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Deutsches Institut für Ernährungsforschung (DIfE), D-14558 Bergholz-Rehbrücke, Germany and * Rowett Research Institute, Bucksburn, Aberdeen, AB295SB, Scotland, UK
2To whom correspondence should be addressed. E-mail: metges{at}www.dife.de
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-15N] lysine. Rats were killed before and 1, 3, 5 and
7 h after meal ingestion and samples of plasma, stomach wall and
contents, small intestine and liver were collected. 13C and
15N enrichments of free and protein-bound amino acids
in plasma and tissues were analyzed by gas
chromatography-combustion isotope ratio mass spectrometry. Urinary
nitrogen excretion was higher (P < 0.05) and
weight gain lower (P < 0.05) in rats given the AA
diet, indicating a lower whole-body net protein synthesis. Free
13C-leucine from the AA diet appeared in the intestinal
mucosa free pool more rapidly (P < 0.05) than the
CAS-13C-leucine, probably due to the faster transit through
the stomach of the AA group. However, the incorporation of dietary
leucine into plasma and liver proteins was higher in the CAS group
7 h after the meal (P < 0.05), whereas lysine
incorporation into liver protein was higher in the AA group
(P < 0.05). We conclude that whole-body
protein homeostasis is better supported by dietary casein-bound
than crystalline free amino acids, and that protein-bound leucine,
but not lysine, is used more efficiently for liver protein synthesis
than dietary free leucine.
KEY WORDS: • 13C leucine • 15N lysine • splanchnic tissue • stable isotopes • mass spectrometry
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Recent
observations that the molecular form of the ingested nitrogen (free
amino acids, peptides or proteins) and the type of dietary protein
(casein vs. whey) affect amino acid absorption kinetics, the degree of
whole-body amino acid oxidation and utilization for protein
synthesis (2
3
4
5
6)
, suggest that these variables may also
require consideration when assessing protein quality. To date, however,
the influence of these factors has been assessed only by examination of
the metabolic utilization of one amino acid, leucine.
Work conducted by Young and colleagues (7)
at the
Massachusetts Institute of Technology has attempted to better define
the amino acid requirement pattern for humans. In large part, these
dietary studies have involved the use of defined L-amino
acid mixtures and assessment of amino acid balance (requirement) based
on intravenous infusion of selected tracer amino acids. In light of the
fact that the molecular form of the dietary nitrogen may affect the
efficiency of amino acid utilization, it is relevant to reevaluate
these amino acid requirement patterns by assessment of dietary
protein-bound amino acid use. Furthermore, results from human and
pig studies also suggest that there is substantial utilization of
dietary amino acids by the splanchnic tissues during the absorptive
process (8
9
10
11
12)
. In humans, first-pass splanchnic
uptake (gut and liver) of leucine was higher when ingested as
casein-bound leucine compared with free crystalline leucine
(2
, unpublished data). Consequently, the use of
the intravenous tracer balance technique may overestimate amino acid
requirements due to the failure to consider first-pass metabolism
(splanchnic uptake), and the degree to which this occurs may differ
among amino acids and with different forms of protein.
The objectives of this study were as follows: 1) to compare whole-body nitrogen homeostasis and growth efficiency of rats offered diets based on either casein or a free amino acid mixture equivalent to the amino acid pattern of casein; and 2) to compare the fate of dietary leucine and lysine in the splanchnic tissues and in plasma when these amino acids are ingested as casein (intrinsically labeled) or as part of a mixture of crystalline free amino acids (extrinsically labeled), simulating the amino acid pattern of casein. We also examined the appearance of a range of other indispensable and dispensable casein-bound amino acids in splanchnic tissues and in plasma.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The experiment was performed in accordance with the guidelines
of the ethics committee of the Ministry of Agriculture, Nutrition and
Forestry (State Brandenburg, Germany, Permission No. 48–3560-0/3). Two
groups (n = 22/group) of male adult rats (Shoe
Wistar, Tierzucht Schoenwalde, Germany) weighing 
170 g were housed
in individual cages under controlled conditions of light (12-h
light:dark cycle) and temperature (22°C). The rats were adapted for
9 d to two adequate isoenergetic and isonitrogenous diets
(Table 1
) containing either casein
(CAS)3
or a free crystalline L-amino acid mixture simulating the
pattern of casein (AA) as the sole dietary nitrogen sources
(Table 2
). The diets were offered as two meals per day (2 x 8 g dry
mass), and the rats were trained to consume the meal within 30 min. To
prevent spillage, food was moistened (food powder/water, 1:2). Rats
consumed water ad libitum.
