Criteria and Significance of Dietary Protein

Sources in Humans

Quantifying the Digestibility of Dietary Protein1

Alison J. Darragh2 and Suzanne M. Hodgkinson
Milk and Health Research Centre, Institute of Food Nutrition and Human Health, Massey University,
Palmerston North, New Zealand

Downloaded from https://academic.oup.com/jn/article-abstract/130/7/1850S/4686199 by guest on 08 April 2020

ABSTRACT 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. J. Nutr. 130:
1850S—1856S, 2000.

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

A significant change in the assessment of dietary protein
quality occurred with the introduction of the protein digest-
ibility– corrected amino acid score (PDCAAS)3 (FAO 1991).
Previously, evaluation of a dietary protein consisted of moni-
toring 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
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.
2
To whom correspondence should be addressed.
3
Abbreviations used: AA, amino acid; ANF, antinutritional factor; EAAL, en-
dogenous amino acid loss; PDCAAS, protein digestibility– corrected amino acid
score; PF, protein-free; PVTC, postvalve T-cecum.
quality for humans (FAO 1991), the advantages and disad-
vantages 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 partic-
ular, 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 re-
placed with the more accurate determination of true ileal AA
digestibility coefficients.
Fecal versus ileal digestibility
As mentioned, to calculate a PDCAAS, the availability of
the AA in a dietary protein is assessed based on the digest-
ibility 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

0022-3166/00 $3.00 © 2000 American Society for Nutritional Sciences.

1850S
 QUANTIFYING DIETARY PROTEIN DIGESTIBILITY 1851S

TABLE 1 cessfully, although the length of time since the original ileos-
tomy operation needs to be carefully considered because gut
Comparison of the ileal and fecal digestibility of dietary microflora readily populate the terminal ileum ( Dowsett et al.
protein for several simple-stomached mammals1 1990, Gorbach et al. 1967), creating an environment not
unlike the large intestine. The technique of entering a tube via
Apparent digestibility the mouth or nose and collecting digesta from different parts
of the digestive tract, including the terminal ileum, has also
Fecal Ileal
been used (Mahé et al. 1992, Modigliani et al. 1973). It is
Piglet 0.97 0.90 highly unlikely, however, that these techniques would be
Growing pig 0.81 0.66 readily accepted as part of a routine method of protein quality
Preruminant calf 0.94 0.88 evaluation.
Adult human 0.89 0.87 The alternative is to use an animal model to test individual
Chicken 0.86 0.78 dietary proteins for digestibility. The use of animal models also
Growing rat 0.78 0.69
allows more flexibility and control over the experimental

Downloaded from https://academic.oup.com/jn/article-abstract/130/7/1850S/4686199 by guest on 08 April 2020

