou met Doreen Uckerman en hulle het vier kinders: Gill, (Gebore 18 Oktober 1934), Myra (Gebore op 07 Julie 1937), Leon (Gebore op ) en Helen (Gebore op 10 Desember 1950)

2. Frederick Gustaff Klingbiel Kok (Gustaf, gebore 12 Mei 1910. Gustaff is jonk dood. Ek kry egter nie die datum en die ouderdom waarop hy dood is nie.

3. Ebenhaezer (Eben, ook latter Kokkie of Kok genoem) Kok, is gebore op 18 Junie 1911en oorlede op 21 Februarie 1981 op Vredefort. Hy trou op 07 Augustus 1939 met Susanna Maria Uys (Gebore 23 April 1911 op Vredefort en oorlede op 04 Januarie 1993 te Warmbad).

Hulle het drie kinders: Susanna Maria (Sannie, ook bekend as Santjie. Gebore op 26 Julie 1940), Johannes Willem (Jan en later Kokkie genoem. Gebore op 03 Mei 1942) en Michiel Eksteen Uys Kok (Uysie, gebore op 28 Februarie 1945)

4. Maria Margaretha KOK (Miempie), gebore op 23 November 1913 en oorlede op 02 April 1956. Sy trou met Adolf Samuel Bosman (Dolf). Hulle het drie dogters: Mariet (Gebore 27 Mei 1935), Ronnie (Gebore 26 November 1936) en Jantjie (Gebore 28 Februarie 1941)

5. Timotius Kok (Timo) KOK, gebore op 05 Augustus 1917. Hy is getroud met Thelma Berriman en hulle het geen kinders gehad nie. Toe die tweede wêreldoorlog uitgebreek het, het Timo by die lugmag aangesluit en is hy , soos hulle daardie jare gesê het, na die Noorde gestuur. Daar was hy betrokke in die oorlog in Egipte en is later na Italië om die stryd daar voort te sit.

Oupa Eben as Boer

Oupa Eben matrikuleer aan die plaaslike skool op Heilbron in 1929 en na skool gaan werk hy by Standardbank. Hy voltooi enkele bankeksamens en werk onder andere op Vrede, Vredefort en Koppies. Om hom te help met sy studies koop hy in 1934 vir hom ‘n Kings English Dictionary wat vandag nog in my besit is. Terwyl hy op Vredefort gewerk het, het hy ouma Susan ontmoet en hulle is op 07 Augustus 1939 getroud. Pa het net ‘n fiets gehad om mee te ry en as hy gaan opsit het, het hy met die fiets Leeuspruit toe gery. Dit was 7 myl van die dorp af. Daar word vertel dat hy een aand baie laat – dit was donkermaan – terug is dorp toe. Hy het nie ‘n bees gesien wat in pad gelê het nie en hy is fiets en al bo-oor die slapende bees. Die storie wil ook dat ouma Susan haar oupa, Piet Rademan, moes help versorg. Hy het op daardie stadium by Oupa Giel en Ouma Santjie gebly en Ma was verantwoordelik vir sy versorging en daarom kon hulle eers trou na sy dood op 99 jarige ouderdom in 1937. Hulle was alby 28 toe hulle getroud is.

Ma se suster, Meraai, was 11 jaar jonger as sy en Eben en Susan wou op ‘n keer vir sy ouers op Heilbron gaan kuier. Op pad na Heilbron, naby Greenlands, het dit vreeslik gereën en hulle kon nie verder op die turfpad ry nie. Meraai was saam in die motor en toe ‘n vriendelike boer hulle nooi om vir die nag by hulle oor te bly het hy gesê: “Ek hoop nie julle gee om dat julle dogter vanaand by julle in die kamer slaap nie, want ons net een kamer beskikbaar”. In daardie jare was so iets ondenkbaar en Pa moes toe maar ewe gedweë die nag op die bank in die sitkamer slaap.

Van Vredefort af is Eben verplaas Koppies toe. Omdat hy in sy hart ‘n boer was het hulle op ‘n plot in die omgewing van Weltevrede net buite die dorp gaan woon. Ons drie kinders is al drie gebore terwyl hulle op die plot gewoon het. Daar was koeie, donkies skape, hoenders en kalkoene op die plot en hoewel ek maar net drie jaar oud was toe ons van daar af plaas toe getrek het,, kan ek vandag nog die reuk van die voerkamer onthou waar al die beeste en ander diere se kos gebêre is.

Terwyl hy op Koppies gewerk het, word hy verplaas na Natal. Ek kan ongelukkig nie die naam van die dorp onthou waarheen hy verplaas is nie. Oupa Giel en ouma Santjie was baie ontsteld omdat hulle kind so ver van hulle af moes weggaan en oupa Giel maak aan pa die aanbod om plaas toe te kom om saam met hom te boer. Hierdie groot skuif Leeuspruit toe vind in 1945 plaas net na Uysie se geboorte.

Pa was vasbeslote om van die boerdery ‘n sukses te maak. Die plaas was selfversorgend. Een keer per jaar in die winter is ‘n bees en ‘n vark geslag en wors en biltong en pekelvleis is gemaak. Daar was nognie yskaste of vrieskaste nie en die nodige verkoeling is gedoen met koelers wat deur middel van water wat verdamp het verkoel is. Verder was daar ‘n sifkas waarin vleis gehang is. Die sifkas (‘n “safe” genoem) is van gaas gemaak om vlieë en brommers uit te hou. ‘n Skaap is elke tweede of derde week geslag, daar was hoenders, eende en makoue wat eiers voorsien het en botter is gemaak van die room van die paar melkkoeie wat gemelk is. Rieme is van die beesvelle gebrei wat gebruik is om die osse in te span. Die hoenders, die eiers, die room en die botter was inkomste om die kruideniersware te koop as daar dorp toe gegaan is.