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-15N lysine (Tables 1
and 2)
. At
1, 3, 5 and 7 h after the complete uptake of their meals, groups
of 4 or 5 randomly selected rats per dietary treatment were killed by
decapitation after injection of barbiturate (Ketamin/Rompun,
Albrecht/Bayer, Aulendorf/Leverkusen, Germany). Four rats in each diet
group were not fed and were used to provide baseline values for
background 13C and 15N
enrichments. Plasma, stomach and small intestinal contents, gastric
tissue, mucosa of the proximal part of the small intestine and liver
were collected immediately. Additionally, as a proxy for amino acid
oxidation, urinary nitrogen excretion and body weight gain were
measured during the experimental period (d 1–10).
13C-labeled casein was derived from goat’s milk.
A lactating goat received nonprimed, continuous intravenous infusion of
a hydrolysate procured from 7 g of uniformly
13C-labeled algae protein (Celtone-C; Martek
Biosciences, Columbia, MD). The milk was collected by hand in several
batches over 24 h after the start of infusion. Casein was
separated as described previously (13)
and the isolated
casein powder was washed several times with deionized water. The casein
batch with the highest enrichment of leucine and lysine (Table 2)
was
used for the feeding experiment (Table 1)
. In the amino acid mixture
[U-13C6] leucine [99
atom % (AP); Mass Trace, Woburn, MA] and
[-15 N] lysine dihydrochloride (95 AP; VEB
Berlin-Chemie, Berlin, Germany) were mixed with unlabeled leucine
and lysine, respectively (Table 2)
.
Sample preparation and analytical methods
Urinary nitrogen excretion, tissue nitrogen contents and amino
acid concentration.
On d 9, the rats were placed in metabolic cages. Urine was collected
over 24 h into HCl, and urinary nitrogen excretion was calculated
on the basis of a micro-Kjeldahl analysis (14)
.
Nitrogen content of tissue and gastric contents was determined by means
of an elemental analyzer (Vario EL N, Elementar Analysensysteme GmbH,
Hanau, Germany). The proximal and distal part of small intestine (upper
and lower third) were removed and contents and empty weights were
weighed for each rat. Plasma free amino acid concentrations were
analyzed after acid precipitation (deproteinization with
5-sulfosalicylic acid) by ion exchange chromatography. Amino acids were
detected by postcolumn ninhydrin detection (TRIONE ninhydrin reagent,
Pickering Laboratories, Mountain View, CA) using HPLC units (Beckman
Instruments GmbH, Munich, Germany) and a step-change elution method
with increasing temperature, lithium-concentration and pH (several
buffers from Laborservice Onken, Gründau, Germany)
(14)
. For calculation of the amino acid concentration, an
external standard solution was used. Measurement of amino acid
concentration of dietary casein and tissue proteins was performed as
previously described (15)
.
Amino acid isolation and determination of 13C and 15N enrichments. After killing, organ tissues were quickly removed, washed with ice-cold NaCl solution (0.15 mol/L), weighed and frozen immediately in liquid nitrogen. Mucosa samples were scraped from the proximal and distal 15 cm of the small intestine using a plastic spatula. Frozen organ and mucosa tissues were lyophilized, homogenized with mortar and pestle and stored in an exsiccator until analyzed.
