Download Review of advances in metabolic bioavailability of amino acids

Livestock Science 133 (2010) 4–9
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Livestock Science
j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / l i v s c i
Review article
Review of advances in metabolic bioavailability of amino acids☆
Crystal L. Levesque a, Soenke Moehn a, Paul B. Pencharz b,c, Ron O. Ball a,⁎
a
b
c
Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, Alberta, Canada T6G 2P5
Research Institute, Hospital for Sick Children, Toronto, ON, Canada M5G 1X8
Departments of Pediatrics and Nutritional Sciences, University of Toronto, Canada
a r t i c l e
i n f o
Keywords:
Amino acids
Bioavailability
Digestibility
Metabolic availability
Indicator amino acid oxidation
Slope ratio
Protein quality
a b s t r a c t
The animal's amino acid requirement and the potential of feedstuffs to supply those amino acids
in metabolically available form (amino acid bioavailability) are intertwined. Although
standardized ileal amino acid digestibility is currently most widely used as an estimate of
dietary protein quality, numerous factors influence these estimates. Slope ratio assays are
considered the standard against which other methods of amino acid availability are judged
because they measure the effect of protein quality on the entire metabolism of the animal.
However, practical use of slope ratio growth assays is limited. Recently, the slope ratio assay
utilizing the indicator amino acid oxidation technique has been developed to determine the
metabolic availability (MA) of amino acids in pigs and humans This review will discuss how the
indicator amino acid oxidation (IAAO) technique can: 1) address some of the problems
associated with the slope ratio assay; 2) be used to determine the metabolic availability (MA) of
amino acids in animal feedstuffs; 3) become a valuable measure of amino acid bioavailability;
and 4) be applied in practical swine nutrition.
© 2010 Elsevier B.V. All rights reserved.
Contents
1.
Introduction . . . . . . . . . . . . . . . . . . . . . . . .
2.
Indicator amino acid oxidation concepts and techniques . . .
3.
IAAO as a measure of metabolic availability of AA in feedstuffs
4.
Diet formulation . . . . . . . . . . . . . . . . . . . . . .
5.
Statistical analysis and standard error . . . . . . . . . . . .
6.
Advantages of metabolic availability . . . . . . . . . . . . .
7.
Practical application of MA . . . . . . . . . . . . . . . . .
8.
Conclusions . . . . . . . . . . . . . . . . . . . . . . . .
Conflict of Interest . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction
The animal's amino acid requirement and the potential of
feedstuffs to supply those amino acids in metabolically
☆ This paper is part of the special issue entitled 11th International
Symposium on Digestive Physiology of Pigs.
⁎ Corresponding author. Tel.: +1 780 492 7151; fax: +1 780 492 4265.
E-mail address: [email protected] (R.O. Ball).
1871-1413/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.livsci.2010.06.013
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available form (feedstuff amino acid bioavailability) are
intertwined. To fully utilize the knowledge of one, we must
also have a complete knowledge of the other. However,
methods used to estimate both amino acid requirements and
amino acid bioavailability are still a matter of considerable
debate in animal and human nutrition research.
Amino acid bioavailability has been defined as the
percentage of amino acids digested and absorbed in a form
available for utilization for the purpose of maintenance or
C.L. Levesque et al. / Livestock Science 133 (2010) 4–9
growth of tissue (ARC, 1981; Batterham, 1992; Fan, 1994;
Danfaer and Fernández, 1999). Amino acid digestibility,
amino acid bioavailability using slope ratio assays, protein
digestibility-corrected amino acid score, net postprandial
protein utilization and postprandial protein utilization have
all been used to estimate protein quality of animal and human
food ingredients (Stein et al., 2007, Elango et al., 2009a).
Each method has its own unique set of advantages and
disadvantages.
