Counting Nitrogen Atoms – The History of Determining Total Meat Content Part 3: Understanding of Protein Metabolism Coming of Age

Counting Nitrogen Atoms – The History of Determining Total Meat Content
Part 3: Understanding of Protein Metabolism Coming of Age
By Eben van Tonder
11 November 2018

For part 1 and 2, click on:

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

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

protein sources

Introduction

The overview of the history of nitrogen as the basis for meat content and nutrition was initiated by interaction between myself and a friend managing one of the largest bacon production lines in Australia.  What exactly is the legislation in New Zealand and Australia related to this?  I asked the help of the imminent meat scientist from South Africa, Dr. Francois Mellette.  The Australia New Zealand Food Standards Code – Standard 2.2.1 – Meat and meat products, states in par 2.2.1-5 with the heading
Requirements for food sold as dried meat or cured and/or dried meat flesh in whole cuts or pieces, manufactured meat or processed meat“, the following:

(2) A food that is sold as cured and/or dried meat flesh in whole cuts or pieces must contain not less than 160 g/kg of meat protein on a fat-free basis.

Dr. Mellette commented that “what this means is that When 80/20 pork is used for bacon, then the 80 has to contain 16% protein. This is just about as much as what it will contain with zero yield. 70/30 has to have a negative yield to achieve this. 90/10 can have approximately a 10% yield to achieve 16% protein in the final product.  To meet 16% protein in the fat-free product, the final total yield can be over 20% addition.”  He did a theoretical calculation with a few assumptions which can be downloaded from this link Australian Bacon 16 Percent Prot in Fat-Free.

I include these practical examples of the application of the work at the beginning of each chapter to interact with the consequences of these historical discoveries as we work our way through history.

Before we commence, lets again plot where we are and where we are going.  We are tracing the development of the scientific understanding of protein metabolism and nutrition, framing the background information to the determination of real meat content and its relationship to nitrogen content.  In my own articles, I follow a chapter on the history of the origin and growth of our present concepts of protein metabolism by Hunro, from the biochemistry department at the University of Glasgow, Scotland, published in 1964 in a book he did with Allison, entitled Mammalian Protein Metabolism. On the subject of the history of our current understanding of nutrition and its relationship to nitrogen content in food, I used as basis an excellent set of articles, done by Carpenter in 2003,  “A Short History of Nutritional Science” from The Journal of Nutrition, Volume 133, Issue 3.

Despite the fact that the development of thought on the two subjects are intertwined, I conclude the historical thread on protein metabolism, do a 4th article on the developments in the field of nutrition and pull it all together for the meat scientists in chapter 6 where we look at the development of the specific thoughts on defining total meat content and its relationship to nitrogen. I add two interesting sections at the end for those who want to know more.

Summary

Having ended the last article by looking at the volcanic nature of the contributions of Justus von Liebig, subsequent years were spent by science still within his gravitational pull.  We now focus exclusively on the development of our understanding of protein metabolism.  Protein metabolism deals with the various biochemical processes responsible for the synthesis of proteins and amino acids.  When we consume proteins, they are broken down in our gastrointestinal tract into individual amino acids by various enzymes and hydrochloric acid.  They are further broken down into α-keto acids which are recycled in the body to generate energy, produce glucose or fat or to form other amino acids, used to build new proteins in the body.  In reviewing this, we will gain a greater understanding of the role of nitrogen in our metabolic processes and the relatively constant nature of nitrogen in the animal body and its various proteins.  This will help us to make sense of the various methods used to determine total meat content.

In particular, we will encounter the contribution of the following scientists and broadening of the following concepts in this article.

Carl Voit (1831 – 1908)  A former pupil of Liebig during the Munich period set out to improve on the methods of measuring nitrogen output in the urine and published extensively on the subject with Bischoff.  After improving on the measurement techniques he was able to demonstrate nitrogen balance determined from intake in the diet and output in the urine and feces.  He, therefore, showed conclusively the fact that, in long-term experiments on healthy experimental subjects, nitrogen intake and output were equal, giving a state of nitrogen equilibrium.

Lyon Playfair (1818-1898).  Professor of chemistry at Edinburgh, this formidable scientist transformed the industrial and educational landscape of the United Kingdom in his time.  He conducted the first scientific studies on the diets of different classes of the population which he reported on in 1853 and in 1865. Playfair arrived at the conclusion that the diet of the average healthy adult should contain 119 gm protein, 51 gm fat and 530 gm carbohydrate.  He states “the opus mechanicum or external dynamical work done by the body of a hard-working labourer, is to be sought in the 3.5 ounces (99.2 grams) of flesh-formers which remain after deducting the amount required for opus vitale from the total plastic food.” Here we can recognize for the first time a subdivision of protein requirements into “wear-and-tear” and other factors.

–  Gelatin and the recognition of the different nutritional value of different proteins.  The Commission de la Gélatine (1841), of which Magendie was a member, reported unfavorably on the nutritive qualities of Gelatin. In 1860, Bischoff and Voit reported that nitrogen equilibrium could not be attained by dogs fed on gelatin as the sole dietary protein. It was discovered that certain amino acids are directly related to nutrition and in 1938, Rose set out a list of amino acids that are essential and nonessential, not only for the rat but also eventually for man. The use of biological methods for the study of differences in protein quality was placed on a firm footing by Karl Thomas.  In 1909, he published the now classical procedure for measuring biological values of proteins by nitrogen balance determinations. This method was used later by Mitchell (1924); from his laboratory since 1924 numerous contributions have demonstrated the precision of this tool in evaluating protein quality. In 1946, Block and Mitchell compared results obtained by this and other biological tests of protein quality with the amino acid composition of the individual proteins, as determined by chemical analysis. They were able to show convincingly that the essential amino acid content of a protein, which they expressed as a “chemical score,” provides a satisfactory indication of the biological value of that protein for man and the rat.

