Started Humapro Today

[Author L. Rea]

Im still kinda laughing my ass off about the 5.6 number they pulled out of air (apparently)!

First, I am just a visitor here and do not mean to bring any drama nor disrespect anyone who does not attack me especially those who pay sponsorship for this board by taking up their marketing space. Second I want to thank you all for bing interested enough to test, flame, or comment. So...

I was hoping someone would open the obvious first crack on the product and my company. lol PsyT, on the real Bro. So...

Techniques for protein quality evaluation: background and discussion
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1. Human protein and amino acid requirements and their relevance to protein quality evaluation
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In this chapter, a brief statement is made concerning the determination of protein and amino acid needs and the estimated requirements for these nutrients in order to provide a basis for establishing the protein quality of food sources and diets. The protein needs of humans have been studied for more than a century, and knowledge of human amino acid requirements dates from the classical studies of Rose and co-workers at the University of Illinois in young men and Leverton and co-workers in young women (1, 2). The amount and proportion of essential amino acids needed at any given age determine the utilization of dietary proteins for maintenance of body protein in adults and for growth in children.
Estimates of the amounts of protein and amino acids needed by humans have been published periodically by the Food and Agriculture Organization (FAO) and the World Health Organization (WHO), and more recently by the National Research Council (NRC) ( 1). The 1973 report Energy and Protein Requirements by an FAO/ WHO Ad Hoc Expert Committee (2) provides a review of knowledge in this field. This report has been extended by a joint FAO/WHO informal gathering of experts in 1975 (3) and by the 1977 first joint FAD/WHO expert consultation on energy intake and protein requirements (4), as well as by the United Nations University publication Protein-Energy Requirements under Conditions Prevailing in Developing Countries (5); so only brief reference to some aspects of human protein and amino acid requirements need be given.
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Protein Requirements
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The requirement for dietary protein in adults has been based both on studies of the amounts of nitrogen lost by subjects receiving very low protein or protein-free diets and on estimates of the amounts of protein from different sources needed to bring adults into nitrogen equilibrium. The first of these two procedures, the "factorial method," estimates the losses of nitrogen (N) from the body by way of the urine, faeces, skin, plus other minor routes in healthy adults following short-term adaptation to essentially proteinfree diets. These summated losses, the obligatory nitrogen loss, represents the minimum need of the body for dietary protein nitrogen. Extensive studies in the United States and elsewhere are in close agreement about the amounts of nitrogen lost under these experimental conditions, with urinary nitrogen output averaging 37 mg/kg body weight/day, faecal nitrogen about 12 mg/kg, and cutaneous losses about 3 mg N/kg {2). When a series of minor routes of nitrogen excretion amounting to 2 mg N/kg are added to these estimates, a total obligatory nitrogen output of 54 mg/kg for an adult is obtained. If the protein of the diet is utilized with complete efficiency, an amount of dietary protein equivalent to the output of 54 mg N/kg, namely 0.34 9 protein/kg body weight should compensate for this obligatory loss and bring the subject into equilibrium.
This thesis has been tested by giving whole egg protein or egg albumin to human adults in quantities sufficient to bring the subjects into nitrogen equilibrium (6-9). These studies demonstrate that, even with a good quality source of nitrogen such as egg or milk, considerably more dietary protein is needed to achieve body nitrogen equilibrium than the estimated 54 mg N/kg/day. For subjects given experimental diets in which the protein is provided by milk, eggs, casein, or mixed diets containing appreciable amounts of animal protein, the amount of dietary protein needed for nitrogen equilibrium averages about 70 mg N/kg daily - that is, 0.44 9 protein/kg (2).
Taking all information then available into account, the 1971 FAD/WHO Expert Committee (2) selected a value of 30 per cent to correct for the difference in efficiency of utilization of protein at low-protein intakes and at requirement levels. More recent data suggest a somewhat higher figure (10).
When a 30 per cent addition was made to the factorial loss figures to allow for this effect, plus another 30 per cent to allow for variation in requirements among apparently similar individuals, the estimate of an intake of high-quality protein sufficient to cover almost all of the population was, for the adult male, 0.57 g/kg/day. Calculations for the adult female, but not including menstrual losses, gave a similar figure of 0.52 g/kg/day. Thus, separate recommendations were not made for men and women.
These allowances for high-quality protein were based on relatively short experimental periods. A systematic attempt to evaluate whether this intake level of egg protein would be adequate for long-term maintenance in healthy young adult males, given adequate dietary energy, revealed changes in body protein status after two to three months ( 11, 12). These included a loss of lean body mass, as judged by changes in 40K and 24-hour urinary creatinine excretion, a fall in serum albumin and haemoglobin, and an increase in serum aminotransferase activity in some individuals. These changes were not observed with the feeding of beef protein at a level of 0.8 g/kg of body weight for three months, but a lower level than this has not been critically tested for its possible adequacy. Thus, there still is a need to define more precisely minimal allowances that are adequate for longterm maintenance.
It was pointed out way back in the 1973 FAD/WHO report (2):
Infections affect protein requirements by inducing some degree of depletion of body N during acute episodes . . . the quantitative effects of infections on the protein needs of an individual cannot by stated, since they are likely to vary with the frequency, severity, and nature of the infection and other host factors, including nutritional status.
Conditions prevailing in developing countries may also increase requirements because of reduced absorption of protein as well as the periodic increases in requirements for recovery from infections.
The UNU working group report Protein-Energy Requirements under Conditions Prevailing in Deve/oping Countries (5) also emphasized the quantitative effects of infections, and the variations in protein and energy digestibility among populations consuming various types of diets.
For the growing child, the criterion of requirement is satisfactory growth. Indeed, where long-term feeding studies can be conducted with a diet providing protein at the level of the estimated requirement together with adequate energy and all essential nutrients, the demonstration of normal growth and maintenance of normal indices of protein nutritional status is a convincing validation of the estimated requirement. For infants, these requirements are approximately 2.4 g/kg up to 3 months of age, 1.85 g/kg at 3-6 months, 1.6 g/kg at 6-9 months, and 1.4 g/kg at 9-11 months. Some investigators, for example Fomon (13), would, however, set these requirements at a lower level, ranging from 1.8 g/kg at less than 4 months to 1.4 g/kg/day between 4 and 12 months of age. During later childhood, the estimates of requirements are less securely based because they are derived using interpolations of average daily growth rates.