In pig nutrition, standardized ileal amino acid digestibility
is currently most widely used as an estimate of dietary protein
quality. Ileal amino acid digestibility is comparatively easy to
determine, and yields results for all amino acids in a
reasonably short time frame. However, amino acid digestibility coefficients are highly dependent on an accurate estimate
of ileal endogenous amino acid losses (Stein et al., 2005).
Numerous factors such as dietary fiber and crude protein
content, antinutritional factors, body weight and composition
of protein-free diet can all influence the estimate of endogenous amino acid losses (Stein et al., 2007). The reader is
directed to Stein et al. (2007) for a detailed description of the
terminology and problems with estimation of ileal endogenous amino acid losses. Amino acid digestibility, in its most
accurate form (true ileal digestibility), can only measure
amino acid disappearance from the gut (Stein et al., 2007) and
thus does not account for first-pass metabolism or amino acids
absorbed in a chemical complex unsuitable for metabolism. If
the degree of first-pass metabolism is different between AA
then the measurement of the pattern of AA in the diet, or the
residual AA in the digesta, will not reflect the availability of AA
to the extra-intestinal tissue (Stoll et al., 1998). Heat
processing of feedstuffs can result in AA forming chemical
complexes (i.e. lysine and Maillard reaction products) that can
be digested and absorbed but cannot be utilized by the animal
for protein synthesis (Carpenter and Booth, 1973).
Assays of amino acid bioavailability that measure parameters of animal growth (slope ratio assays) are considered
the standard against which other methods of amino acid
availability are judged (Lewis and Bayley, 1995). The advantages of slope ratio assays that measure parameters of protein
synthesis are that they measure a response that has practical
and economic consequences and account for the effect of
digestion, absorption and utilization of the amino acids
provided by the protein source (Lewis and Bayley, 1995).
Therefore, such assays measure the effect of protein quality on
the entire metabolism. The importance of this has been shown
by Batterham (1992) who demonstrated that heat-damaged
protein supported lower protein retention than a control diet
despite similar amino acid digestibility. However, practical
use of amino acid bioavailability growth assays is limited by
time, cost and the limitation of examining only one amino acid
at a time (Batterham, 1992).
Recently, the slope ratio assay utilizing the indicator amino
acid oxidation technique as the dependent response criterion
has been developed to determine the metabolic availability
(MA) of amino acids in pigs (Moehn et al., 2005, 2007) and
humans (Humayan et al., 2007). This review will demonstrate
how the indicator amino acid oxidation (IAAO) technique
can 1) address some of the problems associated with the slope
ratio assay; 2) be used to determine the metabolic availability
(MA) of amino acids in animal feedstuffs; 3) become a
5
valuable measure of amino acid bioavailability; and 4) be
applied in practical swine nutrition.
2. Indicator amino acid oxidation concepts and techniques
The IAAO method is based on the principle that amino
acids are either utilized for protein synthesis or must be
oxidized. Therefore, the change in oxidation of the indicator
amino acid is inversely proportional to protein synthesis
(Fig. 1). When one essential amino acid (test amino acid) is
limiting, all other AA are in excess and must be oxidized
(Moehn et al., 2005). As the intake of the limiting amino acid
increases, the oxidation of an indicator amino acid decreases
linearly until the requirement for the limiting amino acid is
reached. Increases of the test amino acid beyond the
requirement do not increase protein synthesis further, and
there is no further change in oxidation of the indicator amino
acid (Fig. 1); indicator amino acid oxidation reaches a plateau.
Ball and Bayley (1986) found that the recovery of
radioactivity in liver protein was greatest at the level of
dietary protein that minimized 1-14C phenylalanine oxidation. The increase in radioactivity of liver protein indicated
that 1-14C phenylalanine was increasingly incorporated into
liver protein, hence protein synthesis, until protein synthesis
was maximized, represented by a plateau in liver protein
radioactivity. Thus the oxidation of the indicator amino acid
indicated that the rate of whole body protein synthesis was
driven by the limiting amino acid. The breakpoint is defined
as the requirement for the test amino acid. The IAAO
technique has been extensively developed to determine
amino acid requirements and was chosen as the gold
standard by the World Health Organization for determination
of amino acid requirements of humans (WHO, 2007;
Pencharz and Ball, 2003).