–  The end of the theory of Liebig that the nonnitrogenous constituents of the diet were the sources of body heat, while dietary protein passed directly into the form of blood protein and tissue protein and the latter was the source of muscular energy.  In consequence, the amount of urea excreted was taken to be a measure of the intensity of muscular activity.  Pettenkofer and Voit (1866) showed that exercise was not associated with a significant increase in urea output. At this time, too, Fick and Wislicenus (1865) performed their historic climb in which they ascended on August 30, 1865, to the top of the Faul horn, on which there was a hotel, and there they made a preliminary examination of their urines, completing the analysis on their return to Zürich. During the ascent, they consumed a diet low in nitrogen, and the urinary analysis showed clearly that the output of nitrogen in the urine corresponded to a breakdown of tissue protein quite insufficient to account for the energy used in their climb of some 6000 feet. These and many other experiments of a less heroic nature demonstrated that protein was not the exclusive fuel for muscle work. We also look at the development and progression of Voit own view of protein metabolism.

– During the second half of the nineteenth century, the breakdown of food protein in the alimentary tract was established and investigations were carried out to determine the form in which the products were absorbed into the body and we look at the emergence of the theory that peptides, rather than amino acids are the normal currency of protein metabolism.

Bollman et al. demonstrated in 1924 that complete removal of the liver results in immediate and total suppression of urea synthesis. This was followed in 1932 by the brilliant studies of Krebs and Henseleit which led to the formulation of the arginine-ornithine cycle as the mechanism of urea synthesis.

– We consider the synthesis of protein within the body.

The Development of Nitrogen Balance as a Technique for the Study of Protein Metabolism:  The Era of Carl Voit

“The idea of balance studies equating income with outgo appears to have been first conceived by Boussingault who took up farming in Alsace after having practiced as an engineer in South America. He was interested in the utilization of foodstuffs by his farm animals, and in 1839 he published the results of studies on milk cows in which the total intake of carbon, hydrogen, oxygen, and nitrogen was compared with the total output of these in the urine, feces, and milk. Boussingault (1844) provides more extensive data of this kind in his book, “Economie rurale.” In the case of nitrogen balance, Boussingault observed a small proportion of the intake which could not be accounted for by excretion in the urine, feces, and milk, and he assumed that this nitrogen must have been lost via the lungs. In this respect, he was following the opinion current at that time. This was based on the experiments of Despretz and Dulong, each of whom had found evidence of excretion of body nitrogen by way of the lungs, in contradiction to the earlier studies of Lavoisier (Lusk, 1922). The ridiculous nature of their data was exposed by Liebig, who pointed out that, if one of the dogs studied by Dulong had, in fact, expired from its tissues the amount of gaseous nitrogen reported, it would in 7 days have eliminated all the nitrogen contained in its carcass, leaving only a mass of mineral ash (Lusk, 1922). Nevertheless, this view continued to hold sway for some considerable time, since it was also concluded by Regnault and Reiset in 1849, in their famous study on the respiration of animals, that the well-nourished animal expires a small quantity of body nitrogen.” (Munro and Allison, 1964)

“The concept of the balance of income and outgo was rapidly adopted and in 1897 Atwater and Langworthy were able to publish a compilation of over 3,600 such experiments which had been published in the literature, most of them dealing with nitrogen. In his book on “Animal Chemistry,” Liebig reports a large-scale study of carbon balance on a company of soldiers of the bodyguard of the Grand Duke of Hesse-Darmstadt over a period of a month; nitrogen was not included in the analyses. More extensive use of the balance experiment was, however, made by Bidder and Schmidt, who worked in the German University in Dorpat, Estonia, and in 1852 published their joint book “Die Verdauungssäfte und der Stoffwechsel.” Schmidt (who had been a pupil of Liebig’s) wrote the section on metabolism. He attempted to compute the total metabolism of animals, combining the results of respiratory gas analysis with data obtained from analysis of urine and feces. This allowed Schmidt to study the fate of both the nitrogen and the carbon of dietary protein and from his findings he was able to draw the following striking conclusion (p. 386): “Wom Gruppen verbande des Eiweisses und Collagens spaltet sich fast sämmtlicher Stick stoff mitseinem Bildung des Atomcomplexes Harnstoff erforderlichen C-, H— and O—Aequivalent ab, während der Rest, circa 5/6 der Gesammt menge warmebildenden Materials betragend, der Oxydation zu Kohlen säure und Wasser anheimfällt und nach Erfüllung seiner calorimetrischen Functionen im Lungengaswechsel ausgeschieden wird.” (In the group of proteins and collagen, almost all nitrogen decomposes with the formation of the atomic complex urea necessary C, H, and O equivalent, while the remainder, about 5/6 of the total amount of thermoforming material, oxidizes the oxidation to carbonic acid and water is lost and excreted after fulfillment of its calorimetric functions in the lung gas exchange).” (Munro and Allison, 1964)

