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1 Clinical Research Center and Laboratory of Human Nutrition, School of Science, Massachusetts Institute of Technology, Cambridge, Massachusetts 02142; and 2 Rowett Research Institute, Bucksburn, Aberdeen AB21 9SB, Scotland
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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In two groups of five adults, each adapted to two
different dietary regimens for 6 days, the metabolic fate of dietary
[1-13C]leucine was examined when ingested
either together with a mixture of free amino acids simulating casein
(extrinsically labeled; condition A), along with the intact
casein (extrinsically labeled; condition B), or bound to casein
(intrinsically labeled; condition C). Fed state leucine
oxidation (Ox), nonoxidative leucine disposal (NOLD), protein
breakdown, and splanchnic uptake have been compared using an 8-h oral
[1-13C]leucine and intravenous
[2H3]leucine tracer protocol while
giving eight equal hourly mixed meals. Lower leucine Ox, increased
NOLD, and net protein synthesis were found with condition C
compared with condition A (19.3 vs. 24.9; 77 vs. 55.8; 18.9 vs.
12.3 µmol · kg
1 · 30 min1; P < 0.05). Ox and NOLD did not
differ between conditions B and C. Splanchnic leucine
uptake calculated from [1-13C]- and
[2H3]leucine plasma enrichments was
between 24 and 35%. These findings indicate that the form in which
leucine is consumed affects its immediate metabolic fate and retention
by the body; the implications of these findings for the tracer balance
technique and estimation of amino acid requirements are discussed.
leucine oxidation; leucine flux; amino acid mixture; intrinsic label
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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PREVIOUS STUDIES CONDUCTED in our laboratory on the relationship between amino acid kinetics and amino acid intake have largely involved use of a diet providing nitrogen in the form of a crystalline L-amino acid mixture (e.g., Refs. 1, 10, 11, 24, 29, 32, 44). This gave us the opportunity to vary precisely the dietary intake of each indispensable amino acid separately, while maintaining total nitrogen intake constant, which is a prerequisite for estimating the amino acid requirement using the tracer-balance technique (48).
However, differences in the time course of absorption have been reported when amino acids are ingested either as free amino acids or small peptides or bound to proteins. Peptide amino acids are known to be absorbed more rapidly than free amino acid mixtures (31, 41, 42), with free amino acids appearing in the peripheral plasma more quickly than amino acids arising from intact proteins (22, 23).
Results on growth and nitrogen utilization from studies comparing whole proteins, peptides, and corresponding amino acid mixtures are, however, conflicting. Nitrogen balance in the rat (19) and in human subjects (35) did not differ between whole protein, hydrolyzed protein, and free amino acid mixtures of identical pattern. Also, no difference in feed efficiency was seen when comparing free amino acids and the equivalent peptide mixture of casein and egg white proteins (8). On the other hand, net protein utilization was observed to be greater with small peptides from milk protein than with an equivalent amino acid mixture in normal rats and animals with a resection of the biliopancreatic duct (34).
Studies by Batterham and Bayley (2) in the pig indicated that oxidation of free phenylalanine, as an index of amino acid utilization, is greater when the diet contains free lysine compared with protein-bound lysine.
The possible effects of the rate and pattern of absorption on the fate of dietary amino acids emerging from different molecular forms of nitrogen intake are important to investigate because 1) our tentative Massachusetts Institute of Technology (MIT) amino acid requirement pattern (48) has been derived, in part, from dietary studies using L-amino acid mixtures, and 2) under circumstances where a net protein catabolism occurs, such as protein-energy malnutrition, renal disease, sepsis and traumatic injury (30), and sarcopenia of the elderly (36), the qualitative nature of the dietary amino acid supply might affect amino acid losses and retention.