Selection of an appropriate indicator AA is an important
component of IAAO. Key criteria when selecting an amino acid
as indicator are 1) an essential amino acid and 2) the labeled C
is irreversibly lost as CO2 during oxidation, preferably during
the first steps of amino acid catabolism. Apart from the
indicator amino acid of choice, phenylalanine, lysine (Ball and
Bayley, 1984a), threonine (Soliman and King, 1969), [14Cmethyl]-methionine (Brookes et al., 1972) and leucine (Hsu et
al., 2006, Kurpad et al., 1998) have been used as indicator
amino acids. The L-[1-13 or 1-14C]-phenylalanine, in the
presence of excess tyrosine, has been used most often because
of its many advantages: the free pool size is comparatively
Fig. 1. Inverse relationship between protein synthesis and oxidation of an
indicator amino acid. Adapted from Ball and Bayley (1986).
6
C.L. Levesque et al. / Livestock Science 133 (2010) 4–9
small with a quick turn-over rate (Neale and Waterlow, 1974),
and the size of the intracellular free phenylalanine pool is
tightly regulated (Flaim et al., 1982). Consequently, phenylalanine oxidation was more responsive to dietary changes
than lysine or leucine (Neale and Waterlow, 1974). The
advantage of the short half-life of the free phenylalanine pool
is that it responds rapidly to changes in test amino acid intake.
Ball and Bayley (1984b) showed in baby pigs that amino acid
requirement could be determined with as little as 4 h of
adaptation to the diet. In sows and growing pigs, phenylalanine oxidation responded within 1–2 days of a change in test
amino acid intake, and remained constant for up to 10 days
after a change in diet (Moehn et al., 2004). Therefore, 2 days of
adaptation to a new dietary test amino acid level is deemed
sufficient. Recently, Elango et al. (2009b) showed in humans
that the adaptation period can be even shorter.
3. IAAO as a measure of metabolic availability of AA
in feedstuffs
The change in appearance of 1-13 or 1-14CO2 in breath
reflects the change of whole body protein synthesis. At
plateau in oxidation, protein synthesis is maximized and
would not be expected to respond to changing supply of test
amino acids of differing availability. However, when the
intake of the test amino acid is limiting, the change in
indicator oxidation reflects the response of whole body
protein synthesis to graded levels (or intakes) of the limiting
amino acid (Moehn et al., 2005). This slope indicates the
change in IAAO per unit change in limiting amino acid. A
shallower slope indicates less amino acid per unit intake is
available to support protein synthesis. Therefore, the ratios of
the slope of the linear response of indicator oxidation can be
used to estimate the MA of essential AA in feedstuffs.
Metabolic availability of AA in feedstuffs is defined as the
ratio of the indicator oxidation response of the addition of
limiting amino acid from test protein source to that of free
amino acid (Elango et al., 2009a). Gabert et al. (2001)
suggested that this comparison of the slope of the response
from a test protein source to the slope of the response to a
defined reference protein source represents relative values
only. However, the true ileal digestibility of crystalline AA is
essentially 100% (Baker, 1992). Thus, the slope of the
indicator amino acid oxidation obtained with the crystalline
form of the test amino acid represents the maximal unit
increase in protein synthesis and is equivalent to 100%
metabolic availability of the test amino acid. The oxidation of
the indicator amino acid is regressed against the amino acid
intake above that provided by the base diet (supplied by free
test amino acid) and the protein source (Moehn et al., 2005).
The ratio of the slope of the indicator oxidation due to the
protein source to the indicator oxidation slope due to the free
amino acid gives the MA of the protein source.