These protein levels assume daily growth at a rate of 1/365 that of annual growth However, it has recently been emphasized (5) that the variations in normal growth rate are such that the diet should allow for normal growth rates three to four times higher than this figure for some periods, and that for recovery from diarrhea, disease and acute infectious diseases of childhood, this figure may be as high as eight or nine times greater in ambulatory village children. For children recovering from severe protein-calorie malnutrition under hospital conditions, growth rate can be up to 18 times greater than that for healthy, well-nourished children.
It would, of course, be desirable to eliminate the causes of the high frequency of infection among children in developing countries, but until this can be accomplished, it must be recognized that their normal growth and development will require the consumption of significantly more protein than is assumed to be a safe allowance for healthy children. How much more will depend on the circumstances of the individual child and cannot be generalized.
Thus, although there is still uncertainty surrounding estimates of human protein requirements and the definition of safe practical allowances for various population groups, current evidence is sufficient to confirm that the major sources of food protein differ in their capacity to meet protein needs.
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Essential Amino Acid Requirements
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Using nitrogen balance and growth as the criteria, and more recently with the aid of studies of blood amino acid levels, the requirements of children and adults for essential amino acids have been estimated. Such estimates show wide variations among
TABLE 1 Protein and Amino Acid Requirements of Human Subjects
Age of subject
3-6 10-12 adult
months years
Protein 1.85 0.80 0.57
Amino acid (mg/kg)
isoleucine 70 30 10
leucine 161 45 14
Iysine 103 60 12
methionine + cystine 58 27 13
phenylalanine + tyrosine 125 27 14
threonine 87 35 7
tryptophan 17 4 4
valine 93 33 10
Total EAA requirement 714 261 84
Ratio of total EAA requirement
to protein requirement 0.39 0.33 0.15
Data taken from FAO/WHO (2). The estimates of requirements are calculated for 0.98 of the population at each age and thus express the upper limit of the range of requirements found in a healthy population Histidine is also an essential amino acid for the infant and is required at a low level by the adult. It was omitted from the pattern because it is limiting only under extreme experimental conditions. individuals and between laboratories, but on careful analysis of the published data it is possible to arrive at values that are reasonably concordant and certainly relevant to utilization of dietary proteins of varying amino acid composition (1, 2). Estimates of needs for the essential amino acids are given in table 1. Similar values for amino acid requirements have been proposed for different age groups by Williams et al. (14). In table 2, the pattern of essential amino acids required is shown, based on the estimates summarized in table 1. In addition the FAO/WHO (2) scoring pattern is given, and the use of such a pattern is discussed in chapter 3.
It is important to emphasize, however, that current estimates of amino acid requirements, including those based on plasma amino acid levels, may reflect the capacity of the organism to adapt maximally to low levels of amino acid intake and, thus, may underestimate the minimum need for long-term protein nutritional maintenance. This point may be more important in relation to adult protein nutrition because the estimates of essential amino acid requirements of children appear to be in good agreement with intakes provided by goodquality proteins when consumed at intakes that are sufficient to support the requirements for growth (15).
The available data demonstrate that the requirements for essential amino acids fall with increasing age (table 1), as do requirements for total dietary protein However, the available data suggest that essential amino acid requirements decline more rapidly than do total protein needs. The proportion of protein needs in infants represented by the content and pattern of the eight essential amino acids in a dietary protein source will be much more significant for the infant and growing child than for theadult.
TABLE 2. Patterns of Amino Acid Requirements and Amino Acid Scoring Patterns for Evaluation of Proteins (mg/g N)
Requirement pattern
Amino acid Scoring
child
infant 10-12 adult
years
Isoleucine 220 230 113 250
Leucine 500 350 156 440
Lysine 325 469 138 340
Methionine + cystine 180 213 150 220
Phenylalanine + tyrosine 394 213 156 380
Threonine 275 275 81 250
Tryptophan 56 30 44 60
Valine 294 256 113 310
Data adapted from FAO/WHO (2).
Similar conditions apply, of course, to the problems of protein quality and formulation of rations in the feeding of commercially important simple-stomached animals and birds.
In any event, it is these current estimates of protein and amino acid requirements that provide the rationale for the determination and assessment of the significance of dietary protein quality in human nutrition.
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References
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1. Committee on Amino Acids, Food and Nutrition Board, National Research Council, Improvement of Protein Nutriture, ed. A.E. Harper and D.M. Hegsted (National Academy of Sciences, Washington, D.C., 1974).
2. Joint FAD/WHO Ad Hoc Expert Committee, Energy and Protein Requirements, WHO Technical Report Series, no. 522; FAO Nutrition Meetings Report Series, no. 52 (WHO, Geneva; FAO, Rome, 1973).
3. Joint FAD/WHO Informal Gathering of Experts, "Energy and Protein Requirements," Food and Nutrition, 1 ( 2): 1 1 ( 1975) .
4. FAD/WHO, "Report of the First Joint FAD/WHO Expert Consultation on Energy Intake and Protein Requirements" (FAO, Rome, 1978).
5. F. Viteri, R. Whitehead, and V. Young, eds., Protein-Energy Requirements under Conditions Prevailing in Developing Countries: Current Knowledge and Research Needs (United Nations University, Tokyo, 1979).
6. H.N. Munro, "Amino Acid Requirements and Metabolism and Their Relevance to Parenteral Nutrition," in A.W. Wilkinson, ea., Parenteral Nutrition (Churchill-Livingston, London, 1972), pp. 34 67.
7. D.H. Calloway and S. Margen, "Variations in Endogenous Nitrogen Excretion and Dietary Nitrogen Utilization as Determinants of Human Requirements," J. Nutr., 101: 205-216 (1971).
8. V.R. Young, Y.S.M. Taylor, W.M. Rand, and N.S. Scrimshaw, "Protein Requirements of Man: Efficiency of Egg Protein Utilization at Maintenance and Submaintenance Levels in Young Men,"J.Nutr., 103: 1164-1174 (1973).
9. K. Kishi, S. Miyatani, and G.Inoue, "Requirement and Utilization of Egg Protein by Japanese Young Men with Marginal Energy Intake," J. Nutr., 108: 658-669 (1978).
10. C. Garza, N.S. Scrimshaw, and V.R. Young, "Human Protein Requirements: The Effect of Variations in Energy Intake within the Maintenance Range," Am. J. C/in. Nutr., 29: 280-287 ( 1 976).
11. C. Garza, N.S. Scrimshaw, and V.R. Young, "Human Protein Requirements: Evaluation of the 1973 FAD/WHO Safe Level of Protein Intake for Young Men at High Energy Intakes," 8rit. J. Nutr., 37: 403420 (1977).
12. C. Garza, N.S. Scrimshaw, and V.R. Young, "Human Protein Requirements: Interrelationships between Energy Intake and Nitrogen Balance in Young Men Consuming the 1973 FAD/WHO Safe Level of Egg Protein, with Added Non-essential Amino Acids," J. Nutr., 108: 90-96 (1978).
13. S.J. Fomon,lnfant Nutrition, 2nd ed. (W.B. Saunders, Philadelphia, Penn., USA, 1974).
14. H.H. Williams, A.E. Harper, D.M. Hegsted, G. Arroyeve, and L.E.J. Holt, "Nitrogen and Amino Acid Requirements," in Committee on Amino Acids, Food and Nutrition Board, National Research Council, Improvement of Protein Nutriture (National Academy of Sciences, Washington, D.C., 1974), pp. 23-63.
15. A.E. Harper, "Amino Acid Requirements - General," in H.L. Greene, M.A. Holliday, and H.N. Munro, eds., Clinical Nutrition Update: Amino Acids [American Medical Association, Chicago, 111., USA, 1977), pp. 58-65.