Therefore, we have conducted a study to explore the prandial metabolic fate of 13C-labeled dietary leucine when it is ingested as a component of mixed meals either bound to a protein (casein; intrinsically labeled) or together with a mixture of crystalline free amino acids (extrinsically labeled) simulating the casein amino acid pattern. As a control, leucine kinetics were measured also when free labeled leucine was given together with the intact protein (casein). In contrast to many published studies (5-7, 9, 21, 45, 46) concerned with prandial and postprandial aspects of amino acid metabolism and the molecular form of the ingested tracer amino acid that did not involve dietary adjustment or adaptation periods to the experimental diets, the present study included a 6-day adjustment period on each experimental diet before the tracer study. Amino acid absorption appears to be readily adaptable to the prevailing protein/amino acid uptake (28), and so it was important to standardize the dietary background of the study population. Additionally, our investigation included an assessment of the splanchnic first-pass uptake of leucine under all three tracer conditions, using a simultaneous intravenous/oral tracer infusion paradigm.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Subjects. Fourteen young adult volunteers, recruited from the student population of MIT and from the community of the Boston-Cambridge area, were randomly assigned to three study groups and were studied as outpatients at the MIT Clinical Research Center (MIT-CRC). They were healthy according to medical history, physical examination, analysis of blood cell count, routine blood biochemical profile, and urinalysis. Subjects who smoked, consumed five or more alcoholic beverages per week, or five cups of caffeinated beverages per day were excluded from participation. Women were studied during the 5- to 10-day period after onset of menstrual bleeding. A negative pregnancy test (on the basis of plasma human choriogonadotropin concentrations) 2-3 days before starting the dietary periods was required from each female subject.
One woman and four men were included in each of groups 1 and 2, whereas two men and two women were studied in group 3. Mean age, weight, and height in groups 1, 2, and 3, respectively, were as follows: age (yr) 20.6 ± 1.7, 21.6 ± 1.1, 26.8 ± 0.5; weight (kg) 69.2 ± 8.2, 74.6 ± 8.3, 71.8 ± 13.9; height (cm) 173.2 ± 11.4, 175 ± 5.4, 176.5 ± 6.4. Written consent was obtained from each subject after explanation of the risks involved and the purpose of the study, which had been approved by the MIT Committee on the Use of Humans as Experimental Subjects and the MIT-CRC Advisory Committee. The subjects, who were paid for their participation in the studies, were instructed to maintain their usual level of activity and were asked to refrain from excessive or competitive exercise.Experimental design and diets. The dietary tracer protocol for groups 1 and 2 consisted of two separate dietary periods of 6 days (adaptation period) with an 8-h tracer ingestion/infusion protocol being conducted on day 7 during the fed state; group 3 was studied on day 7 with tracer-free diets only. The order of the dietary periods was randomized for each subject. Between the two dietary periods, a break of 3 wk occurred when subjects consumed their usual free-choice diets. In group 1, [1-13C]leucine was ingested on one occasion as protein bound to casein (intrinsically labeled; [13C]Leu-casein), and in the second study the free [1-13C]leucine tracer was added to unlabeled casein (extrinsically labeled; casein + [13C]Leu) just before meal feeding. Group 2 subjects were studied after completion of the study in group 1. In group 2, [1-13C]leucine was given orally as either protein bound to casein ([13C]Leu-casein) or with an L-amino acid mixture patterned as in casein (AA + [13C]Leu). Subjects in group 3 participated in tracer-free studies only and received on one occasion either an unlabeled crystalline L-amino acid mixture simulating the casein pattern or unlabeled goat's casein. On the second occasion, they received either an L-amino acid mixture or unlabeled cow's casein. One subject was studied with all three diets.
The diets were isocaloric and isonitrogenous (161 mg N · kg1 · day1)
and contained the same generous amounts of leucine (115 mg
leucine · kg1 · day1;
inclusive of leucine tracers). To keep leucine intake equal on all
days, small amounts of crystalline leucine were given during the first
6 days to adjust for the leucine tracer intake on day 7. Energy
intake was close to 45 kcal · kg1 · day1
(188 kJ · kg1 · day1)
to maintain body weight and consisted of protein-free wheat starch
cookies and a flavored protein-free formula in which the amino acid
mixture or the casein was blended, respectively (Table 1). About 40% of nonprotein energy was
from fat (safflower oil, butter), and 60% was from carbohydrate (beet
sugar, starch). Vitamins and minerals were given to meet or exceed the
recommended allowances or safe and adequate intakes (Table 1 and Ref.