Daar is net met osse geploeg en geplant en geskoffel. Kunsmis was ‘n luukse en ‘n onnodige uitgawe want voor planttyd is die mis uit die bees- en skaapkrale op die lande gegooi as bemesting

Pa was nie bang vir harde werk nie. Oupa het drie plase gehad: Leeuspruit, Stillehoogte en Christina. Laasgenoemde 2 plase was ongeveer 25 Km van Leeuspruit af waar ons gewoon het. As daar op dié twee plase geploeg en geplant is, het hy daar oorgebly van Sondagaand af tot Saterdagmiddag. Daar was geen gebou hier nie en hy het van plastiek kunsmissakke wat hy aanmekaar gewerk het, afskortings gemaak wat om die sleepwa vasgemaak is en daar het hy op ‘n kampbedjie geslaap en sy kos op ‘n oop vuur gemaak. Later is die stoor op Stillehoogte gebou en was dit sy blyplek as die lande daar bewerk is. Uiteindelik het Pa vir Oupa oortuig dat hulle ‘n trekker moet koop. Oupa was nie baie inskiklik nie, maar uiteindelik is daar ‘n Fordsontrekker gekoop. So ‘n mooi bloue.

Soos dit moes gebeur was daar die eerste jaar wat die trekker gebruik is, ‘n totale misoes en Pa en die trekker het die skuld gekry vir die misoes.

Op die plaas was alles nie maanskyn en rose nie. Oupa was ‘n moeilike mens en vir alles wat verkeerd gegaan het het Pa die skuld gekry. Die woordewisselings wat hierop gevolg het was heftig en die enigste ontvlugting was om die vlakte in te loop en daar te gaaan huil.

Ek was nooit bewus van wat die ooreenkoms tussen Pa en Oupa was oor vergoeding nie. Daar is nooit met kinders oor sulke goed gepraat nie. Pa het sy eie beeste en skape gehad en Ma het, so ver as wat ek kan terug onthou met hoenders en kalkoene geboer. Hoedereiers is Vrydae, wat dorpdag was, verkoop en die geld gebruik vir kruideniersware en om ons drie Kokkies se basse toe te hou.
Omdat dit twee huishoudings was, is die kruideniersware se uitgawe tot op die laaste pennie tussen Ma en Ouma gedeel.

Pa was ‘n perdeman en het altyd perde gehad. Op Leeuspruit was dit twee blou skimmelperde met die name Moskou en Breker. Oupa het ook ‘n perekar gehad en die twee skimmels is dikwels voor die kar gespan en dan het ons daarmee by die bure gaan kuier.

Geld vir luukses was daar nooit. Selfs ons kerk en skoolbroekies wat ek en Uysie gedra het, en Sannie se rokkies is deur een of ander “Naaldwerkster” gemaak. Gekoopte klere was ‘n luukse. Een maal per jaar, kort voor Kersfees is ons Potchefstroom toe vir Kersinkopies en dan is daar net by Die Indiërs gekoop, want hulle was die goedkoopste. In daardie jare was dit ‘n vernedering en ‘n skande vir ‘n “Afrikaner” om met ‘n “Koelie” besigheid te doen. My eerste pak klere het ek in 1959 gekry toe ek belydenis van geloof moes aflê. Die pak, ‘n dubbelborspak, is deur ‘n kleremaker op Parys gemaak.

Na ouma Santjie se dood het oom Sypie besluit om af te tree as skoolhoof en het hulle van Welkom af verhuis plaas toe omdat tannie Meraai eendag Leeuspruit sou erf (Leeuspruit was Uysgrond en omdat sy haar ouma Uys se naam gehad het, moes sy Leeuspruit erf). Pa en Ma moes Stillehoogte toe trek. Stillehoogte was Rademangrond en Ma het haar ouma Rademan se naam gehad.

Op Stillehoogte was daar net ‘n stoor en voor hulle kon trek moes daar eers ‘n huis gebou word. Ek onthou nie die jaartal nie, maar dit moes in die omgewing van 1960 gewees het.

Van toe af het dit met die Kokke finasiëel begin beter gaan, want Oupa kon op sy eie manier boer sonder Oupa Giel se inmenging. Uysie skryf in 1963 matriek en vir hom was daar net een ding en dit was “Boer”. Hy doen sy diploma aan die landboukollege op Potchefstroom en na hy daar klaar was, het hy plaas toe gekom en saam met Oupa geboer tot en met Oupa se dood op 21 Februarie 1981. Uysie het natuurlik die plaas Christina ge-erf na oupa Giel se dood omdat hy die eerste naamgenoot van oupa Giel was.

Oupa Eben as gesinsman

Alhoewel geld skaars was, het ons nooit honger gaan slaap nie, maar daar was nooit geld vir luukses nie. Ek wou bitter graag op skool klavierlesse geneem het, maar daar was net nie geld nie.

Op ‘n dag het ek op ‘n brief afgekom wat die bank vir Pa geskryf het om hom te waarsku dat hy baie diep in die skuld is by die bank en hy word gewaarsku om die Bankbestuurder te kom sien in die verband met sy skuld.

As kinders het ons net liefde geken en ek dink nie ek het een dag gehoor dat daar harde woorde tussen my pa en my ma gesê is nie. Saans net voor sononder het ons gaan stap en dan gewedywer om te kyk wie die eerste ‘n aandblom kon sien. Dit was ‘n klein struikie wat net in die nag geblom het en die volgende oggend as die son opgekom het is die blommetjie weer dood.

Saans het ons radio geluister en speletjies gespeel. Pa het ons gereeld laat somme doen en ek en Sannie moes altyd met mekaar meeding om te sien wie die meeste somme reg het. Ons moes ook skrif oefen. Pa het die mooiste handskrif gehad en was baie teleurgesteld dat nie een van ons drie kinders sy mooi handskrif ge-erf het nie. Hy het geglo jy skryf met ‘n vulpen en die balpuntpenne wat toe net in die mode gekom het, was volgens hom die einde van mooi en netjies skryf.

Pa het ook geglo dat ‘n man nie kan trou voor hy nie ‘n skaapboud oordentlik kon sny nie of ‘n hoender kon opsny sonder dat die verskillende dele van hoender stukkend of onherkaanbaar was.

Hy het ook daarvan gehou om party aande kos te maak – dit was wel baie selde. Tamatiebredie was sy forte. Sondae was dit sy werk om die groente uit sy tuin te skil en skoon te maak vir die Sondagmaal.

Sondagaande moes ons na die kerkdiens oor die radio luister en dan het Pa en Ma langs mekaar gesit en hande vasgehou.