 

[Author L. Rea]

2. Analytical methods for the determination of nitrogen and amino acids in foods
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Protein and Other Nitrogen Components of Foodstuffs
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Nitrogen in foods not only comes from amino acids in protein, but also exists in additional forms that may or may not be used as a part of the total nitrogen economy of humans and animals. The nitrogen content of proteins in foods can vary between 150 and 180 g/kg 115-18 per cent), depending on the amino acids they comprise. In addition, purines, pyrimidines, free amino acids, vitamins, creatine, creatinine, and amino sugars can all contribute to the total nitrogen present. In meat, a portion of the nitrogen occurs as free amino acids and peptides; fish may contain these and volatilebase nitrogen and methyl-amino compounds 11). Marine elasmobranchs may also contain urea. Half of the nitrogen of the potato may not be in the form of protein (2), and even in human milk as much as 50 per cent of the total nitrogen may be urea nitrogen (3). Because the nutritional significance of much of the non-amino acid and non-peptide nitrogen is unclear, nitrogen analysis of a food is usually much more precise than the nutritional significance that can be attached to it.
In practice, most biological methods for evaluating protein quality (chaps. 4 and 5) are, in fact, evaluating nitrogen but are expressed as crude protein (N x 6.25). Nitrogen data are also used for amino acid scores (chap. 3) when amino acids are expressed in terms of mg/g N. When results are to be expressed in terms of protein, as for example in protein efficiency ratio (PER) and when amino acid scores are to be expressed per 16 9 N, then the average conversion factor of 6.25, defined as crude protein, is again used (4). Since requirements are also expressed in terms of N x 6.25, conversion factors are not needed and no confusion should exist.
For other purposes, however, such as labelling regulations and food composition data, conversion from nitrogen to protein is wide)y used and a range of conversion factors exists.
Most food composition tables derive estimates of protein content by applying
TABLE 3. Factors Used in Converting Nitrogen to Protein
Foodstuffs Conversion factor for protein content as reported in food Correction factor for conversion of reported value to
composition tables "crude protein"
Cereals
Wheat (hard, medium, or soft)
whole 5.83 1.07
flour (medium or low extraction) 5.70 1.10
macaroni, spaghetti, wheat pastes 5.70 1.10
bran 6.31 0.99
Rice (all varieties) 5.95 1.05
Rye, barley, and oats 5.83 1.07
Pulses, nuts, seeds
Groundnuts 5.46 1.14
Soya 5.71 1.09
Tree nuts
almonds 5.18 1.21
Brazil nuts 5.46 1.14
coconut, chestnuts 5.30 1.18
Seeds - sesame, safflower, sunflower 5.30 1.18
Milk (all species) and cheese 6.38 0.98
Other foods 6.25 1.00
From FAO Nutritional Studies No. 24, "Amino Acid Content of Foods and Biological Data on Proteins" (FAO, Rome, 1970). different factors to the nitrogen content of individual foods. Some of these factors are shown in table 3. These are mostly standard factors; however, some of the values for cereals, legume foods, and oilseed meals reported by Tkachuk (5) are based on direct calculation from amino acid analyses.
To compare the reported protein content of foods with protein requirements, a correction of the reported protein content must be made. The correction factors to convert reported protein to crude protein are also shown in table 3. Considerable care is needed when protein data from food composition tables are used in conjunction with determined protein quality and/or requirement values. Factors different from those in table 3 may have been used, and it is necessary to ascertain how the data were derived.
Some new products such as single-cell protein (SCP) contain high levels of purine nitrogen, some of which may be used, and cell-wall nitrogen, most of which is probably not utilized (6). Recommendations have been made by the Protein-Calorie Advisory Group (PAG) (7) for the calculation of protein nitrogen in SCP products where substantial parts of the total nitrogen may come from nucleic acid. The calculation of "crude protein" by multiplying total nitrogen by 6.25 can give a serious overestimation of protein content. Purine nitrogen should be determined separately and the nucleic acid content estimated by multiplication by a factor to make allowance for the pyrimidine content. As the ratio of the nitrogen in pyrimidine to that in purines approximates 0.40, and both are present in equimolecular amounts in most nucleic acids, the purine nitrogen should be multiplied by 1.4 to obtain nucleic acid nitrogen. Nucleic acid nitrogen multiplied by 9.0 can then be considered as nucleic acids. Consideration of purine nitrogen alone will give an underestimation for nucleic acid nitrogen.
An example of the calculation of protein nitrogen in an SCP product is given below:
Given:
Total N content of a yeast SCP product = 1,000 mg
Purine N =160 mg
Calculation:
Total nucleic acid N = 160 x 1.4 = 224 mg
(allowance made for pyrimidine N)
Corrected protein N = 1,000 - 224 =776 mg
Crude protein content = 1,000 x 6.25 = 6,250 mg
Corrected protein content = 776 x 6.25 = 4,850 mg
Total nucleic acids = 224 x 9.0 = 2,016 mg
For certain foodstuffs, total nitrogen values have sometimes been partitioned into "true protein" and "nonprotein nitrogen." This partitioning has been according to the quantities of nitrogen that were recovered in the precipitate and filtrate, respectively, after extraction of the food with various protein solvents followed by precipitation with trichloroacetic acid or some other protein precipitant. This approach is not recommended, because free amino acid nitrogen may be of the same nutritional value as the protein. Amino acid analyses are now usually feasible for the expression of total amino acids in food. Thus the conventional measure of "protein" or "crude protein" in foods is N x 6.25, and it is recommended that this one factor be used in nutritional studies in which whole diets contain more than one source of nitrogen.
The protein content of foodstuffs is conventionally estimated from the nitrogen content determined by the Kjeldahl technique. Numerous modifications of the original procedure have been proposed (8). A recommended method based on the procedure of the Association of Official Analytical Chemists (AOAC) (9) is fully described in chapter 8 (p. 86).
When the equipment is available, the determination of ammonia in digests may be carried out on an autoanalyser system using a colorimetric method based on reaction with an alkaline phenolate-hypochlorite reagent. This method has proved reliable and can save time. Earlier hopes that food samples could also be rapidly digested with complete recovery in an autoanalyser system have not been supported, and it is still necessary to digest samples prior to autoanalysis.
Other methods, such as those using Biuret and the Folin-Ciocalteu reagent, and fluorimetry have been reviewed by Cole (10). Where many samples of a single, unprocessed material are being screened for their protein content, a dye-binding procedure may be the most appropriate (11, 12).
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Analyses of Individual Amino Acids in Foods
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Hydrolysis Prior to Chemical Analysis
All methods to be discussed require preliminary treatment of the test material to hydrolyse the proteins to the free amino acids. A major problem of amino acid analysis in foodstuffs is the destruction of amino acids during acid hydrolysis. Unfortunately, this problem can be greatest with the essential amino acids likely to be limiting in practical diets: "methionine + cystine," Iysine, threonine, and tryptophan. Proteins and protein foodstuffs differ so widely in their composition that "ideal" hydrolytic procedures would need to be almost specific for each material. Thus, compromises between the ideal and practical are often necessary. Of the many procedures available in the literature, those shown in chapter 8, section B, have proved suitable for routine analysis of the amino acid composition of wide ranges of pure proteins and protein foodstuffs. Many reviews (13-20) exist in the ) literature where analytical problems are discussed.
Amino acids are released and destroyed at different rates that depend upon the amino acid composition and characteristics of the sample. Assessment of amino acid composition has been recommended (21) as being most accurate when derived from five separate hydrolyses - three acid hydrolyses of different time durations (usually 24, 48, and 72 hours) a special acid hydrolysis following performic acid oxidation for cysteic acid and methionine sulphone, and an a)kaline hydrolysis for tryptophan determination. The three hydrolysis times are designed to allow selection of specific times for certain amino acids as well as extrapolation to zero time for the most labile amino acids. Separate procedures for sulphur amino acids and for tryptophan are always essential; however, in most cases a single 24-hour acid hydrolysis can give adequate information for scoring purposes (chap. 3).