37). No other foods or beverages were allowed, except tap water,
decaffeinated tea or coffee with or without artificial sweetener, and
bouillon. Diets were consumed as three daily equal meals (at 0800, 1200, and 1800) in the dining room of the MIT-CRC. Two out of three meals were consumed under supervision of the CRC dietary staff. Every
morning, the subject's body weight and vital signs were recorded. On
the day of the tracer protocol (day 7), oral tracers were given
with the eight small hourly meals, the composition of which was the
same as the 6 days before the tracer protocol (Table 1). Leucine and
energy intake during these 8 h corresponded to two-thirds of the total
daily intake.
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1 · h1.
To achieve this, the labeled goat's casein was diluted with unlabeled
casein. Because unlabeled goat's casein was not available in
sufficient amounts, we used unlabeled cow's casein (New Zealand Milk
Products, Santa Rosa, CA) during diet days 1-6 instead of unlabeled goat's casein. To make sure that the use of unlabeled cow's
casein did not result in a different metabolic response, we compared
the amino acid pattern (Table 2) and
checked the background 13CO2 breath enrichment
(Fig. 1), which were both similar.
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Tracer protocol and sample collection.
On the morning of day 7, the subjects reported to the
outpatient unit of the MIT-CRC at 0630 after an overnight fast. After recording body weight and vital signs, a 20-gauge 5-cm catheter was
placed, under sterile conditions, into an antecubital vein of the
nondominant arm for infusion. A second 20-gauge 3.2-cm catheter was
inserted in a dorsal hand vein for blood sampling. Between samplings of
blood, the intravenous lines were kept open with a slow drip of sterile
physiological saline. Throughout the 8-h tracer protocol, the subjects
remained in bed in a reclined position. The tracer protocol began at
about 0815 with oral prime doses of [13C]sodium
bicarbonate (0.6 µmol/kg),
L-[1-13C]leucine (3.75 µmol/kg),
and
L-[2H2]- phenylalanine
(2.5 µmol/kg) mixed into 24 ml distilled water. Simultaneously, prime
doses of L-[2H3]leucine
(3.75 µmol/kg) and
L-[ring-2H5]phenylalanine
(2.5 µmol/kg) were administered by vein in 10 ml sterile
physiological NaCl solution over a 3-min period. Immediately after the
priming doses were given, the first small hourly meal was ingested
(time 0); the meal consisted of a small portion of cookies and
either the intrinsically [1-13C]leucine-labeled
casein (corresponding to 2.5 µmol · kg1 · h1
[1-13C]leucine), the unlabeled amino acid
mixture plus free [1-13C]leucine (2.5 µmol · kg1 · h1),
or unlabeled casein plus free [1-13C]leucine
(2.5 µmol · kg1 · h1).
These tracers were mixed into the protein-free formula (~150 ml/meal). In addition,
[2H2]phenylalanine (2.5 µmol · kg1 · h1)
was given as oral tracer in distilled water (8 ml/h). Simultaneously, an intravenous continuous infusion of
L-[2H3]leucine (2.5 µmol · kg1 · h1)
and
L-[ring-2H5]phenylalanine
(2.5 µmol · kg1 · h1)
was started. These tracers were continuously infused for 8 h by means
of a screw-driven syringe pump (Harvard Apparatus, Millis, MA). When
[1-13C]leucine bound to casein was given, a
small amount of unlabeled leucine was added to keep leucine intake
equal with all treatments. Leucine intake with each meal was 9.9 mg · kg1 · h1.
Meals were isoenergetic (15.6 kJ · kg1 · h1)
and isonitrogenous (13.3 mg
N · kg1 · h1).
Each meal contained ~11 g of fat, 40 g of carbohydrates, and 6 g of
casein or amino acid mixture. Drinking water was allowed ad libitum. In
the tracer-free studies, the same protocol was performed, but the
leucine and phenylalanine tracers were replaced by the corresponding
unlabeled amino acid, and the amounts of amino acid tracers usually
given by intravenous infusion were given orally. The results of the
labeled phenylalanine studies will be published separately.
