Effect of protein restriction on 15N transfer
from dietary [15N]alanine and
[15N]Spirulina platensis into urea
Mazen J.
Hamadeh and
L. John
Hoffer
Lady Davis Institute for Medical Research, Jewish General Hospital,
Montreal, H3T 1E2; and School of Dietetics and Human Nutrition,
McGill University, Sainte Anne de Bellevue, Quebec, Canada H9X
3V9
 |
ABSTRACT |
Six normal men consumed a mixed test
meal while adapted to high (1.5 g · kg
1 · day
1) and low
(0.3 g · kg
1 · day
1)
protein intakes. They completed this protocol twice: when the test
meals included 3 mg/kg of [15N]alanine
([15N]Ala) and when they included 30 mg/kg of
intrinsically labeled [15N]Spirulina platensis
([15N]SPI). Six subjects with insulin-dependent diabetes
mellitus (IDDM) receiving conventional insulin therapy consumed the
test meal with added [15N]Ala while adapted to their
customary high-protein diet. Protein restriction increased serum
alanine, glycine, glutamine, and methionine concentrations and reduced
those of leucine. Whether the previous diet was high or low in protein,
there was a similar increase in serum alanine, methionine, and
branched-chain amino acid concentrations after the test meal and a
similar pattern of 15N enrichment in serum amino acids for
a given tracer. When [15N]Ala was included in the test
meal, 15N appeared rapidly in serum alanine and glutamine,
to a minor degree in leucine and isoleucine, and not at all in other
circulating amino acids. With [15N]SPI, there was a slow
appearance of the label in all serum amino acids analyzed. Despite the
different serum amino acid labeling, protein restriction reduced the
postmeal transfer of dietary 15N in [15N]Ala
or [15N]SPI into [15N]urea by similar
amounts (38 and 43%, respectively, not significant). The response of
the subjects with IDDM was similar to that of the normal subjects.
Information about adaptive reductions in dietary amino acid catabolism
obtained by adding [15N]Ala to a test meal appears to be
equivalent to that obtained using an intrinsically labeled protein tracer.
humans; stable isotope; fed state; amino acid oxidation
 |
INTRODUCTION |
DIETARY PROTEIN
RESTRICTION induces an adaptive reduction in urea production over
the hours after consumption of a constant-composition test meal
(22). There is also reduced transfer of the
15N in a tracer dose of [15N]alanine
([15N]Ala) included in the test meal into urea,
suggesting that first-pass splanchnic amino acid retention is involved
in the adaptive process (22). This reduction in
15N transfer into urea was less successful in
protein-restricted persons with conventionally treated
insulin-dependent diabetes mellitus (IDDM), raising the possibility
that dietary protein requirements are increased for some persons with
IDDM (21, 22).
However, tracer [15N]Ala is not protein bound, so its
metabolism may not represent the extent to which the amino acids in
dietary proteins are oxidized or conserved for protein synthesis upon their first passage through the splanchnic bed (29). We
have, therefore, conducted a controlled trial of feeding subjects a mixed test meal containing either [15N]Ala or
[15N]Spirulina platensis
([15N]SPI), a 15N-labeled intact protein
tracer (3), before and after protein restriction.
In earlier studies, we used a 0.5 g protein/kg test meal to
examine the effect of previous diet on the efficiency of fed-state protein retention (22, 39). A test meal containing less
protein ought to be a more sensitive tool, because optimal retention of the amino acids in such a meal calls for greater metabolic efficiency. The test meal in the present study contained 0.25 g protein/kg body wt. It was offered to normal research subjects before and after 3 days of adaptation to protein restriction. Each subject underwent the
same protocol twice, the replicate protocols differing only in that a
tracer dose of [15N]Ala was included in the test meals
for three subjects the first time they followed the protocol and a
tracer dose of fully 15N-labeled whole protein,
[15N]SPI, the second time, with the order reversed for
the other three subjects. Measurements were also made in healthy
persons with IDDM receiving conventional insulin therapy while adapted to their customary high-protein intake, and their results were compared
with those of the normal subjects.