Detailed procedures have been described for cereals (21). An ideal analysis for cereal products would use values for threonine and serine from extrapolation to zero time, tyrosine after 24 hours, isoleucine and valine values after 72 hours, and the remaining amino acids as the mean of the three determinations. Samples other than cereals would not necessarily behave in a similar manner. Caution should be used in the interpretation of sulphur amino acid data following oxidation because certain food samples (22) may already contain unavailable oxidized sulphur amino acids as a consequence of procedures used for their processing. Availability, however, is dependent on the degree of oxidation, methionine sulphoxide being generally fully available whereas methionine sulphone is not. As tryptophan is destroyed during normal acid hydrolysis, a separate analytical procedure is needed. Alkaline hydrolysis (p. 92) followed by a special short-column procedure to separate tryptophan from Iysinoalanine and the dibasic amino acids (23) is routinely used in many laboratories. An alternative procedure is to use one of the variants of the Spies and Chambers colour reaction with dimethylaminobenzaldehyde (24-27). It is important with foodstuffs to check the recovery of pure added tryptophan.
Chromatographic Techniques
Most procedures for amino acid analysis depend on the use of chromatography. Early techniques using oneandtwo-dimensional paper chromatography, while at best only semi-quantitative, gave much information on amino acid composition and amino acid metabolism. These procedures have almost all been replaced by column techniques, although thin-layer chromatography has certain applications. Within the various techniques using columns for separation, a major subdivision can be made into preand post-column derivatization procedures. Ion-exchange chromatography uses post-column derivatization in that the amino acids are separated by means of ion exchange, and derivatives are formed after they have emerged from the column so that they may be quantified. The most common derivatization procedure is that using ninhydrin with subsequent determination of optical density. In contrast, pre-column derivatization, as is used for gas-liquid chromatography (GLC) and high-performance liquid chromatography (HPLC), uses columns to separate the amino acid derivatives. These derivatives, after emerging from the column, are then quantified by various detection devices. GLC and HPLC procedures are frequently more rapid than ion exchange procedures, but their major limitations often lie in the preparation of the derivatives rather than in the chromatography as such.
High-Performance Liquid Chromatography
As high pressures are no longer an integral part of HPLC, the general procedure has recently been renamed "high-performance" rather than "high-pressure" liquid chromatography. Pre-column derivatization is required for amino acid analysis. Dansyl chloride (5-dimethylamino-1-naphthalene sulphonyl chloride) is frequently used for this purpose, producing fluorescent dansyl derivatives that are separated by a reversed phase column chromatographic procedure. The column employs silica gel with attached non-polar hydrocarbon functional groups (e.g., octadecyl moieties) as the stationary phase (28) and uses a multi-step non-linear elusion procedure. Among other eluents, acetonitrile and water mixtures have been suggested for the separation of dansylated amino acids (29). These are then detected and measured by a fluorescence detector, which can provide detection limits in the picogram range. It has been demonstrated (28) that HPLC, utilizing various non-polar stationary phases, is superior to ion-exchange chromatography for separating peptides. However, because of limitations in the separation of the polar amino acids, it is at present stir) inferior to ion-exchange for protein compositional studies. Procedures can, however, be extremely rapid and sensitive. Separation of some 24 amino acids in physiological fluids in a 40minute period has been demonstrated (29). Thus, while the procedure currently suffers from some limitations, it seems highly likely that the method will develop into a rapid and sensitive routine procedure for amino acid analysis. One major advantage of HPLC over all other chemical procedures lies in its ability to distinguish D and L forms of amino acids. Therefore, for certain research purposes, its use is already essential.
Ion-Exchange Chromatography lon-exchange chromatography remains the most utilized method of amino acid analysis. Many of the commercial analysers currently available use the automated procedure introduced by Spackman, Stein, and Moore in 1958 (30, 31). Modifications include use of only one column and gradient elusion instead of stepwise elusion (32, 33). With recent advances, such as the use of lithium instead of sodium buffers, higher pressures, narrow columns with fine spherical resin particles, and fluorescamine reagents replacing ninhydrin, the newer commercial equipment will give good separation of the common amino acids in picogram quantities in two hours or less. General reviews of the use of these procedures are available (34, 35). It is essential to introduce an internal standard into each hydrolysate to check the potency of ninhydrin solutions. Norleucine is convenient for acid and neutral runs and for singlecolumn systems. Alpha-amino-beta-guanidino-n-propionic acid is recommended where a separate, short column is employed for basic amino acids. An alternative to the use of internal standards when automated peak area calculations are available is the running of amino acid standards as every third or fourth run.
Automation has progressed to include sequential sample loading and regeneration of the columns. Laborious calculation of peak areas can also be avoided by the integration of photocell signals by computer integration techniques. Methodology of analysis is provided by equipment manufacturers. Some brief notes for users of these techniques are provided in chapter 8 (p. 94).
Gas-Liquid Chromatography
Analysis of amino acids using gas-liquid chromatography requires the quantitative conversion of the amino acids to volatile derivatives (36, 37). The method has the promise of a speedier output and relatively low capital outlay for equipment. Hydroxy acid methyl esters (38), trimethylsilyl esters (39), and n-butyl-N-trifluoroacetyl esters (40) appear to offer some promise. Before their conversion to volatile esters, hydrolysates of foods require a preliminary separation of the amino acid fraction to remove interfering substances. Good agreement with the values obtained by other procedures has been reported with maize and soy bean mea) (41).
Thin-Layer Chromatography
Because of the high expense of modern ion-exchange chromatography equipment, amino acids are often separated by thin-layer chromatography (TLC). TLC techniques are inexpensive, and, although the processing of each plate may be lengthy, many plates can be treated at one time. Hence, output can be quite considerable. The early work (42, 43) has been improved to a level at which the technique may be routinely applied to protein hydrolysates (44, 45). The results agree well with values obtained by ion-exchange chromatography.
Microbiological Assays
Microbiological assays can be extremely useful when other equipment is not available, or when many assays of only one amino acid, for which there is not a convenient, specific colour reaction, are required. An example of the latter is in the preliminary selection of Phaseolus beans for methionine content from large numbers of lines (46).
Detailed procedures for microbiological assays have been published (47-49). Continuous vigilance is required over standardization of organisms, maintenance of cultures, composition of media, and general techniques, because all of these factors have been shown to influence results and to vary from one laboratory to another. High apparent Iysine values for animal products have been traced to a synergistic effect of hydroxylysine; this may be avoided by the inclusion of hydroxylysine in the basal medium 150, 51).
Workers have sometimes used only mild hydrolytic procedures for test materials in order to reduce the risk of losses of amino acids, and have relied on the ability of organisms to utilize soluble peptides. However, this can lead to stimulation of growth with final calculated values being too high, as judged by chemical procedures (52), so the practice needs careful scrutiny.