Pa was ook ‘n harde man wat nie twee keer gepraat het nie. Ek kan nie een pak slae onthou wat hy een van sy kinders gegee het nie, maar as hy gepraat het, het jy geluister. Ouma was die een wat altyd met die slipper of die hareborsel die slaanwerk moes doen.

Pa was ook, soos dit daardie dae maar die gebruik was, baie hard en kwaai met die swart werkers wat op die plaas gewerk het en hy het baie graag sy vuiste gebruik om gesag af te dwing. Hy was self ‘n harde werker, maar het ook van diegene wat vir hom gewerk het dieselfde verwag.

Oupa Eben as familieman

Omdat Pa, nadat hy plaas toe gekom het, baie min kontak met sy eie familie gehad het wat almal baie ver was, het hy vervreemd geraak van baie van hulle. Al kuiers by familie wat ek onthou was by Ouma en Oupa op Heilbron en dan het ons en die Bosmans gereeld oor en weer gekuier. Vakansies het die drie niggies plaas toe gekom en dit was een groot fees en daar moes in elke vakansie wat hulle daar was, ten minste een “middernagfees” gehou word. Lekkers vir die middernagfees is gekoop met geld wat ons weke voor die tyd begin bymekaar maak het. Die kuiers by sy broer Johan in Pretoria kan ek seker op die vingers van my een hand tele en kuiers van hulle op die plaas onthou ek glad nie. Ook ons en Oom Timo en tannie Thelma het selde oor en weer gekuier. Pa, oom Johan en oom Timo het mekaar net oor en weer gebel as hulle verjaar het.

Op Vredefort het ons tussen Ma se baie ooms en tantes en neefs en niggies gewoon, en hulle het uiteindelik Pa se familie geword. In daardie dae het neefs en niggies mekaar nog as neef en niggie aangespreek en Pa en Ma was Neef Kok en nig Susan.

Onder die neefs en niggis met wie daar ‘n besondere band was, tel Rademan en Kitty Marx. Piet en Paula du Plooy, Piet en Baby Dannhauser, Hannes en Janie Goosen, Piet en Chrissie Rademan en Paul en Sannie Zietsman. Ek weet dat toe die berig deurgekom het van die motorongeluk waarin Paul en Sannie en hulle kinders betrokke was, Pa in die nag in sy kar geklim het en deurgery het Kroonstad toe om te gaan hulp verleen.

Oupa Eben en die Politiek

Pa was van huis uit ‘n Sap – Lid van die Verenigde Party – Oupa Jan was ‘n Smuts aanganger as gevolg van sy deelname aan diie Boereoorlog en Oupa Giel en Ouma Santjie was ook Sappe. Toe ek skool toe is in 1949 was dit net ‘n jaar Nadat die Nasinale Party aan bewind gekom het en as ‘n Sap was jy nêrens welkom nie en nêrens gereken nie. Maatjies wie se ouers Nasionaliste was, het nie graag met jou gespeel nie, want jy is beskou as ‘n Volksverraier.

Oupa het ‘n Sap gebly tot die dag van sy dood en was oortuig daarvan dat die Nasionale party met sy Apartheidsbeleid, die ondergang van Suid Afrika sou beteken. Die dag met Gen. Smuts se begrafnis in Pretoria, is Pa en oupa Giel dou voor dag op die plaas weg om in Pretoria te gaan eer betoon aan die Generaal as sy lykstoet verby gekom het waar hulle langs die straat gestaan het.

Oupa Eben as Kerkmens

Gegewe die huis waarin hy groot geword het, was Pa ‘n baie getroue Kerkmens. Vandat ek my verstand gekry het, het ek geweet Sondag gaan jy kerk toe en na kerk gaan jy Sondagskool toe. Dit het die gronslag vir my eie geloof gelê en van kleins af het ek geweet ek wil soos Oupa Jan ook eendag ‘n predikant word.

Die predikante van Vredefort was huisvriende en veral hy en Dr AMH Koornof was baie goeie vriende en ons het baie oor en weer gekuier.

Pa het baie termyne as diaken in die gemeente op Vredefort gedien en later ook as ouderling. Dit was ‘n voorreg om hom, toe hy ouderling was, ‘n wyksbiduur te hoor lei waar die preek wat in Die Kerkbode gepubliseer is, voorgelees is. Veral was dit mooi om te luister hoe hy die liedere wat gesing is, ingesit het, Hy het ‘n pragtige tenoorstem gehad en het graag gesing terwyl hy gewerk het. As kerkraadslid was Pa baie betrokke by die Sendingkommissie van die kerkraad en die blanke Sendelinge wat in Vredefort gewerk het was ook huisvriende en het gereeld buitedienste op die plaas kom hou. Hier dink ek aan mense soos Eerwaarde Breedt, Eerwaarde Smit en Eerwaarde Haasbroek.

Oupa Eben as Gemeenskapsmens

Eben Kok het die gemeenskap van Vredefort op baie maniere gedien. Daar is jaarliks ‘n landbouskou op Vredefort gehou. Hier rondom die jare 1958 – 1960 was Oupa die president van die Skoukomitee en het ook hierdie rol met onderskeiding vervul.

Oupa Eben, die einde van ‘n era

Pa sterf op 21 Februarie 1981 aan ‘n hartaanval – ‘n hartblok, soos dit op sy doodsertifikaat geskryf is – in die spreekkamer van hulle huisdokter, Wilby Turten op Vredefort.

Hy was relatief ‘n gesonde mens maar enkele jare voor sy dood het hy geweldig las gehad van nierstene. So beland hy omtrent ‘n jaar voor sy dood in Die Zuid Afrikaanse Hospitaal in Pretoria met ‘n niersteen wat hulle nie kon uitkry nie. In daardie dae was daar nognie die moderne tegnieke van vandag nie en hulle besluit uiteindelik om die niersteen deur middel van ‘n operasie te verwyder. Toe hy in die teater op die operasietafel lê, kry hy sy eerste hartaanval.

Ek het deurgery van Warmbad af om hom te gaan besoek. Hy het altyd, as hy oor die politeik gepraat het gesê dat hy hoop as die anderskleuriges die beheer in die land oorneem moet hy al liewer by die groot klomp in die hemel wees. Toe ek hom daarna vra in die hospitaal was sy eerlike antwoord: “As ‘n mens daar kom, dink jy anders daaroor”.