1 From Moughan and Donkoh 1991. 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
dietary protein. In some instances, net synthesis of AA in the digestibility in humans (Darragh and Moughan 1995,
large intestine has also been observed (Just 1980, Low 1980, Moughan et al. 1992 and 1994, Rowan et al. 1994). The pig
Rerat 1981), which would lead to an underestimation of and human are similar in several key areas. Both are simple-
protein digestibility. It is obvious, therefore, that a fecal di- stomached, meal-eating, omnivorous mammals. The gastroin-
gestibility coefficient may be unable to accurately differentiate testinal anatomy, physiology and metabolism of the pig are
between dietary proteins due to the confounding and often very similar to those in the human (Moughan et al. 1994). A
equalizing effect of microbial metabolism. comparison of the ileal digestibility of AA by the pig and
Ileal digestibility coefficients, determined after measure- human (Rowan et al. 1994) shows little difference (Table 3),
ment of the quantity of N remaining at the end of the small which offers strong evidence for the validity of using the pig as
intestine or ileum, are thought to provide a much more accu- a model animal for the human in protein digestibility studies.
rate indication of protein availability. Ileal digestibility coef- There are several methods available to collect ileal digesta
ficients should be more sensitive at differentiating between from animal models such as the pig. Sampling from the ter-
dietary proteins because the problems associated with micro- minal ileum after euthanasia is common in both the rat and
bial protein degradation do not arise. Evidence has been pig, whereas other methods, such as cannulation and bypass of
presented to suggest, however, that there is microbial activity the large intestine by ileorectal anastomy or ileostomy, are
in the small intestine (Dierick et al. 1986, Jørgensen and more commonly performed using the pig. There are advan-
Jensen 1994, Knudsen and Jensen 1991). Although there is tages and disadvantages associated with each of these methods.
the possibility that this may affect the accuracy of ileal digest- Sampling at slaughter (Butts et al. 1991, Payne et al. 1968,
ibility values, it would seem reasonable to assume that any
effect would be minimal compared with that observed in the
large intestine. TABLE 2
The impact of microbial degradation in the large intestine Mean apparent fecal and ileal amino acid digestibility
on estimation of protein digestibility coefficients has been
coefficients for adult humans receiving a
observed in several monogastric species, including humans,
with notable differences in fecal and ileal digestibility coeffi- meat/vegetable/cereal/dairy product– based diet1
cients (Moughan and Donkoh 1991), as summarized in Table
Fecal Ileal
1. It should also be noted that the size of the difference digestibility digestibility Statistical
between fecal and ileal digestibility coefficients is not constant Amino acid coefficient2 coefficient3 SE significance4
among the AA. Some AA are more affected by microbial
degradation in the large intestine than are others, as indicated Lysine 0.93 0.94 0.010 NS
by a range of differences observed (Table 2) between fecal and Arginine 0.93 0.90 0.008 *
ileal digestibility coefficients determined for individual AA in Histidine 0.92 0.90 0.014 NS
Aspartate 0.90 0.87 0.009 *
a diet consumed by adult humans (Rowan et al. 1994). Serine 0.92 0.87 0.015 ***
With regard to the calculation of a PDCAAS, resistance to Threonine 0.89 0.85 0.013 **
the introduction of an ileal digestibility coefficient was evident Glutamate 0.95 0.94 0.005 NS
initially, mainly because ileal coefficients, as seen in Tables 1 Proline 0.95 0.90 0.005 **
and 2, are usually lower than the corresponding fecal values, Glycine 0.87 0.72 0.039 ***
which ultimately results in a lower PDCAAS. Opinions are Alanine 0.88 0.88 0.008 NS
changing, however, as the argument builds against the fecal Valine 0.91 0.90 0.007 NS
Isoleucine 0.91 0.91 0.006 NS
digestibility assay, with the ileal digestibility assay being seen Leucine 0.93 0.92 0.005 NS
as more accurate and sensitive. Tyrosine 0.90 0.89 0.007 NS
Unlike the determination of fecal digestibility, which in- Phenylalanine 0.91 0.90 0.007 ***
volves the relatively simple procedure of feces collection, the Cysteine 0.91 0.86 0.017 NS
determination of ileal digestibility requires the more invasive Methionine 0.83 0.93 0.025 ***
technique of sampling digesta from the end of the small Tryptophan 0.83 0.77 0.025 *
intestine. 1 From Rowan et al. 1994.
Measurements of the ileal digestibility of a dietary protein 2 n ⫽ 6.
can be conducted in humans. Techniques such as the recruit- 3 n ⫽ 5.
ment of patients fitted with ileostomies have been used suc- 4 NS, P ⬎ 0.05, *P ⬍ 0.05, **P ⬍ 0.01, ***P ⬍ 0.001.
1852S SUPPLEMENT

TABLE 3 and avoiding ileal transection. With the PVTC cannula, there
is no surgical interference with the small intestine. Also, most
Mean true1 ileal amino acid and nitrogen digestibility of the digesta should pass through the PVTC cannula during
coefficients for ileostomized adult humans (65 kg body sampling, as the ileocecal value protrudes directly into the
weight) and growing pigs (25 kg body weight) receiving a cannula. In practice, mean marker recoveries on the order of
meat/vegetable/cereal/dairy product– based diet2 72–106% have been reported with this method (den Hartog et
al. 1988, Hodgkinson et al. 2000, Köhler et al. 1990 and
Human Pig Statistical 1991). The PVTC cannulation procedure appears to be the
Amino acid (n ⫽ 5) (n ⫽ 6) SE significance3 method of choice for the collection of ileal digesta.
Lysine 0.98 0.98 0.004 NS
Arginine 0.98 0.98 0.005 NS Apparent, true and real digestibility
Histidine 0.99 0.98 0.007 NS
Aspartate 0.99 0.98 0.006 NS When the digestibility of a dietary protein is calculated by
Serine 0.99 1.00 0.006 NS subtracting the amount of nitrogen and AA in ileal digesta

Downloaded from https://academic.oup.com/jn/article-abstract/130/7/1850S/4686199 by guest on 08 April 2020