The goals of this study were 1) to test whether the
reduction in the transfer of 15N added to a meal as
[15N]Ala into urea after protein restriction is
comparable to what occurs after ingestion of [15N]SPI, an
intrinsically labeled protein; and 2) to compare the distribution of the different 15N-labeling vectors in serum
amino acids to gain insight into the validity of using the fate of
[15N]Ala as a marker for dietary free amino N in future studies.
 |
METHODS |
Subjects and protocols.
Six healthy, nonsmoking men using no medications and with normal blood
chemistries were admitted at 0700 to the clinical research unit, where
they consumed a diet providing 38 kcal/kg and 1.5 g protein/kg on
that day. The following morning (day 2) was the first test
meal study, after which the subjects followed a low-protein diet for
the rest of the day for total energy and protein intakes of 38 kcal/kg
and 0.39 g/kg, respectively. Protein restriction (0.31 g/kg, with
maintenance energy) continued on days 3 and 4. The test meal was repeated on the morning of day 5. The
protocol was repeated 10 days later, the replicate protocols differing only in that a tracer dose of [15N]Ala was included in
the test meals for three subjects on the first occasion, and a tracer
dose of fully 15N-labeled whole protein,
[15N]SPI, on the second one. The protocol
order was reversed for the other three subjects. Four healthy men and
two healthy women with IDDM presented to the research unit at 0700 for
a study that they completed the same day. Their blood hemoglobin
AIc concentrations were 6.2 ± 0.7% (mean ± SD;
normal range 3.5-5.5%), and serum insulin C-peptide
concentrations were 0.24 ± 0.04 nmol/l (normal range
0.46-0.72 nmol/l). Their diet was the typical Canadian diet, which
provides ~1.5 g
protein · kg
1 · day
1
(18); they did not change their dietary habit in the days
before the test meal. Their insulin regimen was adjusted so as to
achieve fasting and postprandial plasma glucose levels typical of
conventionally treated diabetes mellitus (9). Fasting and
postmeal capillary blood glucose concentrations were measured using the
Accu-Chek III blood glucose monitor (Boehringer Mannheim, Laval,
QC, Canada). The characteristics of the subjects and details of the
insulin therapy are provided in a companion article (20).
All research volunteers gave their written consent to participate in
the study, which was approved by the Research and Ethics Committee of
the Jewish General Hospital in Montreal.
Test meal.
The test meal was Glucerna (Ross Laboratories, St. Laurent, QC, Canada)
to which beet sugar (Rogers Sugar, Winnipeg, Manitoba, Canada) was
added to provide 0.25 g of protein and 10 kcal of energy/kg body
wt (10% protein, 30% fat, and 60% carbohydrate). Given together with
the test meal was either 3 mg [15N]Ala/kg body wt (99%;
MSD Isotopes, Montreal, QC, Canada) or 30 mg of processed dried
[15N]SPI/kg body wt (99% 15N; Martek,
Columbia, MD). To each meal was also added 200 mg of [13C]urea (99%; MSD Isotopes) with the aim of using the
difference between the amount of tracer administered and the amount of
it found in the serum and urine after the meal as a measure of
extraurinary urea losses.
Intrinsically 15N-labeled Spirulina platensis
was purchased from Martek Biosciences and confirmed to be 99%
15N by isotope ratio-mass spectrometry (IRMS) on a Kjeldahl
digestate. One gram of untreated algae contained 320 mg of glycogen
(26). The following procedure was developed to remove
nonprotein N (free nucleotides, free amino acids, and nucleic acids)
(24). Perchloric acid (30-35 ml of 0.5 N in saline)
was added to each 3-g batch of the dry product, thoroughly mixed, and
incubated in a 70°C water bath for 20 min. The mixture was
centrifuged at 200 g for 10 min at room temperature. The
acid supernatant was decanted, and the procedure was repeated. After
the addition of 40-45 ml of ethanol, the mixture was centrifuged
at 200 g for 10 min at room temperature. The ethanol was
decanted, and the procedure was repeated five times to remove the
perchlorate and chlorophyll, after which the resulting product was oven
dried at 60°C. To determine protein N, 30 mg of the dried algal
protein residue and 10 mg of catalyst (9 g
K2SO4 + 1 g CuSO4) were
digested with 1 ml of acid mixture (7 g SeO2 in 1 liter of
concentrated H2SO4) and 1 ml of H2O
at 125°C for 30 min, then at 250°C for 17 min, and then at 450°C
for 45 min (30). The volume was brought to 40 ml with a
solution of 100 mmol/l sodium phosphate (pH 5.8), and 1 ml was diluted
with 0.8 ml of 1 mol/l NaOH and 0.2 ml of H2O and measured on an Antek 7000 elemental analyzer (Antek Instuments, Houston, TX). A
typical lot of dried algal protein residue contained 98-118 mg N/g
body wt. The administered 15N dose per kilogram of body
weight was 0.470 ± 0.002 mg/kg for [15N]Ala and
3.09 ± 0.08 mg/kg for [15N]SPI, of which ~0.55 mg
was in alanine (3).