“It was, however, in the hands of Carl Voit (1831–1908) that nitrogen balance became a precise tool for the study of protein metabolism. Voit had been a pupil of Liebig during his Munich period. After graduating in medicine at Munich in 1854 he attended classes in science, including the class in chemistry given by Liebig; the instructor in practical chemistry was Pettenkofer, with whom Voit undertook a short study of urea production in patients suffering from cholera (Lusk, 1922). After this period, he spent a year at Göttingen with Wöhler, the organic chemist who had demonstrated a chemical synthesis of urea and who had collaborated with Liebig (Partington, 1951). Voit then returned to Munich to work with Bischoff. Bischoff (1853) had recently published a book with the title “Der Harn stoff als Maass des Stoffwechsels,” (Urea as a measure of metabolism) which was a defense of Liebig’s concept that the amount of urea excreted represented the intensity of tissue break down. Voit applied himself to the improvement of methods of measuring nitrogen output in the urine and, in 1860, he published with Bischoff a book setting down the findings of metabolic experiments using these improved procedures. They describe how dogs were fed with increasing quantities of meat. When the intake was less than 1500 gm of meat, the nitrogen output in the urine and feces exceeded the intake of nitrogen in the meat: there was a loss of nitrogen from the body. When 1800gm of meat were given, output and intake of nitrogen were equal, and with 2000 or 2500 gm of meat per day, a small retention of nitrogen took place. Bischoff and Voit also explored the effect on urea output of feeding carbohydrate and fat along with the meat. More extensive studies of the protein-sparing actions of carbohydrate and fat were later carried out by Voit (1869).” (Munro and Allison, 1964)

“The studies of Bischoff and Voit led naturally on to Voit’s concept of nitrogen balance determined from intake in the diet and output in the urine and feces, and these established the fact that, in long-term experiments on healthy experimental subjects, nitrogen intake and output were equal, giving a state of nitrogen equilibrium. For example, Voit (1866a) found that, after a dog had been consuming meat for 58 days, it had ingested 986 gm of nitrogen, and that the nitrogen of the excreta amounted to 983 gm. From this, it was concluded that other sources of nitrogen loss must be negligible. This thesis was discussed in greater detail by Voit in 1881 when he contributed a section on metabolism and nutrition to Hermann’s “Handbuch der Physiologie” (Manual of Physiology). Evidence obtained since the time of Voit has been summarized in 1928 by Lusk, who concludes “the view that the nitrogen of the urine and faeces could be made a measure for the determination of protein metabolism was thus securely established.” This view has occasionally been challenged (e.g., by Costa, 1960). The use of compounds labeled with heavy nitrogen may finally resolve these doubts.” (Munro and Allison, 1964)

“The use of nitrogen balance in the investigation of protein metabolism was exploited by Voit and his many pupils, who came from an international setting. Among these were Rubner (Germany), Atwater and Lusk (United States), and Cathcart (Britain). With this phase in the history of protein metabolism, we move into modern times, and it is convenient to consider separately the studies made by Voit and his successors in each branch of protein metabolism—on the one hand, nutritional developments; on the other hand, intermediary metabolism of protein. Since both of these are immense topics . . .  only the merest sketch of their recent history will be given, in order to demonstrate the continuity from the time of Voit. . . .  An account of experiments on intermediary metabolism from the time of Voit up to 1921 can be found in the monograph on protein metabolism by Cathcart (1921). Brief mention will finally be made to extension of the study of protein metabolism to disease.” (Munro and Allison, 1964)

The Nutritional Studies of Voit and His Successors: Protein Requirements

“The first scientific studies on the diets of different classes of the population were reported in 1853 and in 1865 by Playfair, who was professor of chemistry at Edinburgh and had been a pupil of Ludwig, the German physiologist. Playfair accepted the doctrine of Liebig that mechanical work was the result of protein breakdown in muscle and his very extensive surveys of dietary intakes among different classes of the population supported him in his supposition that heavy workers eat more protein. He, therefore, set down protein requirements which range from 57 gm in the case of a mere subsistence diet to 184 gm in the case of hard-worked laborers (Playfair, 1865). He states “the opus mechanicum or external dynamical work done by the body of a hard-working labourer, is to be sought in the 3.5 ounces (99.2 grams) of flesh-formers which remain after deducting the amount required for opus vitale from the total plastic food.” Here we can recognize for the first time a subdivision of protein requirements into “wear-and-tear” and other factors, a theme which will recur when the concepts of later investigators in this field are considered.” (Munro and Allison, 1964)

“Playfair arrived at the conclusion that the diet of the average healthy adult should contain 119 gm protein, 51 gm fat and 530 gm carbohydrate. Other estimates made around this time were of similar magnitude, and in 1881 Voit summarized the results of his own researches and of his predecessors and concluded that the protein intake of the average working man should provide 118 gm of protein, as well as 56 gm of fat and 500gm of carbohydrate. For heavy workers, higher intakes of protein were considered necessary. Atwater (1894), a pupil of Voit, supported these figures by surveys of dietaries consumed in the United States. There were, however, other opinions. In 1901, Sivén claimed that a low protein intake (about 30 gm) was adequate to maintain nitrogen equilibrium. This view was supported by the much more extensive experiments of Chittenden (1905), whose interest in the subject grew out of his personal experience of taking a low-protein diet as a treatment for rheumatism (Lusk, 1928). He extended the use of low-protein diets to other subjects, including groups of soldiers, professional men, and athletes. The experiments extended over several months and in consequence of the results obtained, Chittenden (1905) claimed that a protein intake of not more than 50–55 gm daily was sufficient and even desirable for health and vigor. He gained support from the Danish investigator Hindhede (1913), but received much criticism from scientists who had observed lowered resistance to disease on low-protein diets (Cathcart, 1921). The question of recommendations about protein intake has now passed out of the hands of individuals and has become the business of committees of national or international status.” (Munro and Allison, 1964)

The Nutritional Studies of Voit and His Successors:  Recognition of Differences in Protein Quality

“The earlier estimates of requirements did not acknowledge differences in the biological values of different dietary proteins, though these were known to exist in one case: from the earliest studies on dietary protein, it has been recognized that gelatin is not a nutritionally satisfactory protein. The Commission de la Gélatine (1841), of which Magendie was a member, reported unfavorably on its nutritive qualities. In 1840, Liebig had written in “Animal Chemistry” that “animals which were fed exclusively with gelatine, the most highly nitrogenised element of the food of carnivora, died with the symptoms of starvation; in short, the gelatinous tissues are incapable of conversion into blood.” In 1860, Bischoff and Voit reported that nitrogen equilibrium could not be attained by dogs fed on gelatin as the sole dietary protein. However, gelatin remained unique during the rest of the nineteenth century as an isolated example of a food protein whose nutritive qualities were demonstrably inadequate.” (Munro and Allison, 1964)