Threonine 1.05 1.00 0.014 ** from the amount of nitrogen and AA that were ingested, an
Glutamate 0.98 0.98 0.003 NS “apparent” digestibility coefficient is obtained. However, ileal
Proline 1.01 0.98 0.011 NS
Glycine 0.92 0.91 0.013 NS
digesta also contain a significant proportion of nondietary AA,
Alanine 0.99 0.98 0.005 NS from sources such as mucus, cells, digestive enzymes and bile,
Valine 1.00 0.99 0.005 NS and these are called the endogenous AA (Fauconneau and
Isoleucine 1.00 0.98 0.006 NS Michel 1970, Snook 1973). A correction needs to be made for
Leucine 1.00 1.01 0.004 NS these endogenous AA losses (EAAL) when determining di-
Tyrosine 0.99 0.98 0.005 NS gestibility coefficients. To further complicate matters, EAAL is
Phenylalanine 1.00 0.98 0.007 *
Cysteine 1.00 0.92 0.020 ***
not uniform for all diets (e.g., Souffrant 1991), being depen-
Methionine 0.98 0.96 0.006 ** dent on diet composition, particularly the presence of dietary
Tryptophan 0.99 0.97 0.023 NS protein, fiber and antinutritional factors (ANF) (Huisman et
Nitrogen 0.98 0.99 0.012 NS al. 1993). The EAAL, therefore, is made up of two parts: a
basal EAAL that occurs independent of dietary composition
1 Corrected using endogenous amino acid loss for humans and pigs
and an extra EAAL that occurs in response to a specific factor
fed a protein-free diet (Rowan 1989; Rowan et al. 1993). in the diet. Examples of the EAAL that can occur when
2 From Rowan et al. 1994.
3 NS, P ⬎ 0.05, *P ⬍ 0.05, **P ⬍ 0.01, ***P ⬍ 0.001. different protein sources are fed to animals are given in Ta-
ble 4.
The implication of this increased EAAL in terms of human
van Wijk et al. 1998) has the distinct advantage of involving nutrition is that any increase in EAAL brought about by a
minimal disruption to normal digestive function immediately dietary component will increase the consumer’s requirement
before collection. Due to the limited amount of digesta col- for dietary AA to offset the additional gut loss. Because this
lected, there is the possibility of a bias in results due to additional AA requirement cannot feasibly be included in any
unrepresentative sampling of digesta. When the animal is fed single reference AA pattern, the method used to correct for
frequently before slaughter, however, the variability of data the EAAL becomes a crucial determinant in the evaluation of
derived is no greater than that found with cannulated animals the quality of a dietary protein.
(Donkoh et al. 1994). The “slaughter method” has the advan- There are several methods that can be used to quantify the
tage that it may be a more economical option than procedures EAAL. Traditionally, a protein-free (PF) diet was fed to the
that involve the surgical insertion of a cannula. It does, how- animal, and the protein remaining at the end of the small
ever, necessitate the use of a greater number of animals. intestine was assumed to be the basal EAAL. The provision of
Anastomosis (Fuller and Livingstone 1982, Hennig et al. a diet devoid of protein, however, leads to an unphysiological
1986) involves transecting the ileum anterior to the ileocecal state in the recipient animal with the animal being in a
junction and attaching this to the descending colon. This negative body nitrogen balance (Buraczewska 1979, Faucon-
allows a quantitative collection of ileal digesta, but many neau and Michel 1970, Schneeman 1982, Snook and Meyer
studies have shown that anastomosed animals have an altered 1964). Therefore, a method was developed that involved feed-
physiology compared with “intact” animals (e.g., Hennig et al. ing an animal diets containing synthetic AA (Butts et al.
1989, Köhler et al. 1992a and 1992b). It should be noted that
human ileostomates may also have an altered physiology com-
pared with “intact” humans, which could also call into ques-
tion the validity of using human ileostomates for the collec- TABLE 4
tion of ileal digesta. Endogenous nitrogen (EN) flows in the ileal digesta
The insertion of a re-entrant cannula (Cunningham et al. of pigs fed different proteins1
1962, Easter and Tanksley 1973) involves transecting the
terminal ileum and sealing the two ends. A cannula is inserted Protein source Dietary protein
into each end of the sealed ileum and the two cannulae are
joined. This allows a quantitative collection of digesta. The g EN/100 g protein
surgery to insert a re-entrant cannula is complex, however, and
involves total transection of the ileum. Blockages of the can- Skim milk 1.3
Wheat 3.1
nula are common. Soya protein isolate 3.3
Simple T-piece cannulation (Gargello and Zimmerman Barley 4.0
1980 , Livingstone et al. 1977) and postvalve T-cecum Phaseolus beans 10.8
(PVTC) cannulation (van Leeuwen et al. 1991) have the
distinct advantage of maintaining the ileocecal valve intact 1 Adapted from Souffrant 1991.
 QUANTIFYING DIETARY PROTEIN DIGESTIBILITY
TABLE 5
Mean endogenous ileal losses of lysine in the growing pig as
determined using three different methods1
Method
Synthetic Enzyme hydrolyzed
Protein free2 amino acid3 protein4
␮g/g dry matter intake
Lysine5 252a 284a 448b
1 From Butts et al. 1993.
2 Diets devoid of protein, with the lysine present in digesta assumed
to be of endogenous origin.
3 Pigs fed purified diets devoid of lysine. Lysine in digesta assumed
to be of endogenous origin.
4 Pig fed diets containing 10% enzyme hydrolyzed protein (MW
⬍5000 Da) as the sole nitrogen source. Ileal digesta ultrafiltered (MW
cutoff 10,000 Da) to separate endogenous protein.
5 Mean values with a different superscript are significantly different
(P ⬍ 0.05).
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 demon-
strates 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.
Other methods have also been developed using either the
lysine analogue homoarginine (Hagemeister and Erbersdobler
1985, Rutherfurd and Moughan 1990) or the isotope 15N (de
Lange et al. 1990, Souffrant et al. 1982) to simultaneously
measure both the basal EAAL associated with a protein meal
and any extra EAAL that may occur with a specific protein
product.
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 ob-
tained. 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
1853S
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 digest-
ibility (by use of the homoarginine or 15N methods for deter-
mining endogenous loss), the resultant coefficients of digest-
ibility are termed “real” digestibility coefficients. Real
digestibility coefficients reflect the absolute amounts of dietary
N and AA that are absorbed across the intestinal tract.
Downloaded from https://academic.oup.com/jn/article-abstract/130/7/1850S/4686199 by guest on 08 April 2020
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 mea-
sure 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 coeffi-
cients 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.
TABLE 6
The calculation of theoretical apparent, true, and real
digestibility coefficients for the total nitrogen (N) in either a
skim milk or soy protein diet fed to the growing pig
Soy protein
Skim milk isolate
Intake (g N) (I) 40 40
Ileal output (g N)
Undigested dietary N (UN) 2.0 2.0
Basal endogenous N (B) 1.5 1.5
Extra endogenous N (X) 0 2.0
Apparent digestibility
{I ⫺ [UN ⫹ B ⫹ X]}/I 0.91 0.86
True digestibility
{I ⫺ [(UN ⫹ B ⫹ X) ⫺ B]}/I 0.95 0.90
Real digestibility
{I ⫺ [(UN ⫹ B ⫹ X) ⫺ B ⫺ X]}/I 0.95 0.95
1854S SUPPLEMENT

