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Supplement

Quantifying the Digestibility of Dietary Protein¹

Milk and Health Research Centre, Institute of Food Nutrition and Human Health, Massey University, Palmerston North, New Zealand

²To whom correspondence should be addressed.

	ABSTRACT

TOP ABSTRACT INTRODUCTION Fecal versus ileal digestibility Apparent, true and real... Total N versus individual... Special case for heat-treated... REFERENCES

 
The current recommendation, when calculating a protein digestibility–corrected amino acid score, is to determine the digestibility of a dietary protein across the entire digestive tract, using the rat as a model animal for humans. This fecal digestibility value is subsequently corrected for endogenous contributions of protein using a metabolic nitrogen value determined by feeding rats a protein-free diet. The limitations inherent with this method are well recognized, however, and determining the digestibility of a dietary protein to the end of the small intestine is the preferred alternative. Unlike the fecal digestibility assay, which has only one basic methodology, ileal digestibility values can be determined in a number of ways. We discuss the various methods available for determining ileal digestibility values and compare results obtained for dietary proteins using both fecal and ileal digestibility assays. The relative value of using individual amino acid digestibility values as opposed to nitrogen digestibility values is reviewed. In addition, we address issues surrounding measurement of endogenous nitrogen flows, and in particular, the relative merits of determining "true" versus "real" digestibility values.

KEY WORDS: • protein digestibility • apparent digestibility • true digestibility • real digestibility • protein quality evaluation

	INTRODUCTION

TOP ABSTRACT INTRODUCTION Fecal versus ileal digestibility Apparent, true and real... Total N versus individual... Special case for heat-treated... REFERENCES

 
A significant change in the assessment of dietary protein quality occurred with the introduction of the protein digestibility–corrected amino acid score (PDCAAS)³ (FAO 1991 ). Previously, evaluation of a dietary protein consisted of monitoring the metabolic responses of an animal model to subtle differences in the amino acid (AA) composition of a dietary protein. In contrast, the PDCAAS of a dietary protein is calculated by comparing the AA composition of the dietary protein with a reference AA pattern (assumed to represent human nutritional requirements for AA). Each AA is scored relative to the pattern, and the resulting AA score is corrected for availability using a protein digestibility coefficient. The most limiting AA in the dietary protein, which is reflected by the lowest PDCAAS, determines the final score of the dietary protein.

Since its introduction in 1991 as a simple and scientifically sound approach for the routine assessment of dietary protein quality for humans (FAO 1991 ), the advantages and disadvantages associated with the PDCAAS have been extensively reviewed (Darragh et al. 1998 , Barth and Schaafsma 1995 , Fenwick et al. 1995 , Hooydonk 1994 , Sarwar 1997 ). Although a number of concerns have been documented, one in particular, that of quantifying the digestibility of a dietary protein, warrants further debate.

The current PDCAAS methodology prescribes the use of a true fecal nitrogen (N) digestibility coefficient, as determined in the rat. A correction is made for fecal endogenous N to calculate a true N digestibility coefficient. There is sufficient evidence available, however, to have this methodology replaced with the more accurate determination of true ileal AA digestibility coefficients.

	Fecal versus ileal digestibility

TOP ABSTRACT INTRODUCTION Fecal versus ileal digestibility Apparent, true and real... Total N versus individual... Special case for heat-treated... REFERENCES

 
As mentioned, to calculate a PDCAAS, the availability of the AA in a dietary protein is assessed based on the digestibility of total N in that dietary protein. Digestibility is defined as the difference between the amount of N ingested and excreted, expressed as a proportion of N ingested. Although accepted as the recommended procedure, the use of fecal digestibility coefficients to evaluate AA availability is thought to be inherently inaccurate due to the metabolism of both dietary and endogenous proteins by the hindgut microbial population (Lenis 1983 , Sauer and Ozimek 1986 ). As a result of this microbial protein degradation, fecal N digestibility coefficients will tend to overestimate the AA availability in a dietary protein. In some instances, net synthesis of AA in the large intestine has also been observed (Just 1980 , Low 1980 , Rerat 1981 ), which would lead to an underestimation of protein digestibility. It is obvious, therefore, that a fecal digestibility coefficient may be unable to accurately differentiate between dietary proteins due to the confounding and often equalizing effect of microbial metabolism.