20°C until used for analysis. In group 2,
at each time point, an additional 1-ml blood sample was drawn into
prechilled EDTA tubes containing Trasylol (100 µl/ml blood; Bayer,
Kankakee, IL) for insulin and glucagon determination. These plasma
samples were stored at 80°C. In group 3 (tracer-free
studies) no blood was taken. Breath samples were collected into rubber
bags every 30 min, immediately transferred to 15-ml evacuated glass
tubes (Monoject; Cardinal Health), and stored at room temperature until
analyzed. Total carbon dioxide production and oxygen consumption rates
were determined with the aid of the indirect calorimeter (Deltatrac or
Vmax; Sensormedics, Anaheim, CA) by using a ventilated hood system.
Measurements were performed for 20 min during each hour.
Sample analysis. Carbon dioxide from breath was cryogenically trapped, and 13C enrichment was analyzed by isotope ratio-mass spectrometry (Delta E; Finnigan MAT, Bremen, Germany). Plasma concentrations of leucine, isoleucine, valine, phenylalanine, and tyrosine were measured by ion exchange chromatography, postcolumn ninhydrin reaction, with detection at 570 nm for primary amino acids using an HPLC system (Beckman System Gold; Beckman Instruments, San Ramon, CA). Aliquots of plasma samples were deproteinized with 5% sulfosalicylic acid containing a known concentration of norleucine as internal standard. Supernatant (50 µl) was injected on a lithium spherogel column with a buffer flow rate of 0.8 ml/min at a temperature of 40°C. The amino acid pattern of goat's and cow's casein was determined after hydrolysis by a method that we have described recently (40).
The enrichments of plasma [1-13C]- and [2H3]leucine, as well as the corresponding enrichments of the transamination product
-ketoisocaproic acid (-[1-13C]KIC and
-[2H3]KIC), were measured by
electron-impact gas chromatography-mass spectrometry (70 eV) using an
HP 5890 gas chromatograph coupled to an HP 5988 quadrupole mass
spectrometer (Hewlett Packard, Palo Alto, CA). Plasma amino
acids were isolated by cation exchange resin (Bio-Rad AG 50W-X8,
100-200 mesh, H+ form; Bio-Rad, Melville, NY) with 1 ml of 3 M
NH4OH followed by 1 ml double deionized H2O. After the sample was dried under a stream of N2, 50 µl
acetonitrile and 50 µl
N-methyl-N-tert-butydimethylsilyl trifluoroacetamide (Pierce, Rockford, IL) were added, and the tightly capped vial was
heated for 1 h at 60°C to form tert-butyldimethylsilyl
(t-BDMS) derivatives. One microliter was injected in splitless
mode (purge on time 0.5 min) on a DB-1301 fused silica column (30 m × 0.25 mm ID; 0.25 µm; J and W Scientific, Folsom, CA). The
temperature was programmed from 160 to 280°C at 15°C/min and
then from 280 to 300°C at 20°C/min. Leucine eluted at ~5 min
and was monitored for mass-to-charge ratio (m/z)
302-305. Unlabeled leucine was measured at its base peak
m/z 302 [M-57]+,
[1-13C]leucine at m/z 303, and
[2H3]leucine at m/z 305.
-KIC enrichments were measured in quinoxalinol-t-BDMS
derivatives, which were prepared as described previously (12), except that acetonitrile was used instead of pyridine. Separation of -KIC
derivatives was performed on a temperature-programmed DB 1301 column
(30 m × 0.25 mm ID; 0.25 µm; J and W Scientific;
100-280°C at a rate of 30°C/min). -KIC eluted at ~6.9
min, and selected ion monitoring was carried out for
m/z 259 [M-57]+,
m/z 260, and m/z 262 for natural,
-[1-13C]KIC, and
-[2H3]KIC, respectively.