TABLE 7
Digestibility coefficients for total nitrogen and selected amino acids in skim milk and a soy protein product1

Digestibility coefficients for:

Total nitrogen Lysine Methionine Cysteine Threonine Tryptophan

Skim milk 0.95 0.96 0.92 0.94 0.95 0.98

Soybean 0.90 0.87 0.82 0.82 0.84 0.89

1 Adapted from FAO/WHO 1991.

Downloaded from https://academic.oup.com/jn/article-abstract/130/7/1850S/4686199 by guest on 08 April 2020

From the example given in Table 6, it is evident that ically available lysine molecules from the digestive tract. For
calculating real digestibilities failed to identify that the soy unprocessed foods, the digestible reactive lysine content
diet was creating an extra burden on the consumer’s protein should be equivalent to the digestible total lysine content
status by eliciting a greater EAAL. The true digestibility determined using conventional methods, whereas for a pro-
coefficient was able to differentiate between the two protein cessed food, the total lysine content may be higher than the
diets as the extra EAAL associated with the soy diet was reactive lysine content due to the conversion of lysine deriv-
“costed” into the lower digestibility coefficient. By using true atives to lysine during the acid hydrolysis stage of conven-
digestibility coefficients, it is possible, at least in part, for the tional AA analysis and total lysine digestibility will be lower.
PDCAAS to take into account the effect ANF will have on Overall, for the processed food, the digestible available lysine
protein quality. content will be overestimated using conventional procedures.
In severely damaged protein sources, some of the structurally
Total N versus individual AA digestibility coefficients altered lysine derivatives may be acid stable and thus may not
Another area of concern with regard to quantifying protein convert back to lysine during acid hydrolysis. In this case,
digestibility is failure of the current methodology to represent reactive and total lysine values should be more similar. The
the most accurate assessment of AA availability. There can be bioassay has been applied to a range of processed foods (Ruth-
quite striking differences between the digestibility coefficients erfurd et al. 1997), and a comparison of the results from the
for total N and the individual AA in a dietary protein (Table bioassay and conventional AA analysis is shown in Table 8.
7). By using the digestibility coefficient for total N, the avail- Determination of true ileal digestibility using conventional
ability of the tryptophan in skim milk would be underesti- AA analysis will significantly underestimate lysine digestibility
mated, whereas the availability of several AA in the soy
protein diet would be overestimated. It would be more appro-
priate, therefore, to determine individual AA digestibility TABLE 8
coefficients when calculating the PDCAAS for a protein.
Comparison of mean1 true ileal lysine digestibility determined
Special case for heat-treated proteins using conventional amino acid analysis (total) and true ileal
lysine digestibility based on determined reactive lysine
Many foods are processed (exposing them to heat, pressure
and other materials such as alkalis) and/or are stored for long (reactive)2
periods of time with consequent damage to the AA. This may Lysine digestibility
render some of the AA nutritionally unavailable. This is Overall Statistical
particularly so for lysine, which possesses an ⑀-amino group Total3 Reactive4 SE significance
that can react with a wide range of compounds present in the
diet to produce compounds that may be partially absorbed %
from the gut but have no nutritional value to the animal
(Hurrell and Carpenter 1981). A proportion of the reacted Blood meal 96.3 96.7 0.41 NS
Wheat meal 92.6 92.1 0.45 NS
lysine derivatives are acid labile and can revert back to lysine Meat and bone
during the acid hydrolysis step of conventional AA analysis. meal 88.9 91.5 0.76 NS
This does not, however, occur in the digestive tract. Conse- Soybean meal 94.5 96.5 0.41 *
quently, the lysine concentrations of the food and ileal digesta, Dried maize 80.5 84.3 1.54 *