Free amino acids were extracted from 20–30 mg lyophilized tissues
after homogenization using 3 x 2 mL of 0.1 mol/L HCl. The acidic
extracts were centrifuged at 800 x g for 10 min
and the amino acids in the supernatant were purified by cation exchange
chromatography (Dowex AG 50W-X8; Na-form; 200 mesh, Fluka Chemie AG,
Steinheim, Germany) (16)
. Plasma free amino acids were
isolated by ion exchange chromatography as described earlier
(17)
. Protein-bound amino acids in tissues and plasma
were isolated from freeze-dried, finely ground tissues (2–5 mg) or
from 100 µL plasma by acid precipitation with 3.5 mL
of 0.6 mol/L trichloroacetic acid. The protein precipitates were washed
with ethanol and ether and underwent a 24-h acid hydrolysis (6 mol/L
HCl, 110°C). The amino acids from hydrolyzed proteins were purified
by cation exchange before derivatization (16)
.
The 13C and 15N enrichments of the crystalline
leucine and lysine to be mixed into the amino acid mixture were
determined separately using an elemental analyzer (EA 1108, Fisons
Instruments, Italy) coupled to an isotope ratio mass spectrometer
(IRMS; Delta C-IRMS, Finnigan MAT, Bremen, Germany). Enrichments in
free and protein-bound amino acids of tissues, plasma and labeled
goat’s casein were determined after derivatization to their
N-pivaloyl-i-propylesters and were
measured by an IRMS (Delta S-IRMS) coupled on-line to a gas
chromatograph (GC; HP 5890, Hewlett-Packard, Waldbronn, Germany)
via a combustion interface (18
,19)
. Briefly, for
derivatization the amino acid isolates were dried and dissolved in
thionylchloride/i-propanol solution and heated at
100°C. The propylated product was dried and dissolved in pyridine.
After addition of pivaloylchloride, the amino acids were acylated and
methylene chloride was added after cooling. The mixture was then passed
over a silica gel column and the filtrate was dried in a gentle
nitrogen stream and redissolved in ethylacetate for injection. The
chemicals used were all of analytical grade and purchased from several
suppliers (Fluka Chemie, AG, Buchs, Switzerland; Sigma-Aldrich
Chemie GmbH, Steinheim, Germany).
The 15N and 13C abundances, determined by
EA-IRMS or gas chromatography-combustion (GC-C)-IRMS, were
measured against a laboratory standard, which had been calibrated
against international standards (air and PDB Belemnite carbonate,
respectively). 15N or 13C enrichments were
expressed as AP and atom % excess (APE) above baseline as described
recently (19)
. 13C enrichments measured by
GC-C-IRMS were corrected for the contribution of extra carbon
introduced during the derivatization process. On the basis of
stoichiometric mass balance and isotopic fractionation, an empirical
13C correction equation was derived for individual amino
acids (19)
.
Calculations and statistical analysis.
To compare the incorporation of dietary amino acids from either casein
or the amino acid mixture, data were normalized for differences in the
amino acid enrichment in the diet. This was accomplished by calculating
the fractional appearance (FA) from enrichments of lysine and leucine
in tissues (stomach, intestinal mucosa, liver) or plasma, and diet as
follows:
 |
where E is the enrichment of individual amino acids in tissue or plasma and the diet in APE.
For calculation of FA, we assumed that the amino acid enrichment in
meals represented the enrichment at the site of absorption. A direct
comparison of FA from 15N and
13C lysine was possible because rates of carbon
and nitrogen losses during lysine degradation are closely related
(20)
.
Sequestration (S) of dietary leucine and lysine into liver
protein 7 h after meal ingestion was derived from the following
equation:
 |
All results are expressed as means ± (SD). A
nonparametric Mann-Whitney test (Wilcoxon test) was employed to
evaluate the differences between diet groups (21)
. A value
of P < 0.05 was considered to be significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Body weight gains of rats fed the CAS diet were higher than for rats fed the AA diet (20.6 ± 20.0 vs. 12.7 ± 7.4 g/9 d; P < 0.05). Final body weights on d 10 did not differ between the CAS group (191.2 ± 19.8 g) and the AA group (187.0 ± 26.4 g) due to slight differences in initial body weights. Total organ weights (AA vs. CAS; stomach, 1.41 ± 0.15 vs. 1.34 ± 0.27; liver, 7.17 ± 1.10 vs. 7.53 ± 1.08; small intestine, 4.15 ± 0.39 vs. 3.93 + 0.34 g) and total nitrogen content of empty stomach, liver and small intestine were not different between groups and did not change during the 7 h of observation (data not shown). The 24-h urinary nitrogen excretion on d 9 was higher for rats fed the AA diet compared with those fed CAS (204.5 ± 49.5 vs. 154.1 ± 55.0 mg N; P < 0.05). Because dietary nitrogen intake was identical, this indicates higher nitrogen retention in rats fed the diet containing the intact protein.