For purposes of calibration, a training data set was created by
compiling the mass spectral response of graded mixtures of [1-13C]- and
[2H3]leucine and
-[1-13C]- and
-[2H3]KIC together with
unlabeled leucine and unlabeled -KIC, respectively, over a 0-10
mole fractional range for each tracer. Multilinear regression was then
used to generate a prediction equation correlating the ion pair area
ratios (m + 1/m + 0, m + 3/m + 0) obtained spectrometrically on
standards (100% tracee and tracers) as the explanatory variable for
tracer-to-tracee mole ratios. To account for a potential
cross-contribution of one tracer into the target ion of the other
tracer (e.g., [1-13C]leucine contribution to
m + 3), ion pair area ratios
m + 1/m + 0 and
m + 3/m + 0 for both tracers were
considered in the prediction equation. The equation was then applied in
determining ion pair mole ratios from the corresponding spectral
information obtained with the plasma samples, and the resulting mole
ratios were converted algebraically into tracer mole percent excess
(MPE) for each tracer, after subtraction of the corresponding baseline
plasma values for each set of samples. Analysis of replicate standards
(n = 5) under these conditions showed a coefficient of
variation ranging from 2 to 7%, the latter values being those for the
detection of 0.5 MPE of either leucine or -KIC isotopolog in the
presence of a 5 MPE of the other. Also, by way of validation, the
accuracy of estimates for this methodological approach was found to
fall within 5% of expected values on average, based on analysis of sham mixtures prepared gravimetrically and with known
tracer(s)-to-tracee composition (2 MPE).
Hormone concentrations were measured for group 2 only
(AA + [13C]Leu vs.
[13C]Leu-casein). Plasma insulin concentrations
were determined by an enzyme immunoassay (Mercodia Insulin ELISA; ALPCO
American Laboratory Products, Windham, NH). An RIA was used to measure plasma glucagon concentrations (Euro-Diagnostica, Malmö, Sweden).
Background breath 13CO2 enrichments. Under conditions identical to those followed in the main experiment (day 7; 8 small hourly meals), four additional subjects (group 3) were studied without administration of any tracer. Breath samples were taken at 30-min intervals. The aim was to determine the breath 13CO2 background enrichment throughout the 8-h study and to use the values to correct for the 13C due to diet alone (Fig. 1).
Data evaluation.
Leucine oxidation (Leu Ox) was computed for each half hourly interval
during the 8-h tracer protocol. Leucine oxidation during the first hour
of the study was taken to be equal to that measured at the end of the
2-h period to avoid any possible effect of the bicarbonate prime
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1 · 30 min1), Ei is the enrichment in APE of
the administered isotope ([1-13C]Leu,
[2H3]Leu), and Ep is
the enrichment of the respective leucine isotope or of its
transamination product -KIC in plasma at each isotopic steady state.
Leucine splanchnic uptake (Leu Spl Upt) was computed from plasma
enrichments (E) of orally
(E[1-13C]Leu) and intravenously administered leucine
(E[2H3]Leu), at isotopic steady state, normalized for infusion or administration rate (ir) of tracers, as follows
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![= {1 − [(E<SUB>[1−<SUP>13</SUP>C]Leu</SUB>/ir<SUB>[1−<SUP>13</SUP>C]Leu</SUB>)/(E<SUB>[<SUP>2</SUP>H<SUB>3</SUB>]Leu</SUB>/ir<SUB>[<SUP>2</SUP>H<SUB>3</SUB>]Leu</SUB>)]} × 100](/content/vol278/issue6/fulltext/E1000/img008.gif)
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Statistical summary. Values are expressed as means ± SD. All analyses were conducted separately for the two groups of subjects. For outcomes that were measured with two leucine tracers (e.g., rate of appearance: ig [1-13C]Leu or iv [2H3]Leu) the effect of the form of the leucine tracer was determined using a paired t-test. For all outcomes, paired t-tests were used to compare tracer forms within each group. Means were considered to be significantly different at P < 0.05.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Plasma -KIC and leucine enrichments.
Plasma -[2H3]KIC enrichments
were significantly higher than -[1-13C]KIC
enrichments with the exception of
AA + [13C]Leu. Plasma
[13C]leucine and
[2H3]leucine enrichments were
significantly different with all tracer forms (Table
3).
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Leucine oxidation.
The course of total CO2 production did not differ between
the diets in both groups. The pattern of 13CO2
excretion throughout the 8-h tracer study for group 1 receiving diets supplying intrinsically labeled casein or
[1-13C]leucine added to unlabeled casein
(extrinsically labeled) differed only marginally. In contrast,
different patterns of 13CO2 excretion emerged
when subjects (group 2) ingested diets containing [1-13C]leucine added extrinsically to an
L-amino acid mixture simulating the casein pattern (AA + [13C]Leu) compared with the intrinsically
labeled casein ([13C]Leu-casein; Fig.