determined by conventional AA analysis, will be misleading Heated skim milk
and the conventional true ileal digestibility assay will generally powder 69.1 94.0 1.11 ***
Cottonseed meal 62.1 71.9 1.75 **
overestimate lysine availability in heat-treated foods. A differ- Alfalfa-based mix 74.2 86.3 0.63 ***
ent approach is required.
A new bioassay has been developed (Moughan and Ruth- 1 For blood meal, wheat meal, soyabean meal, meat and bone meal,
erfurd 1996) that uses the reaction of O-methylisourea with heated skim milk powder, and cottonseed meal, n ⫽ 8; for the dried
the ⑀-amino of lysine to form the acid-stable derivative ho- maize and alfalfa-based mix, n ⫽ 5.
moarginine. This reaction is coupled with an ileal AA digest- 2 From Rutherfurd et al. 1997.
3 Lysine digestibility was determined using a true ileal amino acid
ibility assay to allow determination of the ileal digestibility of
digestibility assay (rat), and conventional amino acid analysis was used
reactive lysine. The true ileal digestibility of reactive lysine is to quantify total lysine in the diets and digesta.
then calculated. These coefficients can then be used to calcu- 4 Lysine digestibility was determined using a true ileal amino acid
late digestible reactive lysine or available lysine. This new digestibility assay (rat), and the quanidination reaction was used to
approach places emphasis on determining the uptake of chem- quantify reactive lysine in the diets and digesta.
 QUANTIFYING DIETARY PROTEIN DIGESTIBILITY
in foods such as soyabean meal, dried maize, heated skim milk
powder, cottonseed meal and an alfalfa-based mix.
For unprocessed foods, the true ileal digestibility assay is
recommended. For processed feeds, at least in terms of lysine
and possibly other AA, the conventional true ileal digestibility
assay will overestimate the digestible lysine that is available to
the animal. Therefore, other methods, such as the true ileal
reactive lysine digestibility assay, are required to give accurate
results.
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 estima-
tion 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 be-
tween 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 di-
gestibility coefficients for total nitrogen and the individual AA
in a dietary protein. The accuracy of PDCAAS would be
improved by determining individual AA digestibility coeffi-
cients 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 di-
gestibility 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.
LITERATURE CITED
Barth, C. A. & Schaafsma, G. (1995) A critical assessment of the PDCAAS, a
protein evaluation system, issued by FAO/WHO/UNU in 1990/1991. IDF Nutr.
Newslett. 4: 39 – 40.
Buraczewska, L. (1979) Secretion of nitrogenous compounds into the small
intestine of pigs. In: Current Concepts of Digestion and Absorption in Pigs
(Low, A. G. & Partridge, I. G., eds.), pp. 151–156. The National Institute for
Research in Dairying, Shinfield/Hannah Research Institute, Ayr, UK.
Butts, C. A., Moughan, P. J. & Smith, W. C. (1991) Endogenous amino acid
flow at the terminal ileum of the rat determined under conditions of peptide
alimentation. J. Sci. Food Agri. 55: 175–187.
Butts, C. A., Moughan, P. J., Smith, W. C. & Carr, D. H. (1993) Endogenous
lysine and other amino acid flows at the terminal ileum of the growing pig (20
kg body weight): the effect of protein-free, synthetic amino acid, peptide and
protein alimentation. J. Sci. Food Agri. 61: 31– 40.
1855S
Cunningham, H. M., Friend, D. W. & Nicholson, J.W.G. (1962) Note on a
re-entrant fistula for digestion studies with pigs. Can. J. Anim. Sci. 42:
112–113.
Darragh, A. J. & Moughan, P. J. (1995) The three-week-old piglet as a model
animal for studying protein digestion in human infants. J. Pediatr. Gastroen-
terol. Nutr. 21: 387–393.
Darragh, A. J., Moughan, P. J., Rutherfurd, S. M. & Boisen, S. (1995) The
availability of amino acids in feedstuffs for growing pigs. In: Recent Advances
in Animal Nutrition in Australia (Rowe, J. & Nolan, M., eds.), pp. 34 – 40.
University of New England, Armidale, Australia.