Amino acid concentrations in plasma and tissue proteins and gastric nitrogen content.
Concentrations of plasma isoleucine, tyrosine, valine, alanine and
glutamine measured after overnight food deprivation were higher in the
CAS group, but threonine and serine were lower compared with the AA
diet group (Table 3
). At 1 and 3 h after consumption of the CAS and AA meals, plasma
concentrations of almost all indispensable amino acids increased
relative to the baseline values, with the notable exceptions of
phenylalanine, threonine and tyrosine, which did not change after CAS
consumption. For indispensable amino acids, no differences were seen
between diet groups at 1 and 3 h after the meal, although
concentrations tended to be higher after AA consumption (isoleucine,
threonine, tyrosine, valine: 0.05 < P < 0.2).
Variations in plasma leucine and lysine concentrations during the
7 h after meal consumption were of similar magnitude with both
diets (ranges AA vs. CAS; leucine, 92–182 vs. 125–180
µmol/L; lysine, 547–723 vs. 574–725
µmol/L). Most notable among the dispensable amino acids
was the significantly greater increase from baseline for alanine,
aspartic acid, glutamine and glycine at 1 h after consumption of
the AA diet compared with the CAS diet. By contrast, the increase from
baseline for asparagine was greater in rats fed CAS than in those fed
the AA diet. Plasma and tissue protein concentrations of leucine and
lysine were not affected by diet (data not shown). At 3 and 5 h
after meal consumption, rats fed the CAS diet had more nitrogen in
their stomach contents (CAS vs. AA; 3 h, 233 ± 111 vs. 71
± 38 mg N; 5 h, 44 ± 25 vs. 19 ± 6 mg N;
P < 0.05). However, there were no differences in the
dry matter weight and nitrogen level in the intestinal contents between
rats fed the two diets at 7 h postmeal (data not shown).
|
Labeled lysine and leucine from both diets were already incorporated
into stomach tissue by 1 h after meal ingestion, with
incorporation continuing to increase in a curvilinear manner up to
7 h postmeal (Fig. 1A
,
B
). The fractional appearance of dietary leucine into stomach proteins
tended to be higher (P = 0.1) than for lysine,
independent of diet, presumably reflecting differences in leucine and
lysine contents of the diet (Table 2)
and of the stomach proteins
(ranges; leucine, 66–72 vs. lysine, 50–57; mg/g). The appearance of
leucine and lysine from the AA diet into the free pool of the proximal
small intestinal mucosa was more rapid (peaking at 1 h) than for
the CAS diet, whereas appearance of these amino acids from the CAS diet
into the mucosa free pool was much slower (peaking at 3 h)
(Figs. 2A
and 3A
). By 7 h after consumption of the diets, the
differences in free pools of proximal intestinal mucosa between diet
groups had disappeared. The incorporation of dietary leucine and lysine
into protein of proximal mucosa was higher 3 h after AA ingestion
(P < 0.05) (Figs. 2A
and 3A
). In
contrast, by 7 h after meal consumption, 8–10% of the leucine
label, and 4–8% of the lysine label from the CAS diet were recovered
in the distal small intestinal mucosa as free and protein-bound
amino acids, which was significantly higher compared with the AA diet
(data not shown).