“The recognition of amino acids as structural components of proteins opened the way for new concepts of the nutritive values of the proteins. Although individual amino acids had been observed as products of the acid hydrolysis of proteins as early as 1820 (Braconnot, 1820), it was not until 1900 that a partial quantitative analysis of individual isolated proteins for amino acid content was made possible by the method of Kossel and Kutcher (1900), who observed wide divergences between different proteins in their content of individual amino acids. The possibility that gelatin owed its lack of nutritive qualities to deficiency of some of these amino acids was tested by Kauffmann (1905), who demonstrated that he could maintain himself in nitrogen equilibrium if he added tyrosine, cystine, and tryptophan to a diet in which the remaining nitrogen was provided by gelatin; this fell short of a convincing demonstration, since a similar type of experiment yielded negative results in the hands of Rona and Müller (1906). The establishment of a link between defects in the nutritive value of a protein and its amino acid composition was first firmly established in 1906 by Willcock and Hopkins, who found that “. . . a dietary containing zein as its only nitrogenous source is unable to maintain growth of young mice. The addition of tryptophane (an amino acid absent from the decomposition products of zein) to such a dietary does not make it capable of maintaining growth. On the other hand, this addition greatly prolongs the survival of animals fed upon zein, and materially adds to the well-being of such animals. The maintaining growth. On the other hand, this addition greatly prolongs the survival of animals fed upon zein, and materially adds to the well-being of such animals. The addition of tyrosine (which is already present in zein), in equivalent amounts, has no such effect. It is suggested that the tryptophane is directly utilized as the normal precursor of some specific hormone or other substance essential to the processes of the body.”” (Munro and Allison, 1964)

“This work was confirmed and extended by Osborne and Mendel (1914), the latter a pupil of Chittenden. They found that addition of lysine, as well as tryptophan to zein, converted it into a protein capable of sustaining the growth of young animals. The later extensive studies of this group demonstrated that some at least of the amino acids are indispensable constituents of the diet. However, the number of proteins completely deficient in one or more amino acids is limited, and an exploration of the essential amino acids was attempted by chemical deletion of single amino acids from protein hydrolysates. This technique also had considerable limitations, and the definitive solution to the problem had to wait until the 1930’s when Rose, a pupil of Mendel, carried out his painstaking work with diets in which protein was completely replaced by mixtures of purified amino acids. This culminated in the classification (Rose, 1938) of amino acids as essential and nonessential, not only for the rat but also eventually for man. The use of biological methods for the study of differences in protein quality was placed on a firm footing by Karl Thomas, who had studied in Rubner’s laboratory. In 1909, he published the now classical procedure for measuring biological values of proteins by nitrogen balance determinations. This method was used later by Mitchell (1924); from his laboratory since 1924 numerous contributions have demonstrated the precision of this tool in evaluating protein quality. In 1946, Block and Mitchell compared results obtained by this and other biological tests of protein quality with the amino acid composition of the individual proteins, as determined by chemical analysis. They were able to show convincingly that the essential amino acid content of a protein, which they expressed as a “chemical score,” provides a satisfactory indication of the biological value of that protein for man and the rat. With the assurance that the nutritive value of a protein is a function of its content of essential amino acids, and with estimates by Rose and other workers of the essential amino acid requirements of man, we are now, in the second half of the twentieth century, well situated to proceed to a rational determination of the requirements of the body for protein in relation to different types of dietary sources.”  (Munro and Allison, 1964)

Studies on the Metabolism of Nitrogenous Materials since the Time of Voit: General Theories of Protein Metabolism

“At the time when Voit entered the field of protein metabolism, the current view was that of Liebig, who held that the nonnitrogenous constituents of the diet were the sources of body heat, while dietary protein passed directly into the form of blood protein and tissue protein and the latter was the source of muscular energy. In consequence, the amount of urea excreted was taken to be a measure of the intensity of muscular activity. At first, Voit accepted this view (Bischoff and Voit, 1860), but soon Pettenkofer and Voit (1866) carried out experiments which showed that exercise was not associated with a significant increase in urea output. At this time, too, Fick and Wislicenus (1865) performed their historic climb in which they ascended on August 30, 1865, to the top of the Faul horn, on which there was a hotel, and there they made a preliminary examination of their urines, completing the analysis on their return to Zürich. During the ascent, they consumed a diet low in nitrogen, and the urinary analysis showed clearly that the output of nitrogen in the urine corresponded to a breakdown of tissue protein quite insufficient to account for the energy used in their climb of some 6000 feet. These and many other experiments of a less heroic nature demonstrated that protein was not the exclusive fuel for muscle work. The subsidiary question of whether protein metabolism is affected in any way by muscular exertion remained a battle-ground for many years. Cathcart, in reviewing the evidence in 1925, came to the conclusion that a small increment in nitrogen excretion is a necessary concomitant of exercise, but this remains debatable.” (Munro and Allison, 1964)