Ek was bevoorreg om sy begrafnisdiens te hou met Openbaring 14: 13 teks – “Geseënd is die wat van nou af in die Here sterwe. Ja sê die Gees: Hulle sal rus van hulle arbeid, want alles wat hulle gedoen het, volg hulle”.

In honour of him, I will continue to make it with his recipe.

(c)  Eben van Tonder en JW Kok

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Counting Nitrogen Atoms – The History of Determining Total Meat Content
Part 2:  Von Liebig and Gerard Mulder’s theory of proteins
By Eben van Tonder
25 September 2018

Previous Installments in Counting Nitrogen Atoms

Part 1: From the start of the Chemical Revolution to Boussingault

Summary

More men and women who led us to the theory of proteins, understanding their metabolism, digestion, characteristics and how to manipulate them followed the work from the 1600s and 1700s.  Few others had such a profound impact on the progression of the concept of protein and understanding its metabolism than Justus von Liebig.  This chapter mainly deals with his contributions, but also that of Dalton, Wöhler, Berzelius, and Mulder.

John Dalton (1766 – 1844)  – developed the atomic theory.

Friedrich Wöhler (1800 – 1882) – In 1828 he was able to synthesise urea.

Justus von Liebig (1803 – 1873) – studied protein metabolism and placed it on the firm chemical basis;  father of agricultural chemistry.

Wöhler, in collaboration with Liebig, developed the organic chemistry concept that a common radical that would combine with other reagents, but still retain its own nature and be recoverable by further reactions.

Jöns Jacob Berzelius (1779 – 1848) coined the term nitrogen and suggested it to Mulder on 10 July 1838.

Gerard Mulder (1802–1880) – in 1839 established the basic nitrogenous component of a number of organic compounds (fibrin, egg albumin, gluten, etc.) to contain  ~16% nitrogen.   It is the basis of the calculation N x 6.25 (1/0.16 = 6.25) to convert nitrogen content into protein content.  He used the term protein to refer to a protein radical.

In 1842 Liebig also contributed to the study of protein metabolism by drawing attention to urea as an end-product of protein breakdown in the body.

Liebig at first embraces the concept, but after contradictory laboratory results, he rejected the theory in its current form in 1847 (English publication).  The concept of a protein radical disappeared from literature, but the concept of protein as the basic building block of nature and the name were retained.

Despite the fact that almost all his theories have been disproven in subsequent years, Liebig made immense contributions in advancing the study of protein metabolism.

Introduction

In bacon production, one determines the total meat content as follows.  Assume you start with 100kg of meat and inject 20L brine.

Meat weight:  100kg
Brine added:  + 20L (100kg becomes 120kg; added through injection/ tumbling)
Loose 10% in cooking/ smoking: – 12kg (120kg becomes 108kg)
Freezing loss of -1%: – 1.08kg yields total bacon ready for slicing: 106.92kg.

Divide the meat weight you started with by the end weight after processing (100/106.92) = 93.52% total meat content.

According to SA regulations, bacon must be at least 95% total meat content.

One doesn’t lose proteins during steam cooking. Only during water cooking. In the older literature on the subject, when they talk about curing, they mean salt only curing as in dry-curing and in this process, there is a loss of proteins (if done in the traditional way of turning the meat every day and allowing the extracted meat juices to run off). If one, however, cooks the bacon, as in Australia, during the cooking step, fat will melt and drip off. Exactly how much fat is lost is determined through analysis. I am sure the % is small, but surprising results are obtained through analysis.

It will impact the calculation since total meat is defined as lean meat plus fat. Meat weight after the actually visible fat has been trimmed off x 0.9 is a good approximation to determine actual lean meat content. All meat contains fat that can not be seen. Without it, meat will be completely un-edible. Two further ratios we want to become familiar with are the ratio of percentage protein nitrogen to lean meat % being N x 30 = lean meat % and the nitrogen to protein factor which is 6.25 meaning N x 6.25 = total protein.

These ratios are important for meat processors.   Let’s look at our calculation again which we used above.  Note that they only achieve total meat content of 93% in their bacon and they need to have it at 95% or above.  They can now do the following:

Meat weight:  100kg
Brine added:  + 20L (100kg becomes 120kg; added through injection)

In the tumbling stage, add 1kg of pork protein (80% actual protein – the other 20% will be a filler).  Of course, various levels of functionality are commercially available and one must inquire of what the actual protein percentage is to complete the calculation.  This means that the nitrogen added in our example of a product with an 80% functionality is 80% x 1kg = 0.800kg protein / 6.25 – the nitrogen-to-protein ratio to give us the weight of the protein nitrogen x 30 – the protein-to-lean-meat factor = 3.84kg lean meat. In other words, by adding 800g functional protein, they have effectively added 3.84kg to the starting meat weight as lean meat.  There is no fat since the added functional pork proteins do not contain fat.

They can then use their starting ratio as 100kg + 3.84kg = 103.84 which, after injection and tumbling (R100kg plus 20L water less smoking loss) will yield them 108kg.  Dividing the meat weight you started with by the end weight after processing is now 103.84/ 108 = 96.1% total meat content which, if this is in SA, places you well within the legal requirements for bacon.

For those interested in having this in a live spreadsheet I include this sheet, courtesy of Dr Francois Mellett. ED2-8 Cost op Protein, LME, and TME.  Here he compares the cost of different protein sources and uses the conversion factor of 4.8 to move between % protein and TME/ LME.  He derives his conversion factor of 4.8 to move between % protein and LME eqw as follows:  The two equations he works with are:

Protein Nitrogen x 6.25 = Proteins

Percentage Lean Meat = (Percentage Protein Nitrogen × 30 )

Let’s take TVP Soy with a protein content of 50%.  Therefore:

Protein Nitrogen x 6.25 = 50%; Protein Nitrogen % = 50%/6.25 = 8

Percentage Lean Meat = (8 × 30 ) = 240/100 = 2.4.

The same can be achieved by the factor 30/6.25 = 4.8; 4.8 x 50% = 250/100 = 2.4

A very small added benefit for the producer will be that the protein added representing 3.84kg lean meat will be cheaper than the actual meat.  There is, therefore, no financial downside for the producer.  The producer is limited in how much of the protein can be added since it will start to affect the appearance and colour of the bacon.  My suspicion is that in countries like Australia, more can be added due to the fact that the bacon is sold fully cooked which yields a paler bacon as opposed to South African producers where the bacon is sold par-cooked and have a much brighter reddish-pinkish appearance. Adding protein, I suspect, will, therefore, have less of an impact in Australia compared to South Africa. I will not be surprised if some Australian producers add a lot more non-meat and meat protein alike and therefore inject more brine.