Darragh, A. J., Moughan, P. J. & Smith, W. C. (1990) The effect of amino acid
and peptide alimentation on the determination of endogenous amino acid
flow at the terminal ileum of the rat. J. Sci. Food Agri. 51: 47–56.
Darragh, A. J., Schaafsma, G. & Moughan, P. J. (1998) Impact of amino acid
availability on the protein digestibility corrected amino acid score. Bull. IDF
336: 46 –50.
de Lange, C.F.M., Souffrant, W. B. & Sauer, W. C. (1990) Real ileal protein and
amino acid digestibilities in feedstuffs for growing pigs as determined with the
Downloaded from https://academic.oup.com/jn/article-abstract/130/7/1850S/4686199 by guest on 08 April 2020
15
N-isotope dilution technique. J. Anim. Sci. 68: 409 – 418.
den Hartog, L. A., van Leeuwen, P., Huisman, J., Zandstra, T., van Heugten, E.,
van Ommeren, H. J. & van Kleef, D. (1988) Comparison of ileal digestibility
data obtained from pigs provided with a different type of cannula. In: Diges-
tive Physiology in the Pig: Proceedings of the 4th International Seminar
(Buraczewska, L., Buraczewski, S., Pastuszewska, B. & Zebrowska, T., eds.),
pp. 275–282. Institute of Animal Physiology Nutrition, Joblonna, Poland.
Dierick, N. A., Vervaeke, I. J., Decuypere, J. A. & Hendrickx, H. K. (1986)
Influence of the gut flora and of some growth-promoting feed additives on
nitrogen metabolism in pigs. II. Studies in vivo. Livestock Prod. Sci. 14:
177–193.
Donkoh, A., Moughan, P. J. & Smith, W. C. (1994) Comparison of the slaughter
method and simple T-piece cannulation of the terminal ileum for determining
ileal amino acid digestibility in meat and bone meal for the growing pig. Anim.
Feed Sci. Technol. 49: 43–56.
Dowsett, J., Gibney, M. J. & Kennedy, N. P. (1990) Bacterial fermentation
occurs in the terminal ileum of ileostomates. Proc. Nutr. Soc. 49: 110.A. (abs.)
Easter, R. A. & Tanksley, T. D. (1973) A technique for re-entrant ileocecal
cannulation of swine. J. Anim. Sci. 36: 1099 –1103.
Fauconneau, G. & Michel, M. C. (1970) The role of the gastrointestinal tract in
the regulation of protein metabolism. In: Mammalian Protein Metabolism, Vol.
IV (Munro, H. N. ed.), pp. 481–522. Academic Press, London.
Fenwick, R. M., Knighton, D. R. & Moughan, P. J. (1995) Protein quality
measurement by the PDCAAS technique. IDF Nutr. Newslett. 4: 40 – 43.
Food and Agriculture Organization (1991) Protein Quality Evaluation: Report of
the Joint FAO/WHO Expert Consultation, Bethesda, MD, USA, 4 – 8 December
1989. FAO Food and Nutrition Paper No. 51. Food and Agriculture Organiza-
tion of the United Nations, Rome.
Fuller, M. F. & Livingstone, R. M. (1982) Annual Report of Studies in Animal
Nutrition and Allied Sciences, Rowett Research Institute, Aberdeen, Scotland.
Gargallo, J. & Zimmerman, D. R. (1980) A simple intestinal cannula for swine.
Am. J. Vet. Res. 41: 618 – 619.
Gorbach, S. L., Nahas, L., Weinstein, R. & Patterson, J. F. (1967) Studies of
intestinal microflora. IV. The microflora of ileostomy effluent: a unique micro-
bial ecology. Gastroenterology 53: 874 – 880.
Hagemeister, H. & Erbersdobler, H. (1985) Chemical labelling of dietary protein
by transformation of lysine to homoarginine: a new technique to follow intes-
tinal digestion and absorption. Proc. Nutr. Soc. 44: 133.A. (abs.)
Hennig, U., Noel, R., Herrman, U., Wünche, J. & Mehnert, E. (1986) Ernähr-
ungsphysiologische untersuchungen an schweinen mit ileo-rektal-anasto-
mosen. Arch. Anim. Nutr. 36: 585–596.
Hennig, U., Wünche, J., Herrmann, U. & Borgmann, E. (1989) [Nutrition-
physiologic studies in pigs with ileo-rectal anastomoses. 3. Nitrogen retention
and utilization after energy and electrolyte supplementation]. Arch. Anim.
Nutr. 39: 611– 622.
Hodgkinson, S. M., Moughan, P. J., Reynolds, G. W. and James, K.A.C. (2000)
The effect of dietary peptide concentration on endogenous ileal amino acid
loss in the growing pig. Br. J. Nutr. 84: 421– 430.
Hooydonk, A. C. M. (1994) Definition of the nutritional value of dietary proteins.
In: Protein Definition. Proceedings of the 1st IDF Symposium, Minneapolis,
MN, October 1993. International Dairy Federation, Brussels.