“Following the disproof of Liebig’s hypothesis of protein metabolism, Voit advanced his own view of protein metabolism. Voit (1866b) had observed with dogs that the output of nitrogen during the first few days of a fast varied directly with the amount of protein given in the preceding diet. This suggested to him that a labile store of protein was being lost from the body during a period of fasting. He then performed a series of experiments on dogs brought into nitrogen equilibrium at different levels of protein intake (Voit, 1867). When the protein content of the diet was changed, there was at first a period during which the body either gained or lost nitrogen before it attained equilibrium. Ongoing from a low to a higher protein level, some nitrogen was at first retained; alternatively, on passing from a higher to a lower level of intake, the body at first lost nitrogen. Voit, therefore, postulated that there is a variable pool of labile protein in the body, “circulating” or “storage” protein (“Worrathseiweiss”), which is distinct from tissue protein (“Organseiweiss”). The amount of circulating protein in the body is related to the level of protein in the diet. It is readily catabolized, whereas the tissue protein is resistant. A small amount of the tissue protein is constantly renewed from material drawn from the circulating protein. This theory was accepted and elaborated by several of Voit’s contemporaries, and notably by Rubner (1908), one of Voit’s pupils. The separation of protein metabolism into independent compartments reached its nadir with Folin (1905), who studied the effect of nitrogen-rich and nitrogen-poor diets on urinary composition. Folin observed that a change in protein intake affected the output of some urinary components but not of others, and he described “laws governing the chemical composition of urine.” Thus, a reduction in protein intake causes a large reduction in output of urea and inorganic sulfate, but little or no change in output of creatinine, neutral sulfur and only slight changes in excretion of uric acid and ethereal sulfates. He concludes:

“… To explain such changes in the composition of the urine on the basis of protein katabolism, we are forced, it seems to me, to assume that katabolism is not all of one kind. There must be at least two kinds. Moreover, from the nature of the changes in the distribution in the urinary constituents, it can be affirmed, I think, that the two forms of protein katabolism are essentially independent and quite different. One kind is extremely variable in quantity, the other tends to remain constant. The one kind yields chiefly urea and inorganic sulphates, no kreatinin, and probably no neutral sulphur. The other, the constant katabolism, is largely represented by kreatinin and neutral sulphur, and to a less extent by uric acid and ethereal sulphates.”

“If there are two distinct forms of protein metabolism represented by two different sets of waste products, it becomes an exceedingly interesting and important problem to determine, if possible, the nature and significance of each. The fact that the kreatinin elimination is not diminished when practically no protein is furnished with the food, and that the elimination of some of the other constituents is only a little reduced under such conditions, shows why a certain amount of protein must be furnished with the food if nitrogen equilibrium is to be maintained. It is clear that the end-products which tend to be constant in quantity appear to be indispensable for the continuation of life; or, to be more definite, those metabolic processes probably constitute an essential part of the activity which distinguishes living cells from dead ones. I would, therefore, call the protein metabolism which tends to be constant, tissue metabolism or endogenous metabolism, and the other, the variable protein metabolism, I would call the exogenous or intermediate metabolism.”” (Munro and Allison, 1964)

“This separation of protein metabolism into endogenous and exogenous parts was widely accepted, in some cases with the addition of further variations in the theory to allow for certain observations. A very full history of this era is given in Mitchell and Hamilton’s (1929) book “The Biochemistry of the Amino Acids.” (Munro and Allison, 1964)

“In 1935, the Folin theory was seriously challenged by Borsook and Keighley, on the basis of studies on nitrogen and sulfur excretion under various conditions; they concluded that a large part of the daily nitrogen intake is immediately synthesized into a labile pool of body protein, which they designate the “continuing metabolism” of protein. They state: “The continuing metabolism in the normal dietary state in man is quantitatively more important than the endogenous metabolism postulated by Folin. If the metabolism which leads to the urinary creatinine be excluded it is an open question whether the remainder of the endogenous metabolism yielding urea, ammonia, and part or possibly all the uric acid has any physiological reality,” and they propose “much more extensive synthetic processes continually in operation.” Folin’s theory of separate endogenous and exogenous metabolisms was finally discredited as an accurate interpretation of the facts by the isotopic studies of Schoenheimer (1898–1941) who had studied biochemistry under Karl Thomas in Leipzig. The use of isotopes in the study of biological reactions had previously been introduced in 1923 by Hevesy, who employed a natural radioactive isotope of lead (radium D) to study lead metabolism in plants. The extensive application of stable isotopes to the solution of problems in the metabolism of organic molecules was initiated by Schoenheimer and his colleagues in the 1930’s and the results are summarized in Schoenheimer’s (1942) posthumous book “The Dynamic State of Body Constituents.” In this book, Schoenheimer describes experiments with amino acids, labeled with N15, which were fed to rats for 3 days along with an adequate protein intake.

“… According to the concept of independent exogenous and endogenous types of metabolism, most of the dietary nitrogen should have appeared directly in the urine. This was not the case. With leucine less than one-third, with glycine less than one half was excreted; the balance remained in the body. Of the isotopic nitrogen retained, the non-protein nitrogen fraction contained only a small amount. The protein must, therefore, have been involved in very rapid chemical reactions resulting in the fixation of at least half of the nitrogen of the added amino acids. As the weight of the animals had remained constant, the processes in question must have been so balanced as to avoid ultimate change in the amounts of the proteins.” . . . “Different organs are not equally effective in the fixation of dietary nitrogen. The isotope concentration in the protein nitrogen of the various organs indicates the relative activity of the respective proteins in regard to the acceptance of dietary protein nitrogen. The proteins of the internal organs, of serum, and of the intestinal tract are the most active; the proteins of muscles show less activity. . . . As might be expected, the proteins of the skin show least activity.”