 

[Author L. Rea]

Estimation of "Available" Amino Acids in Foods
It cannot be assumed that all the amino acids released from food proteins by digestion in acid (or alkali) will be absorbed and utilized when the food is eaten. This is illustrated by the results in the first and fourth columns of table 4. Excessively high temperatures during the drying of milk caused some destruction of "total" Iysine
TABLE 4. Lysine Levels (mq/q N) in Four Samples of Milk Powder
Sample Total value after acid hydrolysis (104) Lysine reacting with FDNB Lysine released by in vitro enzymatic digestion Value obtained by growth assay with the rat
1. Good quality 500 513 519 506
2. Slightly damaged 475 400 388 381
3. Scorched 425 238 281 250
4. Severelyscorched 380 119 144 125
Source: Ref. 53. content (i.e., the Iysine liberated by acid hydrolysis), but the change in the value of the milk as a source of Iysine for rats (as measured in a growth assay) was very much greater (53) than could be accounted for by this change.
Foods such as milk with a high reducing-sugar content are particularly susceptible to damage when heated through a moisture content range of 50-250 g/kg (54, 55). In such material, even storage at tropical temperatures can be damaging. The dibasic amino acid Iysine is particularly affected by reactions involving the e-NH2 group (56-57). Processing can also damage the sulphur amino acids of foods, and the full extent of the damage through oxidation of the amino acids cystine and methionine will not be detected by specific procedures for measuring the sulphur amino acids in proteins that themselves determine the oxidized forms, cysteic acid and methionine sulphone. Severe heat treatment in some forms of processing can damage the protein of foods, and the availability of al) the essential amino acids may be affected in large part as a result of a decrease in the digestibility of the proteins (57).
In Vitro Enzymatic Methods
The major cause of the reduced nutritional) value of heat-damaged protein foods appears to be impaired enzymatic hydrolysis of the component proteins; thus, much work has been done to estimate the release of amino acids by in vitro enzymic digestion. The methods have been comprehensively reviewed (58, 59). Early experiments showed useful correlation of in vitro results with those from animal feeding experiments, even though the absolute in vitro values were very much lower. Increasingly sophisticated procedures to imitate the action of the mammalian digestive system have been developed. They have been used to demonstrate the physiological basis for some of the differences in the nutritive value of different materials.
However, the tests are, in general, too complex for the routine evaluation of samples.
A procedure suitable for routine screening has been described by Ford (60), who performed microbiological assays for amino acids with Streptococcus zymogenes, an organism that has considerable proteolytic power. When various dried animal protein materials were given a mild pretreatment with papain and then assayed with this organism for methionine activity, the results agreed wel) with results of animal growth assays for methionine (61). S. zymogenes can be used for assay of the other essential amino acids except Iysine, for which the organism does not have a requirement (62). Ford (63, 64) has reviewed the various microbiological assays that are used to screen for proteins for availability of selected essential amino acids, and has compared availability data on the same protein when measured by chemical and microbial assays. Additional comparisons were made, when possible, with chick bioassay data. He concluded that the use of microbiological assays for protein quality grading, and for assessing the biological availability of individual amino acids, can be of great value to the plant breeder and provide a necessary check on the results of total amino acid analysis.
The availability of all of the essential amino acids within a food protein can be measured with the enzymaticultrafiltrate digest (EUD) (65). This assay involves digesting a protein sample with pepsin-trypsin-pancreatin and then determining the available amino acids by analysing the ultrafiltrate of the multi-enzyme digest. This procedure is also briefly considered in chapter 3 (p. 32).
Microbiological techniques such as those using the protozoan Tetrahymena pyriformis W for determination of overall protein quality are discussed in chapter 3 (p. 33). However, T. pyriformis W has also been used for assessment of available Iysine and methionine (63, 64, 66). Animal techniques for available amino acids also exist; these, however, are discussed in chapter 4 (pp. 52-531.
Available Lysine
Processing damage in dried milk seems to be explained by the reaction of lactose with the free e-NH2 groups of Iysine units in the protein, with the result that the affected Iysine units no longer undergo the Van Slyke reaction or form a dinitrophenyl (DNP) derivative with fluorodinitrobenzene (FDNB), and are no longer nutritionally "available" as judged by animal growth assays 153, 54). This loss in availability can be seen by comparing the results for different dried milk samples in the second and fourth columns of table 4.
Fluorodinitrobenzene-reactive Iysine has been measured with many different procedures (57). A suitable procedure is described in chapter 8 (p. 95). The main technical problem is to minimize the loss of DNP-lysine during acid hydrolysis of the treated protein, as the DNP-lysine is susceptible to reduction of nitro groups. The other problem has been to separate and measure the DNP-lysine with an adequate degree of precision with a procedure simple enough for use in a non-specialized quality control laboratory; chromatographic procedures are probably the most specific for this separation (67,68).