|
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). The appearance of lysine in the plasma
free pool reached a quasi plateau between 1 and 5 h after
consumption of both meals, and then decreased at 7 h (Fig. 3B
). In contrast to the mucosa free pool kinetics, the FA of
dietary lysine and leucine in the liver and plasma free pools did not
differ between the diet groups, although both amino acids reached a
peak at 1 h postmeal (Figs. 2
, 3)
. A significantly higher
incorporation of leucine in plasma and liver protein was observed
7 h after CAS ingestion (Fig. B2
and 2C
),
whereas dietary lysine incorporation into liver protein was
significantly higher 3 and 7 h after AA consumption (Fig. 3C
). Consequently, total sequestration of leucine into liver
proteins at 7 h after consumption of the CAS diet was greater than
in rats fed the AA diet (P < 0.05), whereas
sequestration of lysine into liver proteins did not differ between
groups (AA vs. CAS; leucine, 2.66 ± 0.22 vs. 3.83 ± 0.46 mg
(P < 0.01); lysine, 1.62 ± 0.11 vs. 1.31 ± 0.61 mg). Appearance of other dietary indispensable amino acids into small intestinal mucosa and plasma free pools after the casein (CAS) meal.
Except for leucine and lysine, the FA of individual amino acids into
the mucosa and plasma free pools did not reflect the compositional
pattern of these amino acids in the CAS diet (compare Table 2
and
Fig. 4
). Thus, valine, which is similar in concentration to lysine in casein,
appeared in relatively higher amounts in the small intestinal mucosa
and plasma free pools compared with lysine. Although methionine and
alanine have similar concentrations in casein, the FA of alanine was
much lower (Fig. 4)
. The glycine content of casein is lower than that
of alanine, but the FA of glycine in the intestinal mucosa free pool
was higher than that of alanine (Fig. 4)
. An exception to these kinetic
patterns is proline, which is the second highest in concentration in
casein and also had the highest FA in small intestinal mucosa and
plasma free pools (Fig. 4)
. Notably, the FA of phenylalanine, glycine,
aspartic acid, and serine in the mucosa free pool were significantly
greater compared with their FA in the plasma free pool (P
< 0.05).
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|
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Urinary nitrogen excretion, a proxy for whole-body amino acid
oxidation, was higher in rats fed the AA diet. Lower growth rates and
decreased nitrogen retention have been also observed when pigs and rats
are fed diets containing free amino acids compared with those fed whole
proteins (22
23
24
25
26)
. Why free amino acid–based diets are
used less efficiently than diets containing intact proteins is unclear,
but evidence to date suggests that differences in the kinetics of amino
acid absorption and consequently postabsorptive utilization may be
important. The amount of nitrogen in the stomach contents of rats fed
the CAS diet was higher at 3 and 5 h postmeal, suggesting a slower
gastric emptying time after ingestion of the casein meal. This may be
due to the clotting of casein in the stomach and possibly the release
of opioid peptides, leading to a slower gastric emptying
(4
,5
,27)
.
Plasma amino acid concentrations reflect the net difference between the
release of amino acids by the splanchnic bed and their uptake (and
release) by the peripheral tissues. The generally smaller increase of
plasma amino acids after the CAS meal may be indicative of a higher
first-pass splanchnic uptake of casein-derived amino acids
compared with the free amino acid mixture. This interpretation would be
consistent with the finding that splanchnic uptake of
casein-derived leucine was higher than that of crystalline leucine
in humans (2
, unpublished data), resulting in lower plasma
concentration of leucine, as well as isoleucine and valine
(2)
. Similar differences in the kinetics of plasma
concentrations of isoleucine, leucine and lysine were observed when
subjects consumed a meal of cottage cheese compared with one containing
an L-amino acid mixture simulating cottage cheese
(28)
.
Another important aspect in interpreting plasma free amino acid levels
after a meal is the meal composition. Large differences in plasma amino
acid concentrations among dietary proteins were found when only pure
(protein) nitrogen sources were ingested (6
,3
,29)
. Our
data do not show a largely different variation of plasma leucine or
lysine concentrations, respectively, between diets. This is most likely
due to the fact that in this study, as well as in other studies
(2
, unpublished data), mixed meals that also contained
carbohydrates and fat were given. It was shown earlier that the degree
of plasma amino acid concentration enhancement depends on the
composition of the meal (29
,30)
.