The test of the MA concept using the IAAO technique was
conducted by Moehn et al. (2005). Addition of free lysine
from peas, and even more so from heat-treated peas, to a base
diet led to a shallower decrease in oxidation than addition of
free lysine (Fig. 2). This indicated a lower availability of lysine
in peas and heated peas for protein synthesis. The MA of
lysine in peas was calculated to be 88.8% and was similar to
previously published estimates of the true ileal digestibility of
lysine in peas (NRC, 1998). Heating peas using a protocol
similar to that employed by van Barneveld et al (1994), who
used traditional slope ratio assay, reduced the lysine
availability further, to 54.8% (Moehn et al., 2005). Similarly,
Ball et al. (1995) reported impaired lysine availability in
heated cotton seed meal, but found similar oxidation
responses for lysine in soybean meal and for free lysine.
Therefore, using the IAAO in a slope ratio assay allows
determination of the availability of AA in vivo, and is capable,
contrary to digestibility studies, to detect reduced protein
quality of heat-treated feedstuffs.
4. Diet formulation
There are three important dietary considerations for slope
ratio assays: (1) the test amino acid must be first limiting, (2)
the response to changes in test amino acid intake must be
predictable, preferably linear, and (3) the observed response
must not be influenced by other dietary nutrients in the test
feed ingredient (Littell et al., 1995, Batterham, 1992). In
addition, when applying the IAAO in a slope ratio assay, an
isotopic steady state must be achieved.
Batterham (1992) suggested that a basal diet should
contain all nutrients in slight excess, except the amino acid of
interest. This is relatively simple to accomplish with the use of
crystalline AA added alone or in addition to a protein source
in the base diet. All AA except the amino acid of interest must
be set at greater than 100% of requirement while the
maximum intake of the test amino acid should be set at a
level at least 2 SD below requirement. Bertolo et al. (2005)
estimated the CV for inter-animal variability of lysine
requirement was 10%. Restriction of the test amino acid
intake to 80% of the mean requirement, therefore, leaves only
2.5% of the animals with a potentially lower requirement. This
is important because a linear response to changes in test
amino acid intake is critical for calculation of MA. Indicator
oxidation was linear as lysine intake increased up to 80%, 2 SD
below the mean lysine requirement of growing pigs (Moehn
et al., 2005, 2007). Humayan et al. (2007) also noted a linear
decrease in 1-13C phenylalanine oxidation in adult males over
increasing methionine intakes up to the lower 95% confidence
interval of the methionine requirement. Numerous other
studies measuring different response parameters have shown
a linear response to amino acid intakes below requirement:
plasma urea nitrogen and nitrogen retention (Coma et al.,
Fig. 2. Metabolic availability of lysine in peas and heated peas. Adapted from
Moehn et al. (2005).
C.L. Levesque et al. / Livestock Science 133 (2010) 4–9
1995; Dourmad and Étienne, 2002), indicator oxidation (Ball
and Bayley, 1986) and BW and litter gain (Cooper et al.,
2001).
The supply of the indicator amino acid also has to be at a
safe level. If the intake falls short of the requirement, protein
synthesis may be limited by intake of the indicator rather
than the test amino acid. Likewise, a large excess of the
indicator amino acid must also to be avoided. A very large
excess of the indicator amino acid may overwhelm the
animals' capacity to oxidize the amino acid, forestalling a
change in oxidation rate to graded levels of the intake of the
test amino acid. If the animal must oxidize a large excess of
the indicator amino acid sensitivity of the oxidation measurement will be reduced. The test ingredient should be
incorporated at the expense of a non-protein energy source
and at a level to supply a similar quantity of test amino acid to
ensure that the response is due to the test amino acid and not
influenced by other nutrients from the test protein (Batterham, 1992). Addition of the test ingredient will alter the
dietary amino acid profile compared to the reference diets.
However, with the use of crystalline amino acids it is possible
to obtain similar dietary intakes of all essential amino acids
and crude protein in both reference and test protein diets,
thus the only factor driving IAAO becomes the level of test
amino acid intake (Humayan et al., 2007).