Ileal digestibility coefficients, determined after measurement of the quantity of N remaining at the end of the small intestine or ileum, are thought to provide a much more accurate indication of protein availability. Ileal digestibility coefficients should be more sensitive at differentiating between dietary proteins because the problems associated with microbial protein degradation do not arise. Evidence has been presented to suggest, however, that there is microbial activity in the small intestine (Dierick et al. 1986 , Jørgensen and Jensen 1994 , Knudsen and Jensen 1991 ). Although there is the possibility that this may affect the accuracy of ileal digestibility values, it would seem reasonable to assume that any effect would be minimal compared with that observed in the large intestine.

The impact of microbial degradation in the large intestine on estimation of protein digestibility coefficients has been observed in several monogastric species, including humans, with notable differences in fecal and ileal digestibility coefficients (Moughan and Donkoh 1991 ), as summarized in Table 1 . It should also be noted that the size of the difference between fecal and ileal digestibility coefficients is not constant among the AA. Some AA are more affected by microbial degradation in the large intestine than are others, as indicated by a range of differences observed (Table 2 ) between fecal and ileal digestibility coefficients determined for individual AA in a diet consumed by adult humans (Rowan et al. 1994 ).

Unlike the determination of fecal digestibility, which involves the relatively simple procedure of feces collection, the determination of ileal digestibility requires the more invasive technique of sampling digesta from the end of the small intestine.

Measurements of the ileal digestibility of a dietary protein can be conducted in humans. Techniques such as the recruitment of patients fitted with ileostomies have been used successfully, although the length of time since the original ileostomy operation needs to be carefully considered because gut microflora readily populate the terminal ileum (Dowsett et al. 1990 , Gorbach et al. 1967 ), creating an environment not unlike the large intestine. The technique of entering a tube via the mouth or nose and collecting digesta from different parts of the digestive tract, including the terminal ileum, has also been used (Mahé et al. 1992 , Modigliani et al. 1973 ). It is highly unlikely, however, that these techniques would be readily accepted as part of a routine method of protein quality evaluation.

The alternative is to use an animal model to test individual dietary proteins for digestibility. The use of animal models also allows more flexibility and control over the experimental conditions. Although the laboratory rat has traditionally been used in protein quality evaluation, the pig has also been reported as a suitable model for the investigation of protein digestibility in humans (Darragh and Moughan 1995 , Moughan et al. 1992 and 1994 , Rowan et al. 1994 ). The pig and human are similar in several key areas. Both are simple-stomached, meal-eating, omnivorous mammals. The gastrointestinal anatomy, physiology and metabolism of the pig are very similar to those in the human (Moughan et al. 1994 ). A comparison of the ileal digestibility of AA by the pig and human (Rowan et al. 1994 ) shows little difference (Table 3 ), which offers strong evidence for the validity of using the pig as a model animal for the human in protein digestibility studies.

Sampling at slaughter (Butts et al. 1991 , Payne et al. 1968 , van Wijk et al. 1998 ) has the distinct advantage of involving minimal disruption to normal digestive function immediately before collection. Due to the limited amount of digesta collected, there is the possibility of a bias in results due to unrepresentative sampling of digesta. When the animal is fed frequently before slaughter, however, the variability of data derived is no greater than that found with cannulated animals (Donkoh et al. 1994 ). The "slaughter method" has the advantage that it may be a more economical option than procedures that involve the surgical insertion of a cannula. It does, however, necessitate the use of a greater number of animals.

Anastomosis (Fuller and Livingstone 1982 , Hennig et al. 1986 ) involves transecting the ileum anterior to the ileocecal junction and attaching this to the descending colon. This allows a quantitative collection of ileal digesta, but many studies have shown that anastomosed animals have an altered physiology compared with "intact" animals (e.g., Hennig et al. 1989 , Köhler et al. 1992a and 1992b ). It should be noted that human ileostomates may also have an altered physiology compared with "intact" humans, which could also call into question the validity of using human ileostomates for the collection of ileal digesta.