The reality is that actual food legislation in Australia and New Zealand allows for a slightly different approach which we will look at in detail in the next article. For now, it is enough that we start interacting with some of the values we encounter as we learn how they were discovered.

We continue our fascinating journey by looking at the contribution of a formidable man, Justus von Liebig during whose time, protein was identified and named.  We also encounter our first ratio when Mulder estimated that meat proteins contain 16% nitrogen (N).  By multiplying the nitrogen content by 100/16, the protein content is estimated. Therefore, nitrogen x 6.25 is the protein content.

Justus von Liebig

Justus von Liebig’s father was a chemical manufacturer and had a small laboratory attached to his shop.  Here Justus loved performing experiments and an exceptional life was inspired.  After studying pharmacy, he received a doctorate from the University of Erlangen in Bavaria in 1822.  The Grand Duke of Hesse-Darmstadt and his ministers noticed him and funded his further studies in chemistry under Joseph-Louis Gay-Lussac in Paris between 1822 and 1824.  Gay-Lussac himself found all plant seeds “contain a principle abounding in azote.”  It was, in Paris when a meeting with Alexander von Humboldt, according to him, set his career on the path it took.  Thus far, it was the French chemists who were responsible for the progression of protein metabolism.

Humboldt arranged an appointment for Liebig at the small University of Giessen in May 1824. Liebig wrote about this appointment  that “at a larger university, or in a larger place, my energies would have been divided and dissipated, and it would have been much more difficult, perhaps impossible, to reach the goal at which I aimed.”

Applying the techniques that he learned under Gay-Lussac he changed the face of organic chemistry and became the father of agricultural chemistry. With this, the advance in our understanding of protein metabolism shifted to Germany.

“In Giessen, Liebig built up the most thriving school of organic chemistry than in existence, and he perceived that his studies could be logically extended to the chemistry of the living body. His book “Thierchemie in Ihrer Aufwendung auf Physio logie” appeared in 1840, and an English edition entitled “Animal Chemistry, or Organic Chemistry in its Applications to Physiology and Pathology” was published in 1842. Liebig’s main contribution to the study of protein metabolism was to point to its chemical basis, a contribution he was well fitted to make through his training in France and his own studies in organic chemistry. His views on various aspects of protein metabolism can be assessed by quoting some passages from his books. In “Animal Chemistry” (1842), he writes (p. 40):

“… If we hold that increase of mass in the animal body, that development of its organs, and the supply of waste, — that all this is dependent on the blood, that is, on the ingredients of the blood, then only those substances can properly be called nutritious and considered as food which is capable of conversion into blood. To determine, therefore, what substances are capable of affording nourishment, it is only necessary to ascertain the composition of the food, and to compare it with that of the ingredients of the blood. Two substances require special consideration as the chief ingredients of the blood: . . . fibrine, which is identical in all its properties with muscular fiber, when the latter is purified from all foreign matters. The second principal ingredient of the blood is contained in the serum and gives to this liquid all the properties of the white of eggs, with which it is identical. When heated, it coagulates into a white elastic mass, and the coagulating substance is called albumen. Fibrine and albumen, the chief ingredients of blood, contain, in all, seven chemical constituents, among which nitrogen, phosphorus, and sulphur are found. . . . Chemical analysis has led to the remarkable result that fibrine and albumen contain the same organic elements united in the same proportion…. In these two ingredients of blood the particles are arranged in a different order, as shown by the difference of their external properties; but in chemical composition in the ultimate proportion of the organic elements, they are identical. . . . Both albumen and fibrine, in the process of nutrition, are capable of being converted into muscular fiber, and muscular fiber is capable of being reconverted into blood. . . . All part of the animal body which have a decided shape, which forms parts of organs, contain nitrogen; all of them likewise contain carbon and the elements of water.

The most convincing experiments and observations have proved that the animal body is absolutely incapable of producing an elementary body, such as carbon or nitrogen, out of substances which do not contain it; it obviously follows, that all kinds of food fit for the production either of blood, or of cellular tissue, membranes, skin, hair, muscular fiber, etc. must contain a certain amount of nitrogen, because that element is essential to the composition of the above-named organs; because the organs cannot create it from the other elements presented to them; and, finally, because no nitrogen is absorbed from the atmosphere in the vital process.

The nutritive process in the Carnivora is seen in its simplest form. This class of animals lives on the blood and flesh of the graminivora; but this blood and flesh is, in all its properties, identical with their own. . . . In a chemical sense, therefore, it may be said that a carnivorous animal, in supporting the vital process, consumes itself. That which serves for its nutrition is identical with those parts of its organisation which are to be renewed. The process of nutrition in graminivorous animals appears at first sight altogether different. Their digestive organs are less simple, and their food constituents consist of vegetables, the great mass of which contains but little nitrogen. … Chemical researches have shown, that all such parts of vegetables as can afford nutriment to animals contain certain constituents which are rich in nitrogen; and the most ordinary experience proves that animals require for their support and nutrition less of these parts of plants in proportion as they abound in the nitrogenised constituents. Animals cannot be fed on matters destitute of these nitrogenised constituents. . . . These nitrogenised forms of nutriment in the vegetable kingdom may be reduced to three substances, which are easily distinguished by their external characters. Two of them are soluble in water. The third is insoluble.”

He then proceeds to recognize a vegetable fibrin, vegetable albumin and vegetable casein similar in properties to these animal products, and goes on to comment (p. 48):  “How beautifully and admirably simple, with the aid of these discoveries, appears the process of nutrition in animals, the formation of their organs, in which vitality chiefly resides! Those vegetable principles, which in animals are used to form blood, contain the chief constituents of blood, fibrine and albumen, ready formed, as far as regards their composition. . . . From what has been said, it follows that the development of the animal organism and its growth is dependent on the reception of certain principles identical with the chief constituents of blood.”