A further problem lies in the interpretation of the results obtained. FDNB-reactive Iysine appears to be a good indicator of nutritionally available )ysine in oilseed products such as peanut flours (56, 69) and in milk powders (53, 70). Fructose-lysine derivatives give very little DNP-lysine when reacted with FDNB and then hydrolysed with acid (71). With protein-rich materials such as meat and fish products that do not contain appreciable levels of carbohydrates, very severe conditions during the drying process can lead to significant falls in the level of FDNB-reactive Iysine and in the nutritional value of the product. However, such severe treatment results in a large reduction in the availability of all other amino acids in addition to Iysine. Growth assay values for individual amino acids may decrease more than would be expected by the drop in FDNBreactive Iysine (57, 72, 73).
This type of processing damage is thought to be due to the formation of many cross linkages in the protein, involving Iysine and other amino acids, that greatly hinders normal enzymic attack. When digestibility of proteins is reduced, it is not justified to assume that "FDNB-reactive" is synonymous with "nutritionally available" Iysine. On the other hand, measurement of FDNB-reactive Iysine is more sensitive than measurement of total amino acid composition, which may remain less changed despite severe nutritional damage, as can be seen from the dried milk data in table 4.
Although the FDNB procedure has proved to be a useful indicator of protein quality for fish flours (74, 75) and for meat products (56, 76), some results with herring stored and cooked in various ways have not shown a high correlation between FDNB values and nutritional evaluation (77). This is possibly due to partial proteolysis of the raw materials. Ordinary FDNB methods do not measure either free lysine or Iysine that is nitrogen-terminal in a peptide chain. An alternative method, designed to overcome this difficulty, measures "total" Iysine and "FDNB-unreactive" Iysine - i.e., Iysine as such, released from materials after treatment with FDNB and then digestion with acid - (78, 79). Where the Iysine reactions are of a protein-protein nature, the approach generally seems to work well, but even here, measurement of FDNB-reactive Iysine is not always adequate, for example e(gama-glutamyl)-lysine is biologically fully available while e(beta-aspartyl)-lysine is not (80). Unfortunately, the procedure fails to measure adequately the type of damage resulting under mild conditions from reaction of reducing sugars with Iysine units (71). A rapid dye-binding procedure has recently been advocated for the determination of reactive Iysine in foodstuffs (81, 82). Dyebinding procedures are considered in more detail later in this chapter.
Trinitrobenzenesulphonic acid (TNBS) is an alternative reagent to FDNB. TNBS has the technical advantage that it is less dangerous to the user, is water-so)uble, and measures free Iysine. On the other hand, it gives Iysine derivatives that are sensitive to destruction during digestion in acid, and, with some types of material, there is the decisive disadvantage that it still measures a large proportion of the Maillard compounds formed between Iysine and sugar units as "TNBS-reactive" (71).
Finally, the furosine procedure (83) should be briefly described. Lactosyl Iysine in damaged milk upon hydrolysis with HCI is converted to both furosine and Iysine in specific proportions; analysis for furosine can thus be used as a direct indicator of damaged or blocked Iysine.
Chemical methods for determination of available amino acids in foodstuffs have recently been reviewed by Carpenter (82).
Enzymatic Methods for Determining Available Lysine
Because of the inability of chemical assays that use dye-binding procedures or 1-fluoro-2,4-dinitrobenzene (FDNB) to measure accurately available Iysine in many foods, especially high-carbohydrate cereals, alternate methods for estimating available Iysine have been investigated.
Methods for determining the Iysine content of acid hydrolysates using the enzyme Iysine decarboxylase have been described (84, 85). The enzyme is only specific for L-lysine, which has a free e-amino group, and thus can be used to predict biological availability. Lysine decarboxylase from Bacterium cadaveris decarboxylates only L lysine, producing CO2 and cadaverine, both of which can easily be measured and used in determining the Iysine content of a protein hydrolysate. The technique has been modified by immobilizing the Iysine decarboxylase enzyme from B. cadaveris or Escherichia cold B on the surface of a CO2-specific electrode and then determining the quantity of CO: released from a hydrolysate using the electrode response as a measure of the available Iysine (86, 87). Since measurements are made using protein hydrolysates, it is obvious that if the reactive group can be freed from the e-amino group of Iysine by the hydrolysis, then total Iysine rather than available Iysine would be measured by the procedure. insufficient data have as yet been accumulated using this technique to allow its general validity to be ascertained, particularly with respect to how well it compares with bioassay data. The procedure, however, appears to show considerable promise.
Methods for Measuring Available Sulphur-Containing Amino Acids
It has been indicated earlier (p. 11 ) that some of the oxidized forms of cystine and methionine are unavailable, or at best only partially available, for the rat. Methionine and methionine sulphoxide, for example, were shown to be equally available to the rat, but the more highly oxidized methionine sulphone was completely unavailable (88). Many reports have shown that experimental or commercial processes that promote oxidation, for example, drying in the presence of air or bleaching to decolourize, can convert methionine, cystine, and cysteine residues in the protein to their oxidized forms and thereby reduce the nutritive quality of that protein (22, 89-91 ).
In order to measure the available cysteine in processed proteins, Clelands reagent (dithiothreitol, DTT) was used (22) to convert all cystine to cysteine, and the cysteine produced by DTT treatment as well as that inherent in the protein was measured using 5,5'-dithiobis-2-nitrobenzoic acid (DTNB). Cysteic acid does not react with DTNB, and therefore only unoxidized or "available" cysteine is measured.