The insertion of a re-entrant cannula (Cunningham et al. 1962 , Easter and Tanksley 1973 ) involves transecting the terminal ileum and sealing the two ends. A cannula is inserted into each end of the sealed ileum and the two cannulae are joined. This allows a quantitative collection of digesta. The surgery to insert a re-entrant cannula is complex, however, and involves total transection of the ileum. Blockages of the cannula are common.

Simple T-piece cannulation (Gargello and Zimmerman 1980 , Livingstone et al. 1977 ) and postvalve T-cecum (PVTC) cannulation (van Leeuwen et al. 1991 ) have the distinct advantage of maintaining the ileocecal valve intact and avoiding ileal transection. With the PVTC cannula, there is no surgical interference with the small intestine. Also, most of the digesta should pass through the PVTC cannula during sampling, as the ileocecal value protrudes directly into the cannula. In practice, mean marker recoveries on the order of 72–106% have been reported with this method (den Hartog et al. 1988 , Hodgkinson et al. 2000 , Köhler et al. 1990 and 1991 ). The PVTC cannulation procedure appears to be the method of choice for the collection of ileal digesta.

	Apparent, true and real digestibility

TOP ABSTRACT INTRODUCTION Fecal versus ileal digestibility Apparent, true and real... Total N versus individual... Special case for heat-treated... REFERENCES

 
When the digestibility of a dietary protein is calculated by subtracting the amount of nitrogen and AA in ileal digesta from the amount of nitrogen and AA that were ingested, an "apparent" digestibility coefficient is obtained. However, ileal digesta also contain a significant proportion of nondietary AA, from sources such as mucus, cells, digestive enzymes and bile, and these are called the endogenous AA (Fauconneau and Michel 1970 , Snook 1973 ). A correction needs to be made for these endogenous AA losses (EAAL) when determining digestibility coefficients. To further complicate matters, EAAL is not uniform for all diets (e.g., Souffrant 1991 ), being dependent on diet composition, particularly the presence of dietary protein, fiber and antinutritional factors (ANF) (Huisman et al. 1993 ). The EAAL, therefore, is made up of two parts: a basal EAAL that occurs independent of dietary composition and an extra EAAL that occurs in response to a specific factor in the diet. Examples of the EAAL that can occur when different protein sources are fed to animals are given in Table 4 .

There are several methods that can be used to quantify the EAAL. Traditionally, a protein-free (PF) diet was fed to the animal, and the protein remaining at the end of the small intestine was assumed to be the basal EAAL. The provision of a diet devoid of protein, however, leads to an unphysiological state in the recipient animal with the animal being in a negative body nitrogen balance (Buraczewska 1979 , Fauconneau and Michel 1970 , Schneeman 1982 , Snook and Meyer 1964 ). Therefore, a method was developed that involved feeding an animal diets containing synthetic AA (Butts et al. 1993 , Darragh et al. 1990 , Skilton et al. 1988 ). In each diet, selected dietary nonessential or essential AA were excluded; in the latter case, feeding of the diet was accompanied by intravenous infusion of the deleted essential AA. The amounts of the excluded AA in ileal digesta allowed a direct determination of EAAL. With this method, the animals had a readily absorbable source of AA and therefore were not in a negative body nitrogen balance. This should eliminate the unphysiological nature of the PF method. An additional method that has been developed to determine basal EAAL, the enzyme hydrolyzed protein technique, measures basal EAAL under the more physiologically normal conditions of protein alimentation (Butts et al. 1993 , Moughan et al. 1990 ). The latter method involves feeding the test animal an enzyme hydrolyzed protein (usually enzyme hydrolyzed casein) diet with ultrafiltration of the ileal digesta collected to remove any unabsorbed dietary AA. Comparison of these three methods (Table 5 ) has shown that the PF and synthetic AA methods were not significantly different from each other, yet values for both were significantly lower than the EAAL determined using the enzyme hydrolyzed protein method. This demonstrates that the presence of dietary peptides in the gut, as occurs when a normal diet is eaten, provokes a much higher EAAL and that as such, the enzyme hydrolyzed casein method should become the method of choice for determining a basal EAAL correction factor.