Analytical methods.
Serum amino acid concentrations and enrichments were analyzed by gas
chromatography-mass spectrometry (GC-MS), as previously described
(19). To 100 µl of serum were added the internal
standards, 2.5 µg of norleucine (Sigma Chemical, St. Louis, MO), 2.5 µg of L-[3,3,3-2H3]alanine
(99.4% 2H, MSD Isotopes), 2 µg of
[2,2-2H2]glycine (CDN Isotopes,
Pointe-Claire, QC, Canada), 0.5 µg of L-[S-methyl-2H3]methionine
(CDN Isotopes), 0.1 µg of
L-[3,4-13C2]aspartate (MassTrace,
Woburn, MA), and 10 µg of
L-[3,3,4,4-2H4]glutamine (Tracer
Technologies, Somerville, MA), and, after acidification with 1.5 ml of
1 mol/l acetic acid, the sample was applied to 1-ml columns of cation
exchange resin (Dowex 50W-X8, 100-200 mesh, hydrogen form, Bio-Rad
Laboratories, Richmond, CA). The amino acids were eluted into 3.7-ml
flat-bottomed vials equipped with Teflon-lined caps (Du Pont de
Nemours, Wilmingon, DE) with four sequential 1-ml additions of 3 mol/l
NH4OH. The NH4OH fraction was evaporated under
a gentle stream of N2. Tert-butyldimethylsilyl (TBDMS) derivatives were prepared as described by Patterson et al.
(33).
GC-MS analyses were performed using an HP-5890 gas chromatograph
(Hewlett-Packard, Palo Alto, CA) directly coupled to an HP-5988A quadrupole mass spectrometer. Samples were introduced by splitless injection (1.0 µl) from an HP-7673 autoinjector onto a fused silica DB-1 capillary column (30 × 0.25 mm, 0.25-µm film thickness,
J&W Scientific, Folsom, CA) under the following GC conditions: initial column temperature, 110°C (maintained for 2 min); program rate, 6°C/min until 200°C and then 10°C/min to a final column
temperature of 200°C; helium carrier gas column head pressure, 70 kPa; and injector port and transfer line temperatures, 250°C. The
electron impact mass spectrometry conditions were as follows: ionizing energy, 70 eV; emission current, 300 µA; and source temperature, 200°C. The following ions were monitored by selected ion monitoring: TBDMS-alanine [mass-to-charge ratio (m/z) 158.1],
TBDMS-[15N]alanine (m/z 159.1),
TBDMS-L-[3,3,3-2H3]alanine
(m/z 161.1), TBDMS-glycine (m/z 218.1),
TBDMS-[15N]glycine (m/z 219.1),
TBDMS-[2,2-2H2]glycine (m/z
220.1), TBDMS-leucine (m/z 200.2),
TBDMS-[15N]leucine (m/z 201.2),
TBDMS-isoleucine (m/z 200.2),
TBDMS-[15N]isoleucine (m/z 201.2),
TBDMS-norleucine (m/z 200.2), TBDMS-methionine (m/z 218.1), TBDMS-[15N]methionine
(m/z 219.1),
TBDMS-[S-methyl-2H3]methionine
(m/z 221.1), TBDMS-serine (m/z 390.3),
TBDMS-[15N]serine (m/z 391.3), TBDMS-aspartate
(m/z 418.2), TBDMS-[15N]aspartate
(m/z 419.2),
TBDMS-[3,4-13C2]aspartate (m/z
420.2), TBDMS-glutamine (m/z 432.3),
TBDMS-[15N]glutamine (m/z 433.3), and
TBDMS-[3,3,4,4-2H4]glutamine (m/z
436.3).