“These sentences are reminiscent of the concept of the instability of tissue components put forward by Magendie in 1829 on the basis of the evidence then available, and previously quoted; some of his words are worth recalling here to show their similarity to Schoenheimer’s: “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. Nutrition is more or less rapid according to the tissues.” Schoenheimer’s book was a seminal work which announced the new tool of isotopically labeled compounds in metabolic researches, prominent among which was renewed assault on the problem of protein biosynthesis. Schoenheimer’s findings have disposed of Folin’s concept of distinct endogenous and exogenous compartments of protein metabolism. They do not, however, exclude the occurrence of a pool of “storage” or “circulating” protein such as Woit, Rubner, and Borsook and Keighley had envisaged. A pool of “storage” protein which varies with the level of protein in the diet remains for many a convenient concept in the interpretation of experiments on protein metabolism in the whole animal (e.g., Whipple, 1948). The reality of protein stores is a subject which will be considered later in this book.” (Munro and Allison, 1964)

Studies on the Metabolism of Nitrogenous Materials since the Time of Voit: Intermediate Steps in Protein Metabolism

“The role of digestion in protein utilization was unknown to Liebig, who considered that the food proteins were merely solubilized in digestion in order to be used for blood and tissue protein formation (Liebig, 1842, p. 109). During the second half of the nineteenth century, the breakdown of food protein in the alimentary tract was established and investigations were carried out to determine the form in which the products were absorbed into the body. Thus, in 1867 the action of trypsin was studied by Kühne who had been a pupil of Wöhler and with whom Chittenden later worked. The history of this era has been well described by Cathcart (1921) in his monograph on protein metabolism. It culminated in the general conclusion that the absorbed product took the form of free amino acids. This view was particularly suggested by the demonstration by Van Slyke and Meyer (1912) that the concentration of free amino acids in the blood rises after a meal of protein. Nevertheless, as recently as 1954, it was possible for Fisher in his book of protein metabolism to conclude that “evidence for the chemical form of the products of protein absorption is unreliable” and to suggest “that there is enough evidence to consider seriously the possibility that peptides rather than amino acids are the normal currency of protein metabolism.”” (Munro and Allison, 1964)

“As early as 1823, Prévost and Dumas, having disproved the origin of urea from the kidney, had suggested that it was synthesized in the liver and in 1882 Schroeder showed that urea was formed on perfusing the liver with ammonium salts. Nevertheless, exclusive synthesis of urea in the liver remained a battleground for many years (Cathcart, 1921; Mitchell and Hamilton, 1929) until a satisfactory procedure for total hepatectomy was devised. With this tool, Bollman et al. demonstrated in 1924 that complete removal of the liver results in immediate and total suppression of urea synthesis. This was followed in 1932 by the brilliant studies of Krebs and Henseleit which led to the formulation of the arginine-ornithine cycle as the mechanism of urea synthesis. The problem of the mechanism by which the amino groups of the amino acids become available for urea synthesis was materially assisted by the discovery in 1937 of the transaminases by Braunstein and Kritzman.” (Munro and Allison, 1964)

“Braunstein and Kritzman. The fate of the carbon skeleton released from amino acids is a problem more properly belonging to general intermediary metabolism than to the metabolism of proteins. The energy yielded by oxidation of protein in the human body was carefully assessed towards the end of the nineteenth century by Rubner (1885) and by Atwater (see Atwater and Bryant 1899), both pupils of Woit. Atwater’s studies, though exhaustively thorough, are rather inaccessible, but a good review of Rubner’s and Atwater’s work has been provided by Morey (1936). The specific dynamic action following ingestion of food, which had been noted by Lavoisier, was the subject of extensive study by Rubner (1902) and by Lusk (1928), but the reason for the considerable release of energy after consuming protein is still a matter of speculation.” (Munro and Allison, 1964)

“Finally, the synthesis of protein within the body has attracted considerable attention. It was noted soon after the first studies on digestion of protein by gastrointestinal enzymes that, under suitable conditions, these enzymes could produce protein-like products, the plasteins, from partially digested proteins (see review by Wasteneys and Borsook, 1930). This gave rise to speculation as to whether protein synthesis in the tissues is due to reversal of proteolysis catalyzed by means of digestive enzymes present in the cells. This view was eventually rejected, since the products formed by reversal of proteolysis are clearly not similar to tissue proteins. Nevertheless, the synthetic functions of the digestive proteolytic enzymes attracted attention again a few years ago, when it was shown that they were capable of transferring amino acids from one peptide to another—“trans peptidation” (Fruton, 1957). In this way, peptides could be extended and might give rise to proteins. However, this theory has never reached the stage of development at which it can account for the specificity of protein synthesis, and has consequently languished.” (Munro and Allison, 1964)

“In the meantime, a development in the biological study of the cell had disclosed a property which was to become a central tenet in a new and more fruitful theory of protein biosynthesis. Caspersson (1941), working in Stockholm with ultraviolet microscopy, and Brachet (1941), working in Brussels with histochemical techniques, had observed that ribonucleic acid varies in abundance in different cells. Independently in 1941, each of these investigators showed that the intracellular concentration of ribonucleic acid in different cells is proportional to the intensity of protein synthesis in each type of cell and they suggested that ribonucleic acid might play a part in protein formation. Ribonucleic acid occurs as a component of the nucleus as well as of the cytoplasm. In his book, Caspersson (1950) describes from ultraviolet absorption studies how chromosomal deoxyribonucleic acid controls the formation of ribonucleic acid in the nucleolus which then directs the formation of cytoplasmic proteins through subcellular particles containing ribonucleic acid. Attempts to verify this view have been the occasion for much work with cell-free systems, and eventually many aspects of Caspersson’s picture of protein formation would appear to have been justified. In this respect, the studies of Zamecnik and Hoagland on the initial steps from the free amino acid pool to the first stages in protein assembly have been particularly fruitful (Hoagland, 1960). The final answer to the problem of protein biosynthesis will embrace many aspects of biology, from genetics to embryological differentiation.” (Munro and Allison, 1964)

Studies on the Metabolism of Nitrogenous Materials since the Time of Voit: The Study of Protein Metabolism in Pathological Conditions