As mentioned previously, when the digestibility of a dietary protein is calculated by subtracting the amount of AA leaving the terminal ileum from the amount of AA that were ingested by the animal, the apparent digestibility coefficient is obtained. When the basal EAAL, determined using methods such as the PF method or the enzyme hydrolyzed protein method, are corrected for in the calculation of digestibility, the "true" digestibility coefficient is obtained.

True digestibility is a fundamental property of the food, and it is not affected by the dietary conditions under which the food is fed to the animal (e.g., the protein content of the test diet). The apparent digestibility measure, however, will be affected by the assay conditions and is therefore variable and subject to error. True digestibility is a superior measure for determining the AA that are absorbed from the gut and therefore gives a better representation of protein quality than apparent digestibility.

When the "extra" EAAL associated with the presence of ANF and fiber are corrected for in the calculation of digestibility (by use of the homoarginine or ¹⁵N methods for determining endogenous loss), the resultant coefficients of digestibility are termed "real" digestibility coefficients. Real digestibility coefficients reflect the absolute amounts of dietary N and AA that are absorbed across the intestinal tract.

It is possible, therefore, to determine either apparent, true or real ileal digestibility coefficients. Apparent digestibility coefficients will always underestimate AA availability and are also influenced by the digestibility assay conditions (Darragh et al. 1995 ). When protein products do not contain fiber and/or ANF, true and real digestibility are numerically the same, and both will provide an accurate assessment of AA absorption. In protein products that do contain fiber or ANF, however, only real digestibility will provide an accurate measure of AA absorption, with true digestibility underestimating dietary AA absorption.

The suggestion has been made that the calculation of the PDCAAS be altered to include real ileal N digestibility rather than the true fecal N digestibility coefficient currently used (Barth and Schaafsma 1995 ). There may be general agreement that the shift from fecal to ileal digestibility is essential. It should be noted, however, that although able to provide data on absolute absorption of dietary AA, real digestibility coefficients will not be able to differentiate between proteins with or without factors that may increase the EAAL. This is because all EAAL specific to that particular protein being tested would be accounted for when calculating the real digestibility.

True digestibility coefficients, although unable to provide an absolute assessment of AA availability, would be able to differentiate between proteins with or without ANF because the extra EAAL would be factored into a lower digestibility coefficient. True digestibility coefficients, therefore, would more accurately reflect the relative worth of a protein to the consumer and provide a more accurate means of ranking dietary proteins in terms of quality. To illustrate this point, a working example has been included in Table 6 .

	Total N versus individual AA digestibility coefficients

TOP ABSTRACT INTRODUCTION Fecal versus ileal digestibility Apparent, true and real... Total N versus individual... Special case for heat-treated... REFERENCES

 
Another area of concern with regard to quantifying protein digestibility is failure of the current methodology to represent the most accurate assessment of AA availability. There can be quite striking differences between the digestibility coefficients for total N and the individual AA in a dietary protein (Table 7 ). By using the digestibility coefficient for total N, the availability of the tryptophan in skim milk would be underestimated, whereas the availability of several AA in the soy protein diet would be overestimated. It would be more appropriate, therefore, to determine individual AA digestibility coefficients when calculating the PDCAAS for a protein.

	Special case for heat-treated proteins

TOP ABSTRACT INTRODUCTION Fecal versus ileal digestibility Apparent, true and real... Total N versus individual... Special case for heat-treated... REFERENCES