Serum and urinary [15N]urea enrichments were determined
by Metabolic Solutions (Merrimack, NH) by means of a Europa Tracer-Mass IRMS (Europa Scientific, Crewe, UK), with the N2 generated
from ammonium sulfate used as the reference gas, according to the
method of Read et al. (34). This method converts urea N
into NH3, eliminating any contribution from 13C
and 18O. 15N recovery in urea was calculated as
[15N]urea excretion over the 9 h after the test meal
plus the amount of [15N]urea present in total body water
(TBW) at hour 9 divided by 15N intake.
Statistical analysis.
Three-way repeated-measures ANOVA was used to determine
significant differences between data for the normal group. The three factors were diet (high vs. low protein), 15N source
([15N]Ala vs. [15N]SPI), and time for serum
amino acid and urea concentrations. Two-way repeated-measures ANOVA was
used to determine significant differences in 15N enrichment
and recovery, serum postabsorptive and 2-h postmeal amino acid and urea
concentrations, with the two factors being diet and 15N
source. For urea and amino acid enrichments, the two factors were diet
and time within the same 15N source. Within the same
15N source and protein level, serum urea concentration over
time was subjected to one-way repeated-measures ANOVA. When
significance occurred, a Newman-Keuls test was used post hoc to
determine the source of difference. Student's unpaired
t-test and two-way repeated-measures ANOVA were used to
determine significant differences between the normal and IDDM groups,
also using the Newman-Keuls test to determine the source of difference.
Differences were considered significant at P
0.05. Results are presented as means ± SE unless otherwise indicated.
 |
RESULTS |
Three days of protein restriction increased the serum
concentrations of alanine (P = 0.0001), glycine
(P = 0.0001), glutamine (P = 0.0001),
and methionine (P = 0.0001), while decreasing those of
leucine (P < 0.002) (Figs.
1-7).
Whether or not the previous diet was high or low in protein, the test
meal stimulated generally similar increases in serum alanine, leucine,
isoleucine, and methionine and no change in serum glycine, glutamine,
or aspartate concentrations (Figs.
1-7).
The amino acid labeling pattern characteristic of each 15N
tracer was also generally unaffected by whether the previous diet was
high or low in protein (Figs.
1-7).

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Fig. 1.
Serum alanine enrichment and concentration before
{[15N]alanine with high-protein
([15N]Ala-HP), ;
[15N]Spirulina platensis
([15N]SPI)-HP, }; and after
{[15N]Ala with low-protein (LP), ;
[15N]SPI-LP, } protein restriction.
TTR, tracer-to-tracee ratio. Serum concentrations were significantly
higher on the LP diet. Different letters denote significantly different
values. *Significantly different from postabsorptive concentration
(P < 0.05).
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Fig. 2.
Serum glycine enrichment and concentration before
([15N]Ala-HP, ;
[15N]SPI-HP, ) and after
([15N]Ala-LP, ;
[15N]SPI-LP, ) protein restriction. Serum
concentrations were significantly higher on the LP diet. *Significantly
different from postabsorptive concentration (P < 0.05).
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Fig. 3.
Serum leucine enrichment and concentration before
([15N]Ala-HP, ;
[15N]SPI-HP, ) and after
([15N]Ala-LP, ;
[15N]SPI-LP, ) protein restriction. Serum
concentrations were significantly lower on the LP diet. *Significantly
different from postabsorptive concentration (P < 0.05).
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Fig. 4.
Serum isoleucine enrichment and concentration before
([15N]Ala-HP, ;
[15N]SPI-HP, ) and after
([15N]Ala-LP, ;
[15N]SPI-LP, ) protein restriction.
Different letters denote significantly different values. *Significantly
different from postabsorptive concentration (P < 0.05).
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Fig. 5.
Serum methionine enrichment and concentration before
([15N]Ala-HP, ;
[15N]SPI-HP, ) and after
([15N]Ala-LP, ;
[15N]SPI-LP, ) protein restriction. Serum
concentrations were significantly higher on the LP diet. Different
letters denote significantly different values. *Significantly different
from postabsorptive concentration (P < 0.05).