To achieve an isotopic state, either primed i.v. or primed,
frequent oral dosing of the indicator amino acid can be used.
Moehn et al. (2005) showed that either route of isotope
delivery achieved a plateau lasting for 2.5 h, beginning
approximately 1.5 h after the start of isotope delivery. Oral
dosing of isotope circumvents the need for venous catheterization, and assures that the indicator amino acid is subjected
to the same route of application as the test amino acid.
Therefore, oral isotope delivery with frequent, ½ hourly
feeding is the preferred application of the IAAO.
5. Statistical analysis and standard error
As with all statistical analyses, assumptions required to
apply the analysis must hold true and should be tested prior to
final data analysis. The assumptions of linearity of the response
to graded amino acid intakes and intersection of the regression
line are important for calculation of amino acid bioavailability
(Littell et al., 1995). As discussed previously, the assumption of
linearity of the response to graded amino acid intakes holds
true when dietary amino acid intake levels are maintained
below 2 SD of the mean amino acid requirement.
To ensure intersection of the regression lines of the
reference and test proteins, both the crystalline test amino
acid and the test protein source should be added to a semisynthetic or synthetic basal diet containing the lowest level of
test amino acid. The dependent response variable is then
regressed against the amino acid intake above that provided
by the base diet (Moehn et al., 2005).
Estimates of bioavailability usually demonstrate a high
standard error due to the effect of individual variation among
test subjects on the slope of the response and the accuracy of
the total amino acid analysis of the test protein (Batterham,
1992). The short adaptation time required for the IAAO
method (b2 days, Moehn et al., 2004) allows repeated
measures in the same animal at all levels of test amino
7
acid intake, thus reducing the effect of individual variation.
Total amino acid analysis can vary from 5 to 10% between
laboratories (Sarwar et al., 1983; Batterham, 1992). Digestibility experiments require total amino acid analyses to be
conducted on the test diets, the protein-free diet, as well as
the collected digesta; whereas, only the test diets are analyzed
for total amino acid content in MA studies. Additionally, stable
and radioactive isotope analyses are measured with great
precision and are highly sensitive and thus able to detect
smaller differences between treatments than traditional
amino acid analysis. The lower sensitivity of amino acid
analysis compared to isotopic analysis requires additional
numbers of observations to account for differences in the
sensitivity of the response variable.
6. Advantages of metabolic availability
Although the measurement of amino acid bioavailability
by slope ratio assay is regarded as the ‘gold standard’, there
are problems with its practical implementation. Conventional
response criterion, such as comparative slaughter or Nbalance technique, in slope ratio assays requires large number
of animals and prolonged periods of time. These problems can
be addressed by using the IAAO instead of conventional
methods. The short adaptation period to dietary treatments
(b2 days, Moehn et al., 2004) allows repeated measurements
of indicator oxidation within animals in rapid succession.
Therefore, both the number of animals, inter-animal variation
and the time frame of an experiment can be reduced
drastically. Typically, the MA of an amino acid in the test
feed can be tested within 14 days. The IAAO also fulfills the
critical requirement that the response criterion used in a slope
ratio assay should be a measure of protein retention (Batterham, 1992). This has been shown in numerous studies using
the IAAO technique to determine amino acid requirements
of pigs (Ball and Bayley, 1984a; Moehn et al. 2005; Levesque
et al. 2009), chickens (Ewing et al., 2001) and humans (Elango
et al., 2007).
While applying the IAAO in a slope ratio assay is an improvement over conventional techniques, metabolic availability still only provides information for one amino acid at a
time. Determination of ileal digestibility yields values for all
amino acids simultaneously. However, metabolic availability
represents the end result of digestion, absorption and
metabolic utilization of amino acids, while digestibility values
are only a measure of disappearance in the small intestine.