Liebig summarizes his views on the role of nitrogen in nutrition as follows (p. 95): . . . “According to what has been laid down in the preceding pages, the substances of which the food of man is composed may be divided into two classes; into nitrogenised and non-nitrogenised. The former is capable of conversion into blood; the latter incapable of this transformation. Out of those substances which are adapted to the formation of blood are formed all the organised tissues. The other class of substances, in the normal state of health, serve to support the process of respiration. The former may be called the plastic elements of nutrition; the latter, elements of respiration. Among the former, we reckon—vegetable fibrine, vegetable albumen, vegetable caseine, animal flesh, animal blood. Among the elements of respiration in our food are—fat, starch, gum, cane sugar, grape sugar, sugar of milk, pectine, bassorine, wine, beer, spirits.”

These comments do not add appreciably to the concepts which Magendie had propounded 25 years before. It will be particularly noted that Liebig had no conception of the possibility of digestion and reconstruction of proteins taken in the diet.”  (Munro and Allison, 1964)

“Liebig and his students also applied oxidizing agents such as manganese dioxide and chromic acid during acid hydrolysis of proteins, thus obtaining and identifying a series of acids and aldehydes. The idea of studying the degradation products of protein, which was to play such an important role in the next generation, stems from Liebig’s imaginative genius.”  (Sahyun, M. (Editor). 1948)

The atomic theory

“Another important advance in chemistry was taking place that would be put to use in subsequent nutritional studies. John Dalton, a poor and largely self-educated schoolmaster in the north of England, had an important idea. This was that all elements are made up of indivisible particles, or “atoms,” and that for each element every atom is identical. Chemical combination occurs when two or more different atoms form a firm union. These ideas were supported by the proportions of different elements in any compound being fixed and by the different compounds between the same two elements being in simple ratios by weight. Thus the gas we call “carbon dioxide” has exactly twice the weight of oxygen (per unit weight of carbon) that is present in the other gas called “carbon monoxide.” Finally, gases were found to combine in simple relations by volume. Thus 3 volumes of hydrogen combine with 1 volume of nitrogen to form exactly 2 volumes of ammonia gas. From this it also follows that equal volumes of different gases contain the same numbers of molecules, once one accepts that many elements, such as hydrogen, oxygen and nitrogen, have two atoms combined together to form a single molecule.

For some years there was controversy as to whether carbon and oxygen each had one-half of the atomic weights that are now assigned to them, although it is easy to correct molecular formulas obtained in that period. Thus Prout, in England, subjected urea to improved methods of analysis and obtained a molecular formula of C₂H₄N₂O₂, which agrees with the modern formula of CH₄N₂O when we double the atomic weights for C and O.

In the following decade, Friedrich Wöhler in Germany found that he had obtained urea by heating silver cyanate with ammonium chloride. He wrote excitedly to his former professor: “I can make urea without the use of kidneys.” Admittedly, urea was only an excretion product, but the synthesis was one small step in demonstrating that an organic compound produced in living systems could also be produced in the laboratory without the aid of any “vital force.”

Wöhler, in collaboration with Liebig, also developed an important concept in organic chemistry. This was the idea of a common radical that would combine with other reagents, but still retain its own nature and be recoverable by further reactions. The first example was the “benzoyl” radical. Starting with benzaldehyde, one could oxidize it to benzoic acid or form a chlorinated derivative, and so on, and then reproduce the original benzaldehyde by appropriate reduction.”  (Carpenter, 2003)

Gerard Mulder and the nature of animal substance

The Dutch chemist Gerard Mulder (1802–1880) had published a paper in a Dutch journal in 1838 and this was reprinted in 1839 in the Journal für praktische Chemie. Mulder had examined a series of nitrogen-rich organic compounds, including fibrin, egg albumin, gluten, etc., and had concluded that they all contained a basic nitrogenous component (~16%)  to which he gave the name of “protein” (Munro and Allison, 1964) from a Greek term implying that it was the primary material of the animal kingdom.

The term protein was coined by Jöns Jacob Berzelius, and suggested it to Mulder who was the first one to use it in a published article. (Bulletin des Sciences Physiques et Naturelles en Néerlande (1838); Hartley, Harold (1951) “Ueber die Zusammensetzung einiger thierischen Substanzen” 1839)). Berzelius suggested the word to Mulder in a letter from Stockholm on 10 July 1838. (Vickery, H, B, 1950)

Mulder also suggested using the symbol “Pr” for the radical, that egg albumin could be expressed as “Pr₁₀ · SP” and serum albumin as “Pr₁₀ · S₂P,” and that the radical itself had the molecular formula “C₄₀H₆₂N₁₀O₁₂.  (Carpenter, 2003)

“”This common nucleus was united to phosphorus and sulfur to give the various compounds referred to above. “Die organische Substanz, welche in allen Bestandtheilen des thier ischen Körpers, so wie auch, wie wir bald sehen, im Pflanzenreiche Vorkommt, könnte Protein von Tporetos primarius, genannt werden. Der Faserstoff und Eiweissstoff der Eierhaben also die Formel Pr + SP, der Eiweissstoff des Serums Pr + SP.” (The organic substance which is found in all the constituents of the animal body, as well as, as we shall soon see, in the vegetable kingdom, might be called protein of Tporetos primarius. The fiber and protein of the eggs thus have the formula Pr + SP, the protein of the serum Pr + SP)  (Munro and Allison, 1964)

“This concept was seized upon by Liebig, who elaborated it thus (p. 104): “… When animal albumen, fibrine, and caseine are dissolved in a moderately strong solution of caustic potash, and the solution is exposed for some time to a high temperature, these substances are decomposed. The addition of acetic acid to the solution causes, in all three, the separation of a gelatinous translucent precipitate, which has exactly the same characters and composition, from whichever of the three substances above mentioned it has been obtained. Mulder, to whom we owe the discovery of this compound, found, by exact and careful analysis, that it contains the same organic elements, and exactly the same proportion, as the animal matters from which it is prepared; insomuch, that if we deduct from the analysis of albumen, fibrine, and caseine, the ashes they yield, when incinerated, as well as the sulphur and phosphorus they contain, and then calculate the remainder for 100 parts, we obtain the same result as in the analysis of the precipitate above described, prepared by potash, which is free from inorganic matter.”  (Munro and Allison, 1964)

Viewed in this light, the chief constituents of the blood and the caseine of milk may be regarded as compounds of phosphates and other salts, and of sulphur and phosphorus, with a compound of carbon, nitrogen, and oxygen, in which the relative proportion of these elements is invariable; and this compound may be considered as the commencement and starting-point of all other animal tissues because these are all produced from the blood. . . . Mulder further ascertained, that the insoluble nitrogenised constituent of wheat flour (vegetable fibrine), when treated with potash, yields the very same product, protein; and it has recently been proved that vegetable albumen and casein are acted on by potash as animal albumen and casein are. The true starting-point for all the tissues is, consequently albumen; all nitrogenised articles of food, whether derived from the animal or from the vegetable kingdom, are converted into albumen before they can take part in the process of nutrition.”