“Mention should also be made of the stimulus given by Voit to the study of protein metabolism under pathological conditions. Work in this field was stimulated by the possibility of making nitorogen balance measure were very thoroughly surveyed by von Noorden in 1907. Later studies are too numerous to consider here. Many of these phenomena are readily explicable on the basis of our knowledge of the control of protein metabolism in the normal subject, but one still preserves its mysterious nature. That is the increased nitrogen output and negative nitrogen balance which follows injury. A number of authors writing during the latter years of the nineteenth century had suggested that trauma was followed by an increased output of urea (e.g., Malcolm, 1893), but it received only sporadic attention until Cuthbertson, a pupil of Cathcart’s, initiated a systematic study of the phenomenon from 1930 onwards. As a result, it became recognized that, following a fracture or other severe injury, there is marked and early loss of nitrogen, sulfur and phosphorus. The importance of this phenomenon was recognized during the Second World War, but the reason for its occurrence is still unsolved.” (Munro and Allison, 1964)

Conclusion

“From this outline of the history lying behind our present concepts of protein metabolism, certain salient features emerge. The most striking of these is the unbroken tradition handed down in continuity from one investigator to the next for a period of 2 centuries. Joseph Black’s discovery of carbon dioxide in 1756 was the first step towards the understand ing of oxidation and thus was the foundation of modern chemistry. It opened up the possibility of the existence of a variety of gases in the atmosphere, among which nitrogen was separated in 1772 by Daniel Rutherford.

Family Tree of Protein Metabolism.png

The work of Black was acknowledged as an inspiration by Lavoisier, who carried out the first experiment to determine whether atmospheric nitrogen was exchanged with the nitrogen of the body. Lavoisier’s work with the gaseous elements led to the supplanting of the phlogiston theory by the modern theory of oxidation and through this to the chemical analysis of compounds of biological interest. In particular, 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; it will be noted that these analyses were based on procedures in which gases were evolved or were absorbed and thus were direct consequences of the earlier studies on the properties of atmospheric gases. 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 Munro who wrote the chapter quoted here.

Lavoisier, Liebig, and Voit were all systematic and industrious. This devotion to their subject not only allowed them to develop a theme, but it also gave them the authority which goes with lengthy experience in a subject. This authority continued to be effective after they had ceased to carry out active experimental work. When Liebig went to Munich in 1852, he had already ceased to perform experiments but was still able to inspire Voit in 1854 to enter the field of protein metabolism. A century before, Joseph Black made his only investigation into the atmospheric gases, but he continued to take an active interest in the development of the new chemistry and in 1772 this led his pupil Daniel Rutherford to the isolation of nitrogen. Magendie on physiology, Liebig on animal chemistry, Voit on protein and nutrition in Hermann’s Handbuch, Rubner on energy exchange, and even in modern times, Schoenheimer on the “Dynamic State of Body Constituents,” had a considerable influence on contemporary scientific thinking.”  (Munro and Allison, 1964)

Want to know more?  Further Study with Kendra Sticka and Zach Murphy

Want to know more?  A Short Current Description of Protein Metabolism

From BC Open Textbooks

“Much of the body is made of protein, and these proteins take on a myriad of forms. They represent cell signaling receptors, signaling molecules, structural members, enzymes, intracellular trafficking components, extracellular matrix scaffolds, ion pumps, ion channels, oxygen and CO2 transporters (hemoglobin). That is not even the complete list! There is protein in bones (collagen), muscles, and tendons; the hemoglobin that transports oxygen; and enzymes that catalyze all biochemical reactions. Protein is also used for growth and repair. Amid all these necessary functions, proteins also hold the potential to serve as a metabolic fuel source. Proteins are not stored for later use, so excess proteins must be converted into glucose or triglycerides, and used to supply energy or build energy reserves. Although the body can synthesize proteins from amino acids, food is an important source of those amino acids, especially because humans cannot synthesize all of the 20 amino acids used to build proteins.

The digestion of proteins begins in the stomach. When protein-rich foods enter the stomach, they are greeted by a mixture of the enzyme pepsin and hydrochloric acid (HCl; 0.5 percent). The latter produces an environmental pH of 1.5–3.5 that denatures proteins within food. Pepsin cuts proteins into smaller polypeptides and their constituent amino acids. When the food-gastric juice mixture (chyme) enters the small intestine, the pancreas releases sodium bicarbonate to neutralize the HCl. This helps to protect the lining of the intestine. The small intestine also releases digestive hormones, including secretin and CCK, which stimulate digestive processes to break down the proteins further. Secretin also stimulates the pancreas to release sodium bicarbonate. The pancreas releases most of the digestive enzymes, including the proteases trypsin, chymotrypsin, and elastase, which aid protein digestion. Together, all of these enzymes break complex proteins into smaller individual amino acids (Figure 1), which are then transported across the intestinal mucosa to be used to create new proteins, or to be converted into fats or acetyl CoA and used in the Krebs cycle.

The left panel shows the main organs of the digestive system, and the right panel shows a magnified view of the intestine. Text callouts indicate the different protein digesting enzymes produced in different organs.
Figure 1. Digestive Enzymes and Hormones. Enzymes in the stomach and small intestine break down proteins into amino acids. HCl in the stomach aids in proteolysis, and hormones secreted by intestinal cells direct the digestive processes.

In order to avoid breaking down the proteins that make up the pancreas and small intestine, pancreatic enzymes are released as inactive proenzymes that are only activated in the small intestine. In the pancreas, vesicles store trypsin and chymotrypsin as trypsinogen and chymotrypsinogen. Once released into the small intestine, an enzyme found in the wall of the small intestine, called enterokinase, binds to trypsinogen and converts it into its active form, trypsin. Trypsin then binds to chymotrypsinogen to convert it into the active chymotrypsin. Trypsin and chymotrypsin break down large proteins into smaller peptides, a process called proteolysis. These smaller peptides are catabolized into their constituent amino acids, which are transported across the apical surface of the intestinal mucosa in a process that is mediated by sodium-amino acid transporters. These transporters bind sodium and then bind the amino acid to transport it across the membrane. At the basal surface of the mucosal cells, the sodium and amino acid are released. The sodium can be reused in the transporter, whereas the amino acids are transferred into the bloodstream to be transported to the liver and cells throughout the body for protein synthesis.