A new bioassay has been developed (Moughan and Rutherfurd 1996 ) that uses the reaction of O-methylisourea with the -amino of lysine to form the acid-stable derivative homoarginine. This reaction is coupled with an ileal AA digestibility assay to allow determination of the ileal digestibility of reactive lysine. The true ileal digestibility of reactive lysine is then calculated. These coefficients can then be used to calculate digestible reactive lysine or available lysine. This new approach places emphasis on determining the uptake of chemically available lysine molecules from the digestive tract. For unprocessed foods, the digestible reactive lysine content should be equivalent to the digestible total lysine content determined using conventional methods, whereas for a processed food, the total lysine content may be higher than the reactive lysine content due to the conversion of lysine derivatives to lysine during the acid hydrolysis stage of conventional AA analysis and total lysine digestibility will be lower. Overall, for the processed food, the digestible available lysine content will be overestimated using conventional procedures. In severely damaged protein sources, some of the structurally altered lysine derivatives may be acid stable and thus may not convert back to lysine during acid hydrolysis. In this case, reactive and total lysine values should be more similar. The bioassay has been applied to a range of processed foods (Rutherfurd et al. 1997 ), and a comparison of the results from the bioassay and conventional AA analysis is shown in Table 8 . Determination of true ileal digestibility using conventional AA analysis will significantly underestimate lysine digestibility in foods such as soyabean meal, dried maize, heated skim milk powder, cottonseed meal and an alfalfa-based mix.

Several concerns still remain regarding the calculation of PDCAAS for the evaluation of dietary protein. At the present, when calculating PDCAAS values, corrections are made for the availability of nitrogen in the protein using true fecal nitrogen digestibility coefficients with metabolic fecal nitrogen determined after feeding a test animal a PF diet. There are several concerns with this methodology.

There is strong evidence that fecal digestibility coefficients do not accurately differentiate between dietary proteins due to the effects of microbial metabolism in the large intestine. Therefore, it is recommended that digesta collected at the end of the small intestine, the terminal ileum, be used for the determination of digestibility coefficients. This will increase the accuracy and sensitivity of the assay.

When calculating true ileal digestibility coefficients, EAAL should be determined using a method that allows the estimation of basal endogenous losses only. The PF method is not recommended for the determination of EAAL, because this method results in an unphysiological state in the animal. The enzyme hydrolyzed protein method is more appropriate for the determination of EAAL. True digestibility should be used to calculate PDCAAS, as opposed to real digestibility, because only true digestibility coefficients allow a differentiation between proteins that contain factors that may increase EAAL, e.g., ANF and fiber. True digestibility coefficients provide a more accurate ranking of dietary proteins in terms of quality.

At the present, PDCAAS values are corrected for the digestibility of total nitrogen as opposed to individual AA. However, there often are quite large differences between digestibility coefficients for total nitrogen and the individual AA in a dietary protein. The accuracy of PDCAAS would be improved by determining individual AA digestibility coefficients as opposed to only that for total nitrogen.

It should also be noted that when a dietary protein has undergone some form of heat treatment, the availability of some AA, particularly lysine, might be underestimated using the conventional true ileal digestibility assay. The assay must be modified to account for the damage to heat-sensitive AA.

These recommendations relating to quantifying protein digestibility should be considered, and changes should be made to the PDCAAS system to improve the accuracy of PDCAAS for the differentiation between dietary proteins in terms of nutritional quality.

	FOOTNOTES

 
¹ Presented at the symposium "Criteria and Significance of Dietary Protein Sources in Humans," held in San Francisco, CA, on October 4, 1999. The symposium was sponsored by the National Dairy Council; International Dairy Federation; United Kingdom Dairy Association; Dairy Farmers of Canada; Davisco Foods International, Inc.; New Zealand Milk; CAMPINA MELKUNIE, Zaltbommel, The Netherlands; Land O’Lakes; and CERIN. Published as a supplement to The Journal of Nutrition. Guest editors for this publication were Gregory D. Miller, National Dairy Council, Rosemont, IL, and Daniel Tome, Institut National Agronomique, Paris, France.

³ Abbreviations used: AA, amino acid; ANF, antinutritional factor; EAAL, endogenous amino acid loss; PDCAAS, protein digestibility–corrected amino acid score; PF, protein-free; PVTC, postvalve T-cecum.

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TOP ABSTRACT INTRODUCTION Fecal versus ileal digestibility Apparent, true and real... Total N versus individual... Special case for heat-treated... REFERENCES

 

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