Before the first decade or so of the twentieth century, it was thought that proteins were all of generally similar nutritional value, despite recognition of very obvious differences in their chemical and physical properties.
Rubner, however, in the late 1890s, observed that proteins of varying origins were not of the same value in nutrition, and the earlier work of the Magendie Commission on Gelatin had demonstrated that gelatin could not support life (McCollum 1957). But these efforts and numerous nitrogen balance studies performed after the 1850s were essentially disregarded until the beginning of the next century (see Munro 1964a) when work reported from England confirmed that the different values resulted from lack of amino acids (Willcock and Hopkins 1906-7).
Sherman (1914), in the first edition of his textbook (the eighth edition appeared in 1952), defined protein in basically modern terms: “The word protein should designate that group of substances, which consist, as far as at present is known, essentially of combinations of alpha-amino acids and their derivatives” (1914: 26). By 1914, Lusk, in a lecture to the New York Academy of Sciences on the importance of proteins, could provide an almost complete list of those amino acids recognized today as constituents of proteins, with methionine and threonine the most notable absentees.
Within a decade of Lusk’s lecture, Thomas B. Osborne and Lafayette B. Mendel at Yale University had convincingly demonstrated that there were major differences in the quality of proteins and had established the basis of our present evaluation techniques. Mendel suspected that the concentration of protein in a diet was not a completely satisfactory indicator of its ability to support growth, whereas Osborne had isolated a number of pure proteins. Thus began a most fruitful collaboration whereby Mendel studied the growth response of rats that were fed the proteins isolated by Osborne.
They found, quite early in their studies, that the various isolated proteins differed in their potential to support growth because the weight gain (of rats) per unit of protein consumed varied significantly. Hence was born the PER or protein efficiency ratio (Osborne, Mendel, and Ferry 1919), which remained the official method for the evaluation of protein in the United States (Pellett 1963) until the protein quality evaluation report by the FAO/WHO in 1991. From these collaborative studies came the early conclusions that rats grew better on animal proteins than on plant proteins, but that adding some of the then newly isolated and chemically identified amino acids to plant proteins could make them equal to animal proteins in their growth-promoting ability. The broad principles of protein nutrition that we accept today were well known by the second decade of the twentieth century (Mendel 1923).
Whether, however, there were fundamental differences between animal and vegetable proteins remained an important issue. It was a debate in which agreement was reached much earlier in scientific circles than in the public arena, where it remained an issue at least until the 1950s (Block and Bolling 1951). By that time it was agreed that an essential amino acid from a plant was the same as one from an animal but that the proportions present in plant and animal foods could differ and that mixing proteins in the diet (complementation) could confer nutritional benefits.
Nitrogen balance techniques had been in use at least since Boussingault in 1839, but the determination of the biological values of proteins from nitrogen balance data is generally attributed to K. Thomas (1909).The ability to scale the nutritional differences in proteins numerically arose in the first and second decades of the twentieth century with the introduction of the PER by Osborne, Mendel and E. L. Ferry (1919) and the refinement of Thomas’s nitrogen balance techniques by H. H. Mitchell in 1924.
Protein and Amino Acid Analysis
Before nitrogen balance and other biological methods for the evaluation of protein quality could be used in any routine manner, improvements in procedures for the analysis of protein were required. Although the Dumas procedure (which converted the nitrogen of protein to gaseous nitrogen that could then be measured) had been available since 1835, it was not until 1883 that J. Kjeldahl described a convenient procedure for converting the nitrogen of most organic materials into ammonia. The procedure involved boiling the material in the presence of concentrated sulfuric acid and a catalyst. After release with a strong base, the ammonia could be determined by acid-base titration. Numerous modifications of the method have been proposed, but it remains remarkably similar in its essentials to the original procedure and is still widely used.
Nitrogen in foods comes not only from the amino acids in protein but also from additional forms that may, or may not, be used as part of the total nitrogen economy in humans and animals. The nitrogen content of proteins in foods can vary from between about 15 to 18 percent, depending on the amino acids in these proteins. In addition, purines, pyrimidines, free amino acids, vitamins, creatine, creatinine, and amino sugars all contribute to the total nitrogen present. Urea is also a major contributor in foods as important as human milk.
Because the average content of nitrogen (N) in protein is 16 percent, the nitrogen content multiplied by 6.25 is frequently used to convert N to protein. This should then be termed “crude protein.” It is not always appreciated that the amino acids themselves show a very large range in their content of nitrogen. Whereas tyrosine has less than 8 percent N, lysine has 19 percent and arginine contains 32 percent. The presence of an amide group in an amino acid can double the N content; aspartic acid contains 10 percent N, but asparagine contains 21 percent. For other N-containing compounds, values can even be higher, with 47 percent N in urea and some 80 percent in ammonia. In practice, most protein evaluation techniques are actually evaluating the nitrogen present but are calling it protein (N x 6.25). Because a number of other factors from 53 to 6.38 (Jones 1941) may be used to convert food nitrogen to protein in different foods, considerable potential for confusion exists (Pellett and Young 1980).
Early in the twentieth century as the ability developed to detect and identify amino acids, it was realized that they were present in living materials, not only as components of their proteins but also as free amino acids. It also became clear that there were many more amino acids in living systems than the 20 or so known to be present in proteins. Because all amino acids behave in a rather similar chemical manner, additional techniques, often of a physicochemical nature, such as column chromatography, became necessary to solve the complex problems of separating and determining their presence in foods (Moore and Stein 1954; Moore, Spackman, and Stein 1958).
A major milestone was the description (Spackman, Stein, and Moore 1958) of an automatic analyzer allowing separation and quantitative analysis of amino acids based on ion-exchange column chromatography. As a result, modern dedicated amino acid analyzers quantitatively separate protein hydrolysates into their constituent amino acids in 1 hour or less and physiological fluids in about 2 hours.
In the introduction to the first edition of their major work on the amino acid composition of food proteins, R. J. Block and D. Bolling wrote:
It has been our experience that a food protein may be a good source of those nutritionally valuable amino acids which are most commonly estimated (i. e., cystine, methionine, arginine, histidine, lysine, tyrosine and tryptophan) and yet be deficient in one or more of the other essentials for which analytical methods are more difficult and often less accurate (1951: xiv).
Threonine was not listed, but the other essential amino acids considered at the time to be difficult to analyze (leucine, isoleucine, valine, and phenylalanine) have rarely proved to be in deficit in normal foods or diets. Analytical techniques for amino acids at the time were chemical and followed in a direct manner from those developed by Fischer (1901). Large quantities of protein were required before amino acid composition could be undertaken, and accuracy was poor.
Even today, when techniques such as ion-exchange chromatography are in use, the accuracy and reproducibility of analyses for cystine, methionine, and tryptophan are problematic, not least because of destruction of amino acids during the hydrolysis stage of analysis (see FAO/WHO 1991). Current views are that although the supply of any of the essential amino acids and of total nitrogen may theoretically limit protein utilization, only lysine, threonine, tryptophan, and the sulfur-containing amino acids are likely to be limiting in normal foods or diets. Even further, we now believe that in diets based on predominantly cereal protein sources, lysine may be the only amino acid that conditions overall protein quality (Pellett and Young 1988,1990; Pellett 1996;Young, Scrimshaw, and Pellett 1997).
World History