Liebig then (p. 131), like Mulder, ascribes the formula C4s H36N6O14 to protein, and albumen becomes C18H38N6014 + P + S, fibrine is C48E36. N6014 + P + 2 S, and so on. Liebig continued to explore the field of protein chemistry and eventually came to reject Mulder’s original concept of the nucleus of “protein.”

He sets forth his arguments against Mulder at some length in his book “Researches on the Chemistry of Food,” published in an English edition in 1847. Here Liebig indicates that several chemists were unable to repeat some of Mulder’s basic experiments and that his formulas for fibrin, albumin, etc., as compounds of protein with sulfur and phosphorus in specific relations, do not agree with the results of more recent analyses of these substances. In this, he conveniently forgets his own earlier enthusiasm for Mulder’s view and says (p. 18): “… A theoretical view in natural science is never absolutely true, it is only true for the period during which it prevails; it is the nearest and most exact expression of the knowledge and the observations of that period. It ceases to be true for a later period, inasmuch as a number of newly acquired facts can no longer be included in it. . . . But the case is very different with the so-called protein theory, which cannot be regarded as one of the theoretical views just mentioned, since, being supported by observations both erroneous in themselves and misinterpreted as to their significance, it had no foundation in itself, and was never regarded, by those intimately acquainted with its chemical groundwork, as an expression of the knowledge of a given period.” (Munro and Allison, 1964). “Mulder was enraged by the tone of the criticism from Liebig, who was now denying what he himself had previously asserted.” (Carpenter, 2003)

“In the midst of this destructive criticism, however, Liebig is constructive enough to suggest the lines along which research has ultimately resolved the structure of the protein molecule.

He says (p. 27) : … The study of the products, which caseine yields when acted on by concentrated hydrochloric acid, of which, as Bopp had found, Tyrosine and Leucine constitute the chief part, and the accurate determination of the products which the blood constituents, caseine, and gelatine, yield when oxidised, among which the most remarkable are oil of bitter almonds, butyric acid, aldehyde, butyric aldehyde, valerianic acid, valeronitrile, and valeracetonitrile, have opened up a new and fertile field of research into numberless relations of the food to the digestive process, and into the action of remedies in morbid conditions.” (Munro and Allison, 1964)

“It is ironical to think that, in using the word “protein” to denote the most important class of body constituents, we are commemorating an erroneous oversimplification of protein structure, and furthermore are using the word in a meaning different from that originally intended. It is significant that the German word for protein, as English-speaking people now use the word, is “Eiweiss.” This may well be a tribute to Liebig’s eventual rejection of Mulder’s hypothesis.” (Munro and Allison, 1964). Dennis M Bier states that despite these nuances, Berzelius and Mulder were, in the most basic analysis, right: “Protein is the essential general principle of the constituents of the animal body. Thus, one might briefly summarize the physiological roles of protein in metabolism as “responsible for just about everything.” (Bier, D. M., 1999). The concept of a protein radical disappeared from the literature and the term “Protein” gradually began to be applied to all the materials previously described as “animal substance.”   (Carpenter, 2003)

Is Protein the only true nutrient?

In his book, Animal Chemistry or Organic Chemistry in its Application to Physiology and Pathology, Liebig argued that, “because his analyses of muscles failed to show the presence of any fat or carbohydrate, the energy needed for their contraction must come from an explosive breakdown of the protein molecules themselves, resulting in the production and excretion of urea. Protein was therefore the only true nutrient, providing both the machinery of the body and the fuel for its work.

If that was true, what role was left for the other constituents of the diet, and why did carbonic acid production increase so greatly during exercise? Liebig’s explanation was that increased respiration was needed to keep the heart and other tissues from overheating. However, this, unfortunately, led to more oxygen gaining access to the tissues, which could cause oxidative damage and loss of protein tissue. It was the function of the fats and carbohydrates to mop up this excess by being themselves preferentially oxidized.

Liebig’s book was at first generally regarded as a giant intellectual synthesis, and many people were converted to his ideas. For example, when the Professor of Medicine at Edinburgh University was called in to investigate a serious and unexpected outbreak of scurvy in a Scottish prison, his immediate conclusion was that it must be the result of an inadequate intake of protein. However, his calculations indicated that the average daily protein intake was an ample 135 g. But only 15 g of this quantity were from animal sources and 102 g were from gluten.

He suggested that the power of the body to convert gluten to animal protein was limited and that the level of milk in the diet should be increased so as to raise the intake of animal protein.

Another Scottish physician replied that the value of lemon juice in the prevention of scurvy was well established and could not possibly be attributed to its protein content, given that a curative dose contained only a negligible amount of nitrogen.

Another difficulty in believing that muscular work required the breakdown of protein was that the traditional diet of labourers was of lower protein content than of the less active rich. Edward Smith, a British physician and physiologist who was interested in the welfare of prisoners, and was concerned at the stressfulness of their having to work on a treadmill, measured their urea excretion in the 24 h during and after their 8 h of work, and again on their subsequent rest days, and found no difference. This was, of course, quite contrary to what Liebig would have predicted on the basis that the energy expended all came from the breakdown of protein that resulted in the production of urea.”  (Carpenter, 2003)

Liebig and Urine

“Liebig also contributed to the study of protein metabolism by drawing attention to urea as an end-product of protein breakdown in the body. Here again, however, he appears to have been the author of some misconceptions, for in his “Animal Chemistry” (1842), (p. 62) he says: “… We know that the urine of dogs, fed for three weeks exclusively on pure sugar, contains as much of the most highly nitrogenised constituent, urea, as in the normal condition. Differences in the quantity of urea secreted in these and similar experiments are explained by the condition of the animal in regard to the amount of the natural motions permitted. Every motion increases the amount of organised tissue which undergoes metamorphosis. Thus, after a walk, the secretion of urine in man is invariably increased.