2). Figure 3
shows the course of the whole body leucine oxidation derived from the
13CO2 output and plasma
-[13C]KIC enrichments for groups 1 and 2.
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-[13C]KIC and
-[2H3]KIC enrichments are
summarized in Table 3 for groups 1 and 2. Leucine
oxidation was significantly higher when free leucine was given together
with the crystalline amino acid mixtures compared with the diet with
the intrinsically labeled casein. For tracer AA + [13C]Leu, total leucine intake was
37.14, and for intrinsically labeled casein it was 36.69 µmol · kg1 · 30 min1. Hence, in comparison with the leucine
oxidation data (Table 3), this suggests a substantial leucine retention
during this 8-h period by both experimental groups.
Rate of appearance, nonoxidative leucine disposal, protein degradation, and splanchnic uptake. The rate of appearance was lower for the intravenously administered [2H3]leucine tracer (Table 3) with the exception of the AA + [13C]Leu tracer. The rate of appearance measured after the intake of amino acid mixture plus free [13C]leucine was significantly lower than after the intrinsically labeled casein (Table 3; P < 0.05).
With the use of plasma-[13C]KIC enrichment,
nonoxidative leucine disposal and also the difference between
nonoxidative leucine disposal and protein degradation (net protein
synthesis) was significantly higher, whereas the fraction of the
turnover oxidized was significantly lower when the intact intrinsically
labeled casein was ingested (group 2; Table
4).
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Hormones and leucine plasma concentrations.
Plasma insulin showed a meal-driven pattern, whereas the amplitude
appeared to be smaller with the casein diet compared with the free
amino acid mixture. The mean overall plasma insulin level did not
differ significantly between the two diets (Fig.
4A). No systematic differences
appeared to occur for plasma glucagon concentration (Fig. 4B),
and the insulin-to-glucagon ratio (data not shown) was not
significantly different between the intact protein and amino acid
diets.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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This study has revealed that, during the fed state, leucine oxidation is higher when a [13C]leucine tracer is ingested as free [13C]leucine together with a free amino acid mixture compared with an intrinsically [13C]leucine-labeled casein (Table 3). Together with the finding of a higher NOLD with the intrinsically labeled casein (Table 4), this suggests that a higher proportion of dietary leucine derived from intact casein is utilized during the absorptive phase for whole body protein synthesis in comparison with that from an equivalent intake of free L-amino acids.
These observations have a number of important implications, as follows.
The first implication is the impact of the present results on leucine requirements as estimated by the tracer-balance technique. Previously, we conducted 24-h tracer studies in which the minimal physiological requirement for leucine and other indispensable amino acids was estimated by the tracer-balance technique (e.g., see Refs. 1, 11, 12, and 29). The requirement values derived from these studies are two to three times higher than the current recommendations by the Food and Agricultural Organization (FAO)/World Health Organization (WHO)/United Nations (UNU) (15), which were based on results from nitrogen balance experiments. Although a more recent UN expert group has acknowledged that these latter values are no longer nutritionally relevant (14), the UN has not yet adopted a new set of tentative new values for adult human requirements for indispensable amino acids such as those proposed by the MIT group (48). Detailed discussions of this ongoing debate have been published (20, 33, 47, 49).
As noted in the RESULTS, all of the subjects were in a marked positive leucine balance during the 8-h tracer period, although balance was lower when the L-amino acid mixture was consumed (Table 3). However, because the subjects were receiving a generous intake of leucine, about eight times the required intake according to the FAO/WHO/UNU (15) or three times that which we have proposed as the leucine requirement (11, 12, 48), it would be reasonable to predict that a daily body leucine equilibrium would prevail for all of the diets in essentially all of these healthy adults. On this basis, the rate of leucine oxidation during the postabsorptive period would probably compensate for fed-state differences, and the rate at this time would be predicted to be lower when [1-13C]leucine plus the L-amino acid mixture was given compared with the casein-based diets. The investigations by Pacy et al. (38) on the diurnal rhythm of leucine and nitrogen metabolism in subjects consuming different intakes of protein would appear to support this prediction. To test this hypothesis, it will be necessary to conduct continuous 24-h tracer studies, as previously carried out in our laboratory. This too has implications for the design of studies concerned with establishing the whole body mechanisms responsible for determining body protein balance at different energy and protein intakes (5-7, 21, 38).