Huisman, J., Verstegen, M. W. A, van Leeuwen, P. & Tamminga, S. (1993)
Reduction of N pollution by decrease of the excretion of endogenous N in
pigs. In: Nitrogen Flow in Pig Production and Environmental Consequences.
pp. 55– 61. Pudoc Scientific Publishers, Wageningen.
Hurrell, R. F. & Carpenter, K. J. (1981) The estimation of available lysine in
foodstuffs after Maillard reactions. Prog. Food Nutr. Sci. 5: 159 –176.
Jørgensen, H. & Jensen, B. B. (1994) The effect of dietary fiber on digestibility,
microbial activity, and microbial gas production in various regions of the
gastrointestinal tract of pigs. VIth International Symposium on Digestive Phys-
iology in Pigs (Souffrant, W.-B. & Hagemeister, H., eds.), pp. 273–276. EAAP
Publication No. 80. European Association of Animal Production, Germany.
Just, A. (1980) Ileal digestibility of protein: applied aspects. In: Current Con-
cepts of Digestion and Absorption in the Pig (Low, A. G. & Partridge, I. G.,
eds.), pp. 66 –72. National Institute on Research in Dairying, England/Hannah
Research Institute, Scotland.
Knudsen, K.E.B. & Jensen, B. B. (1991) Effect of source and level of dietary
fibre on microbial fermentation in the large intestine of pigs. In: Digestive
1856S SUPPLEMENT
Physiology in Pigs. Proceedings of the Vth International Symposium on Di-
gestive Physiology in Pigs, April 24 –26, 1991 (Huisman, J., den Hartog, L. A.
& Verstegen, M.W.A., eds.), pp. 389 –394. EAAP Publication No. 54. Puduc,
Wageningen, the Netherlands.
Köhler, T., Huisman, J., den Hartog, L. A. & Mosenthin, R. (1990) Comparison
of different digesta collection methods to determine the apparent digestibili-
ties of the nutrients at the terminal ileum in pigs. J. Sci. Food Agri. 53:
465– 475.
Köhler, T., Mosenthin, R., Verstegen, M. W. A., Huisman, J., den Hartog, L. A. &
Ahrens, F. (1992a) Effect of ileo-rectal anastomosis and post-valve T-
caecum cannulation on growing pigs. 1. Growth performance, N-balance and
intestinal adaptation. Br. J. Nutr. 68: 293–303.
Köhler, T., Verstegen, M. W. A., Huisman, J., van Leeuwen, P. & Mosenthin, R.
(1991) Comparison of various techniques for measuring ileal digestibility in
pigs. In: Digestive Physiology in Pigs. Proceedings of the Vth International
Symposium on Digestive Physiology in Pigs, April 24 –26, 1991 (Huisman, J.,
den Hartog, L. A. & Verstegen, M.W.A., eds.), pp. 296 –303. EAAP Publication
No. 54. Puduc, Wageningen, the Netherlands.
Köhler, T., Verstegen, M. W. A., Mosenthin, R., Wensing, T., den Hartog, L. A. &
Huisman, J. (1992b) Effect of ileo-rectal anastomosis and post-valve T-
caecum cannulation on growing pigs. 2. Blood variables and mineral bal-
ances. Br. J. Nutr. 68: 305–315.
Lenis, A. (1983) Faecal amino acid digestibility in feedstuffs for pigs. In:
Metabolisme et Nutrition Azotes (Arnal, M., Pion, R. & Bonin, D., eds.), pp.
385–389. INRA, Paris.
Livingstone, R. M., Fowler, V. R., White, F. & Wenham, G. (1977) Annealed
glass cannulae for use in digestion studies with pigs. Vet. Record 101: 368.
Low, A. G. (1980) Nutrient absorption in pigs. J. Sci. Food Agri. 31: 1087–1130.
Mahé, S., Huneaum, J. F., Marteau, P., Thuillier, F. & Tomé, D. (1992) Gas-
troileal nitrogen and electrolyte movements after bovine milk ingestion in
humans. Am. J. Clin. Nutr. 56: 410 – 416.
Modigliani, R., Rambaud, J. C. & Bernier, J. J. (1973) The method of intralu-
minanl perfusion of the human small intestine. 1. Principle and technique.
Digestion 9: 176 –192.
Moughan, P. J., Birtles, M. J., Cranwell, P. D., Smith, W. C. & Pedraza, M. (1992)
The piglet as a model animal for studying aspects of digestion and absorption
in milk-fed human infants. World Rev. Nutr. Diet. 67: 40 –113.
Moughan, P. J., Cranwell, P. D., Darragh, A. J. & Rowan, A. M. (1994) The
domestic pig as a model for studying digestion in humans. In: Digestive
Physiology in Pigs. Proceedings of the Vth International Symposium on Di-
gestive Physiology in Pigs, April 24 –26, 1991 (Huisman, J., den Hartog, L. A.
& Verstegen, M.W.A., eds.), pp. 389 –396. EAAP Publications No. 80. Puduc,
Wageningen, the Netherlands.