Furthermore, ileal digestibility usually is determined using
surgically modified animals, while metabolic availability can
be measured in intact pigs. Surgical modification (e.g. Tcanula, re-entrant canula, ileal–rectal anastomosis) may
disrupt the regular function of the gastro-intestinal tract
(Fuller, 1991), and thus yield digestibility values that may
deviate from those applicable to intact animals. However, ileal
digestibility is the most common measure of protein quality in
pig nutrition and the basis for most recommendations for
dietary amino acid supply; given its comparatively easy
determination, it can be expected that ileal digestibility will
be the measure of choice for the foreseeable future. In contrast,
Batterham (1992) suggests that amino acid availability values
are the most useful for creating amino acid availability
reference tables, evaluating the effect of processing conditions,
8
C.L. Levesque et al. / Livestock Science 133 (2010) 4–9
and as reference values in the development of new techniques.
The metabolic availability method becomes the method of
choice to determine amino acid (e.g. lysine) availability if losses
during and/or after absorption are suspected, e.g. lysine in
heat damaged proteins. The metabolic availability method also
becomes the method of choice if surgical modification is either
unethical, e.g. in humans, or impractical.
7. Practical application of MA
Practical diet formulation relies on the additivity of the
estimates of feed amino acid content when creating mixed
feed diets. Fan (1994) argued that protein retention is affected
by the diet balance of AA, diet level of protein:energy and
energy sources, chronological appearance of AA at the tissue
level, genotype, and physiological state. Based on this
argument, Stein et al. (2007) regarded the metabolic
availability estimate as relevant only under the same conditions (dietary, genetic, and physiological state) that the slope
ratio study was conducted and deem the availability estimates
as not likely additive. However, the factors described by Fan
(1994) do not affect the availability estimate by slope ratio if
the experiment is properly conducted. Ball and Bayley (1986)
showed that at constant phenylalanine intake, the ratio of
protein bound to free phenylalanine in the liver was not
affected by protein intake ranging from deficient to excess.
This lack of influence of dietary protein on the distribution of
label between protein bound and non-protein bound fractions
suggests that MA estimates would be additive. Additionally,
the efficiency of use of protein above maintenance is not
affected by pig breed (Kyriazakis and Emmans, 1995). Similar
to any digestibility studies, diet formulation and animal
selection are used to control potential dietary influences,
other than the test amino acid level, on observed responses.
Acknowledging the relationship between amino acid
requirement of the animal and feedstuff amino acid bioavailability, it has been suggested that methods used to express
both requirement and AA availability should be the same
(Lewis and Bayley, 1995; Stein et al., 2007). Advancements in
amino acid digestibility methods have improved the relationship between amino acid requirements and the estimation of
feedstuffs to meet these requirements; however, amino acid
requirements are a reflection of the effect of genetics,
physiological state and dietary factors. By definition, amino
acid bioavailability insinuates that ingredient characteristics,
genetics, physiological state and dietary factors influencing
protein retention should be considered. Controlling for the
effect of these influences can be accomplished through
experimental design but to fully understand how these factors
might influence the supply and utilization of AA for protein
synthesis appropriate experimental techniques must be
utilized. The non-invasive IAAO technique provides a unique
opportunity to understand metabolically how the supply of
AA for protein synthesis changes with physiological state,
ingredient or genetics.
8. Conclusions
The development of the IAAO method can address many of
the limitation of previous bioavailability studies using a slope
ratio growth assay.
Ileal digestibility will continue to be the most common
measure of choice for the foreseeable future. However, the
minimally invasive IAAO technique becomes the method of
choice when evaluating feedstuffs during sensitive physiological states, when losses during and/or after absorption are
expected, when surgical modification is unethical or impractical and when an integration of factors on amino acid
utilization is desired.
The development of MA using the IAAO technique also has
the potential to ensure that advancements in amino acid
bioavailability will keep pace with improvements in knowledge of amino acid requirement.
Conflict of Interest
We have no conflict of interest.
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