Because almost all of the enterally delivered glutamic acid and most of
the glutamine is taken up during the first pass by gut and liver
(11)
, the difference seen in postmeal plasma glutamine
concentration (Table 3)
points to either a decreased splanchnic uptake
or an increased endogenous glutamine production when glutamine is
ingested as a free amino acid. However, it cannot be excluded that the
high plasma glutamine concentration was due to the fact that no
glutamic acid was included in the L-amino acid mixture
(Table 2)
, which may affect splanchnic amino acid transporter
activities and the glutamate-glutamine metabolic pathway
(11)
.
There were no differences in organ weights and nitrogen (protein)
content, nor were leucine and lysine concentrations in tissue proteins
different between rats fed the AA or CAS meals. Thus, FA could be
compared between rats fed the two diets without the confounding effect
of changes in amino acid pool size. The appearance of
dietary-derived labeled lysine and leucine in stomach protein
within 1 h after meal consumption suggests that these dietary
amino acids are rapidly taken up by the stomach mucosa and either
incorporated directly into gastric proteins (first-pass use) or
incorporated into gastric proteins (second-pass use) subsequent to
intestinal absorption into the blood (31
32
33)
. However, it
cannot be excluded that in spite of careful rinsing of the splanchnic
organs, some food particles continued to adhere to the mucosa, carrying
with them labeled amino acids. The lower incorporation of dietary
lysine into stomach tissue protein in comparison to leucine,
independent of the nitrogen source, is likely to be due to a lower
lysine content in stomach tissue protein.
Comparison of the FA of amino acids in free and protein-bound pools
allowed an assessment of the efficiency of transfer and use of amino
acids derived from the two diets. During the early postprandial period
(0–1 h) dietary labeled amino acids appeared more rapidly and to a
greater extent in plasma and tissue free pools (15 times higher than in
protein-bound pools), whereas in the later postprandial period (7
h), these dietary amino acids had been incorporated mainly into
proteins. Compared with rats fed the AA diet, the FA of leucine derived
from CAS were higher in plasma and liver proteins. Total sequestration
of leucine into liver constitutive proteins was also higher in rats fed
the CAS meal. Stoll et al. (8)
reported that in contrast
to the small intestinal mucosa, in which the majority of the
metabolized amino acids were catabolized (i.e., oxidized), the liver
utilizes a nutritionally important amount of dietary amino acids for
protein synthesis. Our results indicate that the availability of
dietary leucine and lysine for protein synthesis in the liver
(constitutive and plasma export proteins), but not in the small
intestinal mucosa, depends on the molecular form of the ingested
nitrogen source. Presumably, the slower gastric emptying after
consumption of the CAS diet, and thus a slower rate of amino acid
absorption, prevents the rapid increase in intracellular free leucine
concentration in splanchnic tissues, resulting in less induction of
catabolism. Consistent with this view are the observations that leucine
oxidation is lower, splanchnic (gut plus liver) uptake of dietary
leucine is higher and nonoxidative leucine disposal (protein synthesis)
is greater in humans consuming a diet containing casein vs. one
containing crystalline amino acids (2
, unpublished data).
The differences in absorption rate of amino acids derived from dietary
protein-bound vs. free amino acids is analogous to the "slow"
and "fast" protein concept of Boirie et al. (6)
. These
authors observed that leucine derived from dietary casein was used for
tissue protein synthesis and supported higher postprandial protein gain
in humans than leucine derived from whey proteins. The authors
attributed these results to the slower rate of digestion and absorption
of the casein than the whey proteins. Our study offers the additional
opportunity to examine the metabolic fate of two amino acids, lysine
and leucine, at the same time. Dietary lysine and leucine displayed
differential incorporation kinetics into liver and plasma proteins, and
furthermore, these differences were dependent upon whether the CAS or
AA diet was fed (Figs. 2
, 3)
. These results not only suggest
differences in the sites of catabolism of these amino acids but also
that the form of dietary nitrogen influences the efficiency of use of
these amino acids. This result is not surprising given the known
differences in the major sites of catabolism of leucine (muscle) and
lysine (liver) in the body. Our results also support the long-held
view that it is not possible, and certainly misleading, to extrapolate
metabolic results from one amino acid to another. Future studies will
be necessary to examine a larger range of amino acids and whether the
form and composition of protein-amino acids in the diet have
similar or different effects on general amino acid utilization. Such
results have important implications for the establishment of
nutritional requirements for amino acids and the system by which
protein sources are scored for quality.