Freely available amino acids are used to create proteins. If amino acids exist in excess, the body has no capacity or mechanism for their storage; thus, they are converted into glucose or ketones, or they are decomposed. Amino acid decomposition results in hydrocarbons and nitrogenous waste. However, high concentrations of nitrogen are toxic. The urea cycle processes nitrogen and facilitates its excretion from the body.

Urea Cycle

The urea cycle is a set of biochemical reactions that produces urea from ammonium ions in order to prevent a toxic level of ammonium in the body. It occurs primarily in the liver and, to a lesser extent, in the kidney. Prior to the urea cycle, ammonium ions are produced from the breakdown of amino acids. In these reactions, an amine group, or ammonium ion, from the amino acid is exchanged with a keto group on another molecule. This transamination event creates a molecule that is necessary for the Krebs cycle and an ammonium ion that enters into the urea cycle to be eliminated.

In the urea cycle, ammonium is combined with CO2, resulting in urea and water. The urea is eliminated through the kidneys in the urine (Figure 2).

This image shows the reactions of the urea cycle and the organelles in which they take place.
Figure 2. Urea Cycle. Nitrogen is transaminated, creating ammonia and intermediates of the Krebs cycle. Ammonia is processed in the urea cycle to produce urea that is eliminated through the kidneys.

Amino acids can also be used as a source of energy, especially in times of starvation. Because the processing of amino acids results in the creation of metabolic intermediates, including pyruvate, acetyl CoA, acetoacyl CoA, oxaloacetate, and α-ketoglutarate, amino acids can serve as a source of energy production through the Krebs cycle (Figure 3). Figure 4 summarizes the pathways of catabolism and anabolism for carbohydrates, lipids, and proteins.

This figure shows the different reactions in which products of carbohydrate breakdown are converted into different amino acids.
Figure 3. Energy from Amino Acids. Amino acids can be broken down into precursors for glycolysis or the Krebs cycle. Amino acids (in bold) can enter the cycle through more than one pathway.
This diagram shows the different metabolic pathways, and how they are connected.
Figure 4. Catabolic and Anabolic Pathways. Nutrients follow a complex pathway from ingestion through anabolism and catabolism to energy production.
Disorders of the…

Metabolism: Pyruvate Dehydrogenase Complex Deficiency and Phenylketonuria

Pyruvate dehydrogenase complex deficiency (PDCD) and phenylketonuria (PKU) are genetic disorders. Pyruvate dehydrogenase is the enzyme that converts pyruvate into acetyl CoA, the molecule necessary to begin the Krebs cycle to produce ATP. With low levels of the pyruvate dehydrogenase complex (PDC), the rate of cycling through the Krebs cycle is dramatically reduced. This results in a decrease in the total amount of energy that is produced by the cells of the body. PDC deficiency results in a neurodegenerative disease that ranges in severity, depending on the levels of the PDC enzyme. It may cause developmental defects, muscle spasms, and death. Treatments can include diet modification, vitamin supplementation, and gene therapy; however, damage to the central nervous system usually cannot be reversed.

PKU affects about 1 in every 15,000 births in the United States. People afflicted with PKU lack sufficient activity of the enzyme phenylalanine hydroxylase and are therefore unable to break down phenylalanine into tyrosine adequately. Because of this, levels of phenylalanine rise to toxic levels in the body, which results in damage to the central nervous system and brain. Symptoms include delayed neurological development, hyperactivity, mental retardation, seizures, skin rash, tremors, and uncontrolled movements of the arms and legs. Pregnant women with PKU are at a high risk for exposing the fetus to too much phenylalanine, which can cross the placenta and affect fetal development. Babies exposed to excess phenylalanine in utero may present with heart defects, physical and/or mental retardation, and microcephaly. Every infant in the United States and Canada is tested at birth to determine whether PKU is present. The earlier a modified diet is begun, the less severe the symptoms will be. The person must closely follow a strict diet that is low in phenylalanine to avoid symptoms and damage. Phenylalanine is found in high concentrations in artificial sweeteners, including aspartame. Therefore, these sweeteners must be avoided. Some animal products and certain starches are also high in phenylalanine, and intake of these foods should be carefully monitored.


For part 4, click on

Counting Nitrogen Atoms – The History of Determining Total Meat Content
Part 4: Being written

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References:

Ahren, Kevin, PhD was a Professor in the Department of Biochemistry and Biophysics at Oregon State University.  His lectures are available on line at https://www.lecturio.com/medical-courses/history-introduction-to-biochemistry.lecture, on Youtube.  He is a co-author on three popular Open Educational electronic textbooks. They are 1) “Biochemistry Free and Easy,” 2) “Biochemistry Free For All,” and 3) “Kevin and Indira’s Guide to Getting Into Medical School.” Each of these books can be downloaded for free at http://biochem.science.oregonstate.edu/content/biochemistry-free-and-easy.

Australia New Zealand Food Standards Code – Standard 2.2.1 – Meat and meat products

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

Millett, F.  Private Communication

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

Murphy, Zach: A co-founder of Ninja Nerd Science and is responsible for preparing, drawing and presenting the scientific information. Zach attended Misericordia University where he received his Bachelors of Science degree in Biology and a minor in chemistry.

Sticka, Kendra. Received her Ph.D. in Biochemistry and molecular biology from the University of Alaska, Fairbanks in 2016. She currently is an Assistant Professor at the University of Alaska, Anchorage.

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