A method for differentiating between methionine and its oxidized forms (methionine sulphone and sulphoxide) was first described by McCarthy and Sullivan (92). These authors described a colorimetric assay that could detect the loss of methionine in proteins due to heat treatment by specifically determining the actual methionine content of the protein before and after heat treatment. This procedure was later modified (93) to eliminate the possible interference from histidine in the measurement of methionine. It was also demonstrated (22, 90) that the early colorimetric method (92) for available methionine could explain the poor nutritional quality of heat-treated casein measured by rat assay, as the oxidized methionine was non-available.
Because the colorimetric assay (92) for available methionine required an enzymatic hydrolysis to release the methionine prior to colorimetric analysis, methods to measure the amount of methionine directly using the intact protein have been sought. Inasmuch as the methionine residue on a protein was capable of reducing dimethyl sulphoxide (Me2SO) to dimethyl sulphide (Me2S), the methionine content of various peptides could be measured (94) by exposing them to Me2SO and measuring the amount of Me2S formed. The Me2SO was evolved into the headspace above the peptide solution and was then measured using GLC techniques. This procedure was then applied to food proteins that had part of their methionine in the oxidized form, either from H202 treatment or from typical industrial processing procedures. The results for available methionine correlated well (94) with rat PER data.
An alternative procedure for measuring the available methionine content of intact proteins was developed by Ellinger and Duncan (95), who treated the protein with cyanogen bromide (CNBr) and then measured the amount of methylthiocyanate (MeSCN) produced as a consequence of the CNBr reacting with methionine residues. Again, only unoxidized or "available" methionine was measured, as the oxidized forms were unreactive. Mackenzie (96) utilized an improved CNBr assay to determine the available methionine in pea protein.
Of the procedures so far described for determining the methionine content of food proteins, those that determine methionine directly on the intact protein, and therefore that eliminate the variable and timeconsuming enzymic hydrolysis step, have a definite advantage. For any of these procedures to be fully acceptable, however, as techniques for assaying available methionine, cysteine, and cystine in foods, compara tive data between available su)phur amino acids and biological assay values on the same samples are essential. Despite the paucity of data, it is now c)ear that the sulphur amino acids are liable to oxidation during the processing of food proteins, and this reactivity can be a major factor leading to loss of nutritional quality in some foods.
While not a measure of available sulphur amino acids, the use of total sulphur determination as an indicator of the combined level of the two sulphur-containing amino acids, cystine and methionine, has been studied by several workers (97-99). In genera), significant correlations have been found between the contents of "cystine + methionine" and total sulphur. However, a large part of the correlation derives from each measure's being correlated with the total nitrogen (99, 100). The usefulness of the S:N ratio in the prediction of "cystine + methionine" (g/kg crude protein) is less certain; within the limited range of such values found in breeding studies of grain legumes (101), high and significant correlation coefficients have not been established.
Dye-Binding Procedures
Dye-binding procedures are rapid and inexpensive methods of analysis that can be successfully semiautomated (102) and promise to be very useful when applied for protein quality process control where damage involving the binding of Iysine amino groups may occur (81).
Azo dyes combine with the free basic amino groups of Iysine, histidine, and arginine, and with terminal amino groups of the protein chain (103). Good correlations were obtained between dye-binding capacity (DBC) with Acid Orange 10 and chick growth (104) measured with heated soy bean meal, and between Acid Orange 12 binding and mouse growth with a series of heated barleys ( 105). Although positive correlations have been obtained between DBC and animal growth tests or FDNB-reactive Iysine with fish and meat meals, the scatter is still considerable (76, 104, 106). The use of DBC values as indicators of heat damage requires constant amino acid composition of the raw material, and this composition can vary in fish and meat meals (107, 108). Oilseeds tend to have a more constant amino acid composition.
A measure specific for lysine is obtained from the difference in the DBC of a sample before and after treatment with propionic anhydride, which inactivates the lysine groups but has no effect on arginine and histidine (109).
A disadvantage of azo dyes is that they fail to detect damage from protein-sugar reactions under mild conditions (108), probably because the early Maillard products, such as deoxyfructosyl Iysine units, are still basic in nature. Azo dyes cannot therefore be relied on for process control of milk powders.
The reactive dye Remazol Brilliant Blue R has been used as an indicator of the reactive Iysine content of heated milk (110) and whey protein (111). Under the conditions of the test, this dye is thought to react only with the free amino acid group of Iysine and the thiol group of cystine and may detect even early Maillard damage ( 108). However, the method involves gel filtration and is less convenient than azodye procedures.
The phthalein dye Cresol Red shows increased binding with soy bean meals subjected to increasing heat treatment (112), i.e., the opposite result to that obtained with azo dyes and reactive dyes. Good correlations have been found between the dye absorption of overheated soy bean meals and their protein quality for chicks ( 113, 114). However, the test is an empirical one and cannot be used as a general indicator of processing damage to protein foods (108).
Other Methods
With some materials such as cottonseed meals, simple tests of nitrogen solubility have proved useful indirect indicators of the amount of nutritional damage caused by processing (76, 115). With other materials, such as soy milks, this approach has not proved useful (116).