An important and as yet unanswered question is whether similar results
would have been obtained at the test leucine intake level of 40 mg · kg1 · day1
and/or when evaluated using a 24-h protocol. This would be difficult to
test with the present paradigm, because, for a diet containing an
intrinsically labeled protein to provide peptide-bound leucine at a
level of 40 mg
leucine · kg1 · day1
or less, this would require supplementation with a leucine free amino
acid mixture so that an adequate total nitrogen intake is given. Thus,
although giving the tracer in the form of an intrinsically labeled
protein would appear to be the closest simulation of a normal dietary
amino acid intake, there are clearly some limitations to this model.
Furthermore, the marked differences between the MIT estimates of
requirements using the tracer-balance concept and those of Rose (43),
which have served as an important data base for the proposed UN
requirement values (15), would not be explained by differences in the
molecular form of the amino acid ingested, because all of these studies
essentially involved use of free amino acid-based diets.
An additional issue with respect to the present and our previous studies on the leucine requirements of healthy adults (12) is that the [13C]leucine tracer was given via the intravenous route in contrast to the oral route in the present study. However, we do not think that this limits the significance of the present findings and their possible implications for the evaluation of amino acid requirements, because we have not found any important differences between estimates of leucine oxidation caused by the route of administration of the [13C]leucine tracer (25).
The second implication is the influence of the nature of intrinsically
labeled protein on protein metabolism. We observed a significantly
lower leucine oxidation and higher NOLD when the intrinsically labeled
casein was given in comparison with the L-amino acid diet
(group 2; Table 4). First-pass splanchnic extraction of leucine
was between 24 and 35%, confirming earlier results (4, 10, 25, 26).
Leucine rate of appearance calculated from
-[2H3]KIC and
-[1-13C]KIC enrichments were comparable with
results of an earlier study where a 1.5 g
protein · kg1 · day1
diet was provided (26).
Although we did not find an effect of the forms of dietary nitrogen or of the leucine tracer administered on estimates of splanchnic extraction, it would be of interest to examine the effect of the molecular form of nitrogen intake on the possible site of leucine oxidation and of NOLD (splanchnic or peripheral). In this context, therefore, the increase of the mean total plasma amino acid concentration in humans was significantly higher after consuming a meal containing a free amino acid mixture simulating cottage cheese protein in comparison with the intact protein control (22). Here, the isoleucine, leucine, and lysine plasma concentrations peaked earlier (30 min after the meal) and higher and declined more rapidly than after the cottage cheese meal was ingested (60 min after the meal; see Ref. 23). We found in the present study a higher mean plasma leucine, isoleucine, and valine concentration during the ingestion of the free amino acid diet that may be causally related to the observed higher rate of leucine oxidation.
The extent to which the difference in leucine oxidation between the L-amino acid mixture and the intrinsically labeled casein is due to a difference in the rate of amino acid absorption per se is not clear. In the pig, peak absorption of amino acids from a milk enzyme hydrolyzate occurred earlier and at a higher level than with an amino acid mixture of identical pattern (41), although the differences disappeared ~1 h after feeding (42). In general, it appears that amino acid absorption from peptides is more rapid than from amino acid mixtures (31), and a recent report showed that feeding of oligopeptides (casein hydrolyzate) induced a higher oxidation of leucine but also a higher rate of protein synthesis and a lesser inhibition of protein breakdown compared with intact casein (9). Furthermore, Beaufrere and co-workers [Boirie et al. (5)] recently proposed the concept of "slow and fast dietary proteins." Their investigation revealed that two major milk proteins (casein and whey) have different metabolic fates related to the apparent rate of amino acid absorption when a single protein meal, without addition of energy substrate, was given. From their results, the slowly absorbed dietary protein promoted postprandial protein deposition by an inhibition of protein breakdown, whereas the "fast" dietary protein stimulated protein synthesis, as well as oxidation. However, the significance of these findings for our understanding of the metabolic basis of the requirements for indispensable amino acids is unclear for a number of reasons, including the fact that a single meal of protein alone was given. Also, from the leucine oxidation data, it appeared that, in their experiment, there was essentially no prandial retention of the dietary leucine. This is not consistent with the apparently high nutritional quality of whey protein, based on nitrogen balance measurements (17, 18). Minimally, studies involving more complete meals would be desirable. Furthermore, intestinal amino acid absorption may adaptively respond to alterations in amino acid/protein intake (28), so a period of adjustment to the test dietary interventions compared would constitute a further improvement in the experimental approach. In summary, the available literature does not permit a clear determination as to whether differences in the rates of amino acid and/or tracer absorption, per se, are responsible for the present findings on the differences in rate of leucine oxidation among the diet/tracer.