Moughan, P. J., Darragh, A. J., Smith, W. C. & Butts, C. A. (1990) Perchloric
and trichloroacetic acids as precipitants of protein in endogenous ileal digesta
from the rat. J. Sci. Food Agri. 52: 13–21.
Moughan, P. J. & Donkoh, A. (1991) Amino acid digestibility in non-ruminants:
a review. Recent Adv. Anim. Nutr. Australia 172–184.
Moughan, P. J. & Rutherfurd, S. M. (1996) A new method for determining
digestible reactive lysine in foods. J. Agri. Food Chem 44: 2202–2209.
Payne, W. L., Combs, G. F., Kifer, R. R. & Snyder, D. G. (1968) Investigation of
protein quality: ileal recovery of amino acids. Fed. Proc. 27: 1199 –1203.
Rérat, A. A. (1981) Digestion and absorption of nutrients in the pig. World Rev.
Nutr. Diet. 37: 229 –287.
Rowan, A. M. (1989) A Study of the Digestion of Protein in Humans using Ileal
and Faecal Assays. Master’s Thesis, Massey University, Palmerston North,
New Zealand.
Rowan, A. M., Moughan, P. J. & Wilson, M. N. (1993) Endogenous amino acid
flow at the terminal ileum of adult humans determined following the ingestion
of a single protein-free meal. J. Sci Food Agri. 61: 439 – 442.
Rowan, A. M., Moughan, P. J., Wilson, M. N., Maher, K. & Tasman-Jones, C.
(1994) Comparison of the ileal and faecal digestibility of dietary amino acids
in adult humans and evaluation of the pig as a model animal for digestion
studies in man. Br. J. Nutr. 71: 29 – 42.
Rutherfurd, S. M. & Moughan, P. J. (1990) Guanidination of lysine in selected
dietary proteins. J. Agri. Food Chem. 38: 209 –211.
Rutherfurd, S. M., Moughan, P. J. & van Osch, L. (1997) Digestible reactive
lysine in processed feedstuffs: application of a new bioassay. J. Agri. Food
Chem. 45: 1189 –1194.
Sarwar, G. (1997) The protein digestibility-corrected amino acid score method
Downloaded from https://academic.oup.com/jn/article-abstract/130/7/1850S/4686199 by guest on 08 April 2020
overestimates quality of proteins containing antinutritional factors and of
poorly digestible proteins supplemented with limiting amino acids in rats. J.
Nutr. 127: 758 –764.
Sauer, W. C. & Ozimek, L. (1986) Digestibility of amino acids in swine: results
and their practical application: a review. Livestock Prod. Sci. 15: 367–388.
Schneeman, B. O. (1982) Digestive enzyme activities from the pancreas in
response to diet. In: Physiologie Digestive Chez le Porc (Laplace, J. P.,
Corring, T. & Rerat, A., eds.), pp. 125–131. Institut National de la Recherche
Agronomique, Paris.
Skilton, G. A., Moughan, P. J. & Smith, W. C. (1988) Determination of endog-
enous amino acid flow at the terminal ileum of the rat. J. Sci. Food Agri. 44:
227–235.
Snook, J. T. (1973) Protein digestion. World Rev. Nutr. Diet. 18: 121–176.
Snook, J. T. & Meyer, J. H. (1964) Factors influencing the significance of
endogenous nitrogen to the non-ruminant. In: The Role of the Gastrointestinal
Tract in Protein Metabolism (Munro, H. N., ed.), pp. 97- 116. Blackwell
Scientific Publications, Oxford.
Souffrant, W. B. (1991) Endogenous nitrogen losses during digestion in pigs.
In: Digestive Physiology in Pigs. Proceedings of the Vth International Sympo-
sium on Digestive Physiology in Pigs, April 24 –26, 1991 (Huisman, J., den
Hartog, L. A. & Verstegen, M.W.A., eds.). EAAP Publication No. 54. Puduc,
Wageningen, the Netherlands.
Souffrant, W. B., Köhler, R. & Gebhardt, G. (1982) [Determination of endoge-
nous nitrogen in the digestive contents by the isotope technique (15N)]. In:
Physiologie Digestive Chez le Porc (Corring, T., Rerat, A. & Laplace, J. P.,
eds.), pp. 176 –187. INRA Publications (Les Colloques de l’INRA No. 12).
.Institut National de la Recherche Agronomique, Paris.
van Leeuwen, P., van Kleef, D. J., van Kempen, G.J.M., Huisman, J. & Verstegen,
M.W.A. (1991) The post-valve T-caecum cannulation technique in pigs
applicated (sic) to determine the digestibility of amino acid in maize, ground-
nut and sunflower seed. J. Anim. Phys. Anim. Nutr. 65: 183–193.
van Wijk, H. J., Moughan, P. J., Hodgkinson, S. M., Jansen, P. P. & Pearson, G.
(1998) Variation in apparent and true ileal amino acid digestibility in barley
using a rat model. Anim. Feed Sci. Tech. 76: 9 –22.