This study also provided the opportunity to trace the metabolic fates
of other dietary protein (casein)-bound indispensable amino acids in
plasma and splanchnic tissues. Differences in leucine and lysine
concentrations in dietary casein were largely reflected in their FA in
the intestinal mucosa, possibly indicating similar pool sizes and/or
similar turnover rates of these amino acids in the intestinal mucosa.
By contrast, although valine has a concentration similar to that of
lysine in casein, the transfer of dietary valine into the small
intestinal mucosa occurred at a higher rate than for lysine. Again,
these differences may reflect a larger free lysine pool and/or a higher
turnover rate for lysine than for valine in the small intestine.
Interestingly, almost 50% of the proline enrichment in the dietary
casein was recovered in the mucosa, which probably indicates that de
novo synthesis from glutamate and proline oxidative breakdown during h
1 after meal ingestion were very low. The rather low FA for alanine,
relative to its composition in casein, may reflect a substantial net
production of alanine by intestinal tissue (9)
. With the
exceptions of alanine and proline, however, the FA of other dispensable
amino acids monitored was lower into plasma compared with the mucosa
(Fig. 4)
. One explanation for this may be a greater splanchnic uptake
of glycine, aspartic and glutamic acids, and serine in comparison to
alanine, proline and the other indispensable amino acids. Consequently,
a greater splanchnic removal of these amino acids is compatible with
the unchanged or lower plasma concentrations of glycine, aspartic and
glutamic acids, and serine after the CAS meal. Furthermore,
phenylalanine showed a 100% greater FA into the mucosa than into
plasma, which is supported by observations of Stoll et al.
(34
,35)
in which 76% of enteral phenylalanine in pigs was
extracted by splanchnic tissues and 59% of dietary phenylalanine was
used for mucosal protein synthesis.
In summary, we have shown that dietary protein-bound amino acids (casein), as opposed to crystalline amino acids, support higher rates of weight gain and nitrogen retention in rats. In part, this appears to be related to a slower gastric emptying rate and slower rates of appearance of free amino acids in the blood circulation when rats are fed casein, all of which result in a greater efficiency of transfer of amino acids into tissue and plasma proteins. Further, differences were noted in the FA of amino acids derived from casein into tissues, with leucine transfers into liver and plasma proteins occurring at a higher efficiency than for lysine. We conclude that the molecular form in which dietary amino acids are consumed influences their oxidation as well as their peripheral and splanchnic availability for protein synthesis, and that differences in the metabolic utilization of dietary derived amino acids makes it unjustifiable to extrapolate results from one amino acid to another in assessing overall protein efficiency and quality.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
3 Abbreviations used: AA, L-amino acid mixture; AP, atom %; APE atom % excess; CAS, casein; EA-IRMS,
elemental analyzer isotope ratio mass spectrometry; FA, fractional appearance; GC-C-IRMS, gas chromatography-combustion isotope ratio mass spectrometry.
Manuscript received December 1, 2000. Initial review completed January 20, 3001. Revision accepted March 30, 2001.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
1. FAO/WHO Protein Quality Evaluation. Report of joint FAO/WHO expert consultation. FAO Food and Nutrition Paper 51 1992:1-66 Rome, Italy.
2.
Metges C. C., El-Khoury A. E., Selvaraj A. B., Tsay R. H., Atkinson A., Regan M. M., Bequette B. J., Young V. R. Kinetics of L-[1-13C] leucine when ingested with free amino acids, unlabeled or intrinsically labeled casein. Am. J. Physiol. 2000;278:E1000-E1009
3.
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