Another consideration, with reference to a full interpretation of the nutritional significance of the present findings, is that of the meal pattern. Here we used the frequent, small, and equal meal paradigm that has been applied in most of our amino acid kinetic/requirement studies (11, 29) as well as by other authors (21, 39). However, meal patterns can affect the fate of absorbed amino acids, because we showed that urinary excretion of total nitrogen and leucine oxidation were lower when an isonitrogenous isocaloric intake of three meals per day was given compared with multiple small meals (13). For comparison, in the pig, phenylalanine oxidation was found to be higher when the mixed diet containing free lysine plus grain was given once daily compared with the administration of six equal small meals (2). It is interesting to speculate that the amino acid supply during parenteral nutrition, with the prompt rise in plasma and tissue amino acid concentration, mimics the situation of "fast" protein absorption and possibly limits the efficiency of amino acid retention caused by promotion of amino acid oxidation. Furthermore, to finally assess the nutritional significance of tracer studies of the kind used here and by others, it seems likely that the paradigm might usefully include relatively long-term tracer infusions lasting for at least one complete day.
Also, the question arises as to whether the nature of the specific
labeled protein chosen for our study may have influenced the results.
That is, would a different intrinsically labeled protein, such as a
highly digestible vegetable protein, have demonstrated a lower rate of
leucine oxidation and a higher nonoxidative leucine disposal compared
with the respective amino acid mixture? Thus a recent study (39) in
elderly women showed that when dietary protein intake was increased
through addition of vegetable protein, postabsorptive protein breakdown
was not inhibited to the same extent as that occurring when animal
protein was given. The study also showed that net protein synthesis
during the fed period of the day was less with feeding high vegetable
protein vs. a high animal protein diet. These kinetic or leucine
turnover differences were observed despite the fact that both
high-protein diets supplied a generous total nitrogen intake
(201-209 mg
N · kg1 · day1),
and the subjects were in daily body nitrogen balance. Again, we make
these points not only to underscore the possibility that the present
findings with casein may not necessarily predict those with other
good-quality protein sources but also to emphasize caution when drawing
nutritional interpretations from leucine kinetics that apply to
relatively brief windows of time during the day.
In conclusion, the present and earlier findings reveal that the immediate metabolic fate of absorbed amino acids is determined by a complex interaction of factors, including the molecular form of the amino acid ingested, the amino acid profile, the composition of the meal, the level of intake, and the pattern of meal ingestion.
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ACKNOWLEDGEMENTS |
|---|
This research was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-42101 and P-30-DK-40561 RR8 and by a grant of the Deutsche Forschungsgemeinschaft, Bonn, Germany.
| |
FOOTNOTES |
|---|
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: C. C. Metges, German Institute of Human Nutrition, Arthur-Scheunert-Allee 114-116, 14558 Bergholz-Rehbrücke, Germany (E-mail: metges{at}www.dife.de).
Received 13 May 1999; accepted in final form 29 November 1999.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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) or casein (cow
and goat; 
and 
) but without the leucine, phenylalanine, and
bicarbonate tracers. Values are expressed as difference from the
prefeeding values [atom percent excess (APE) × 1,000].
). B: group 2 (n = 5) receiving, as a tracer, either intrinsically
[1-13C]leucine-labeled casein (
) or intrinsically labeled casein (
).