Later (p. 245), he says: “The amount of tissue metamorphosed in a given time may be measured by the quantity of nitrogen in the urine.” This statement reflects Liebig’s view that protein in muscle was the fuel for muscular exercise, and he believed that the nitrogenous components of the diet must first be converted to living tissue before being broken down to yield urea. “There can be no greater contradiction, with regard to the nutritive process, than to suppose that the nitrogen of the food can pass into the urine as urea, without having previously become part of an organized tissue.” (p. 144).” (Munro and Allison, 1964)

Liebig’s Contribution to Protein Metabolism

“It may appear in these quotations from Liebig’s writings that he did not contribute much of permanent value to the study of protein metabolism. This is not so. Through his vigorous application of organic analysis to compounds of biological interest, of which he identified several, he laid the foundations of intermediary metabolism and made advances possible for his successors. Thus, although he did not resolve any major problems, he pointed the way to their ultimate solution. Of intermediary metabolism, he says prophetically (“Researches on the Chemistry of Food,” (1847) p. 10): “The intermediary members of the almost infinite series of compounds which must connect Urea and Uric acid with the constituents of the food, are, with the exception of a few products derived from the bile, almost entirely unknown to us; and yet each individual member of this series, considered by itself, inasmuch as it subserves certain vital purposes, must be of the utmost importance in regard to the explanation of the vital processes, or of the action of remedies.”

He was also aware that some chemical reactions only occur in biological systems and suspected that these were dependent on the presence of proteins. The following passage (p. 7) from his book on food chemistry shows how close he comes to our modern concept of enzymes:  …There is, probably, no fact more firmly established as to its chemical signification, than this, that the chief constituents of the animal body, albumen, fibrine, the gelatinous tissues, and caseous matter, when their elements are in a state of motion, that is, of separation, exert on all substances which serve as food for men and animals, a defined action, the visible sign of which is a chemical alteration of the substance brought in contact with them. That the elements of sugar, of sugar of milk, or starch, etc., in contact with the sulphurised and nitrogenised constituents of the body, or with analogous compounds which occur in plants, when these are in a state of decomposition, are subjected to a new arrangement and that new products are formed from them, most of which cannot be produced by chemical affinities, this is a fact, independent of all theory.”

Finally, Liebig contributed to the development of protein metabolism by founding a school of biochemical studies, first in Giessen, and later in Munich, where he became professor of chemistry in 1852. From this school emerged a number of distinguished exponents of metabolism, chief among them being Carl Voit, whose researches in protein metabolism placed the concept of nitrogen balance on a firm footing.

He too had become interested in the subject of “animal chemistry,” and wrote that Dumas must be wrong because it was well known that pigs would fatten when fed on potatoes that were rich in starch, but contained only a negligible level of fat. This meant that animals must be able to convert carbohydrates to fat even though the conversion required “reduction” rather than oxidation.

This was a challenge to the French workers who had been the undisputed authorities in the field, and Boussingault put the matter to the test in another pioneering study. He killed and analyzed the carcass of a young pig, while feeding a littermate of the same starting weight on measured amounts of feed for an additional 3 months. Carcass analysis of the second pig showed that it contained an additional 13.6 kg fat, whereas the feed it had eaten had only contained 6.8 kg.

This careful work had therefore shown that the French school was in the wrong on this point. Boussingault and Dumas both retired from working with animals, and Liebig became the new authority, even though he had never actually carried out a feeding trial. He continued to push his ideas on physiology and nutrition. Most of these were gradually shown to have been completely wrong, but at least they stimulated others to do research, putting them to the test.”  (Munro and Allison, 1964)

Conclusion

Neither Mulder nor Liebig illuminated protein or its metabolism fully, but we gain a great appreciation for the work done by these men in the early 1800s. I wonder how many of today’s researchers would do as much as these men did with the scant knowledge they had.

Nitrogen, key to the art of bacon curing takes front and centre stage in the formulation of the theory of animal proteins and nutrition. It becomes essential, not just in preserving meat, but in defining it. Its chemistry is important, not just to meat processing, but to life itself. It is astounding to recognize a man like Edward Smith as a contemporary of Liebig who would pen one of the most authoritative works on food and nutrition.

The development of the art of meat curing and understanding its chemistry and processes is intimately connected to our most basic understanding of life itself.

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(c) eben van tonder

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References:

Bier, D. M.; The Energy Costs of Protein Metabolism: Lean and Mean on Uncle Sam’s Team, Protein and Amino Acids, 1999, Pp. 109-119. Washington, D.C., National Academy Press

Bulletin des Sciences Physiques et Naturelles en Néerlande (1838). pg 104. SUR LA COMPOSITION DE QUELQUES SUBSTANCES ANIMALES.

Carpenter, K. J.; A Short History of Nutritional Science: Part 1 (1785–1885), The Journal of Nutrition, Volume 133, Issue 3, 1 March 2003, Pages 638–645, https://doi.org/10.1093/jn/133.3.638

Hartley, Harold (1951). “Origin of the Word ‘Protein. Nature 168(4267): 244–244. Bibcode 1951Natur.168..244Hdoi10.1038/168244a0.

Munro, H. N., and Allison, J. B..  1964.  Mammalian Protein Metabolism.   Academic Press.

Vickery, H, B; The origin of the word protein” Yale journal of biology and medicine vol. 22,5 (1950): 387-93.

“Ueber die Zusammensetzung einiger thierischen Substanzen”. Journal für Praktische Chemie (in German).16: 129–152. 1839.doi10.1002/prac.18390160137

Featured Image: Venison Sausage Catalan Style, Robert Goodrick.

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)

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.   (Galloway, 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.

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(c) eben van tonder

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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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References

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Berthollet (1785) “Analyse de l’alkali volatil”(Analysis of volatile alkali), Mémoires de l’Académie Royale des Sciences, 316–326.

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https://kb.osu.edu/dspace/bitstream/handle/1811/28946/Pictorial%20Life%20History_Scheele.pdf?sequence=1