Conclusions
________________________________________
The total amino acid profile of a food protein expressed in relation to some standard is a good indicator of the potential nutritive value, but it may at times be misleading if one or more of the essential amino acids are only partially available. If this is true, the "actual" nutritive value of the protein will be less than its "predicted" value, obtained from its amino acid profile. It is therefore critical that the availability of the reactive, but most frequently limiting essential amino acids, Iysine, methionine, and cystine,* be known. This is especially true for proteins present in processed foods and food ingredients.
Before amino acid data, whether "available" or "total," can be used as indicators of potential nutritional value, they must be expressed in relation to some reference standard. Such standards and the calculation of amino acid score are discussed in detail in the following chapter.
________________________________________
References
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[Author L. Rea]

Hope that helps answer your question Bro and thanks again for asking.

After final patent revisions and locks the first set of independent clinical studies on HumaPro by Bio-Analytical Research group LLC will be posted then presented for peer review and publishing. The second sets underway next moth at University of Omaha and University of OK will start and go straight to peer review next by 2011. The error may make when reading about HumaPro is that it was some sort of design intended for hard core bodybuilding. The project began in 1998 for far superior OTC option for pharmaceutical use for CKD, autism, ADHD, renal disease, diabetics and aging where the highest level of positive nitrogen (most of you use anabolics and know that work as well as I do) must be obtained with the very least negative catabolite waste products needed to prevent killing the patients faster. Many test subjects are well past their death dates now. I am one of them. That test results showed the product to so damn anabolic and effective lead to offering it into a much smaller market of bodybuilding because the results are very unique, helps prevent many of these diseases from attacking my brothers and sisters in what I love most, and works. Your call.


Thanks for your time and again my apology if I was a rude visitor in any way.

Author L. Rea

 

[Tom]

much appreciated

thanks for posting such complete and detailed feedback. i look forward to trying humapro in the near future as part of my leaning out phase.

 

[TeknoViking]

hahah^^^^ bump

 

[chris250]

Magnum,

I know you said you where going to continue using Humapro...so any updates on what is going on with your body/training/weight/ect ??? and how are you using Humapro ?

thanks

chris

 

[TeknoViking]

i would like to know as well.

 

[hank]

I'm using it at 40 pills a day AND LOVING IT!

 

[HUMALOG]

i saw Magnum at the mall. he was so large it was like he was a planet and everyone else around him was just little moons orbiting around him

 

[Tom]

I'm using it at 40 pills a day AND LOVING IT!

Hank....can you elaborate a little more? More strength, endurance, positive changes in body composition? Thank you.

 

[hank]

Im currently dieting and my strength has goig back up to what I was doing when I was 30 pounds heavier. The Muscle fullness is unreal like your pumped above normal! People around here ask me why I'm so pumped! I am leaning out but I'm following a strict diet and doing cardio. Only drawback it makes me VERY HUNGRY! Hope this helps!

 

[Tom]

Im currently dieting and my strength has goig back up to what I was doing when I was 30 pounds heavier. The Muscle fullness is unreal like your pumped above normal! People around here ask me why I'm so pumped! I am leaning out but I'm following a strict diet and doing cardio. Only drawback it makes me VERY HUNGRY! Hope this helps!

yes, thank you. i also want to use this in my leaning out phase.

 

[chris250]

Im currently dieting and my strength has goig back up to what I was doing when I was 30 pounds heavier. The Muscle fullness is unreal like your pumped above normal! People around here ask me why I'm so pumped! I am leaning out but I'm following a strict diet and doing cardio. Only drawback it makes me VERY HUNGRY! Hope this helps!

hmmm interesting...

 

[BigJB]

I suppose this whole argument comes down to whether these are better than LBAs as a supplemental protein source.

 

[Magnum]

Magnum,

I know you said you where going to continue using Humapro...so any updates on what is going on with your body/training/weight/ect ??? and how are you using Humapro ?

thanks

chris

Yes, at the moment I am subing two meals at 8 tabs each. I'm around 286 at the moment. Trying to get down to 168 for the Boston Marathon.

 

[Magnum]

i saw Magnum at the mall. he was so large it was like he was a planet and everyone else around him was just little moons orbiting around him

I'm more like Jupiter, a gaseous planet.

 

[ICU11b]

At 40 a day(2 week now) I am getting leaner w/ what seems like no loss in strength. I feel like I recoup better, not as sore as long. No bloat or indegestion w/ these. Diet has been a little off due to family death... I use 10 when I wake, 10 pre, 10 post, 10 before bed. I work in two real meals & also two shakes(3 omega raw, 1/2c oats, 1 bananna, 1 scoop fiber). I paid 38& change w/ allstar health.

 

[Knight9]

Back in stock! For a while at least..

 

[Thebigone]

I've been hearing A LOT of good feedback about this stuff from a few guys at my gym> These are long time bodybuilders as well with a lot of expierence. They havent lowered their protien intake at all but added 20pills per day of humanopro. They said 5-10 is a waste, you need a good 20-30 per day. They have reported being heavier and fuller while dieting and burning fat faster than normal. 20pills per day, thats a good $70-$80 for a month supply. Wondering if its worth giving it a shot.