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Fig. 6.
Serum glutamine enrichment and concentration before
([15N]Ala-HP, ;
[15N]SPI-HP, ) and after
([15N]Ala-LP, ;
[15N]SPI-LP, ) protein restriction. Serum
concentrations were significantly higher on the LP diet. *Significantly
different from postabsorptive concentration (P < 0.05).
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Fig. 7.
Serum aspartate enrichment and concentration before
([15N]Ala-HP, ;
[15N]SPI-HP, ) and after
([15N]Ala-LP, ;
[15N]SPI-LP, ) protein restriction.
*Significantly different from postabsorptive concentration
(P < 0.05).
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|
By contrast, the form of 15N-labeled amino acid tracer used
with the test meal greatly affected serum amino acid labeling. After [15N]Ala, label appeared rapidly in serum alanine and
glutamine, to a minor degree in leucine and isoleucine, and not at all
in other circulating amino acids. After [15N]SPI, there
was a slow appearance of the label in all serum amino acids analyzed,
with no preference for alanine or glutamine.
As measured 9 h after the test meal, protein restriction reduced
the transfer of 15N into urea by 38% with the use of
[15N]Ala and by 43% with the use of
[15N]SPI. This difference was not statistically significant.
The IDDM subjects consumed the test meal with [15N]Ala
while adapted to a conventional, high-protein diet. Their serum urea and amino acid concentrations were close to normal, except that postabsorptive serum leucine and isoleucine concentrations were subnormal and glutamine concentrations were higher than normal (Table
1). Postmeal 15N-labeled
amino acid enrichments (including those in alanine) were similar to
those of the normal subjects (data not shown). Their 15N
transfer into urea was normal (Table
2).
 |
DISCUSSION |
The main conclusion of this study is that addition of
[15N]Ala to a test meal provided information about an
adaptive change in fed-state amino acid catabolism equivalent to what
was obtained using an intrinsically labeled protein tracer. This was
obtained despite very different serum amino acid 15N
labeling and different absolute recoveries of the administered 15N dose in urea.
A trace amount of [13C]urea was included in the test
meals to verify the accuracy of the [15N]urea recovery
results. Measurement of [15N]urea production involves
adding the amount of the tracer in urine collected over the 9-h
postmeal observation period to that in TBW at the final time point, as
described by Fern et al. (12) in their single-dose
[15N]glycine end-product model for whole body N turnover.
The calculation ignores any loss of urea (and urea tracer) in the gut
or elsewhere (16, 27, 38, 44, 46). It also ignores the
possibility that diet or disease could change renal urea clearance,
which in turn could introduce systematic error by changing the
distribution of urea between TBW and urine (17). Our
finding that [13C]urea recovery was unaffected by diet or
IDDM allays concern that these may have had a distorting effect on
[15N]urea recovery. As is noted in the companion article
(20), the [13C]urea recoveries were higher
than anticipated, perhaps due to an imprecision in our mathematical
correction for the contribution of [15N]urea to the
[M+1]urea mass. Although it is possible that
[15N]urea synthesis partially distorted our
[13C]urea recovery measurement, the reverse could not
occur, because the IRMS analysis converts urea to ammonia, eliminating
any contribution from 13C or 18O.
Figures 1-7 illustrate that serum amino acid concentration
profiles were closely similar when the dietary protocol was repeated and that the postmeal serum amino acid 15N-labeling pattern
was similar for a given 15N tracer whether the preceding
diet was high or low in protein. However, the labeling patterns were
very different with the different tracers. After
[15N]Ala, the 15N tracer can be assumed
to have been rapidly absorbed and a large amount of it deaminated
(2, 25, 45). After [15N]SPI,
15N-labeled amino acids were released into the circulation
slowly. There was greater total [15N]urea production
after [15N]SPI; this can be attributed to its larger
15N dose. The greater recovery of 15N from
[15N]Ala in urea is a consequence of its rapid
deamination compared with other amino acids (2, 25, 45).
The same phenomenon is observed when whole body N flux is measured
using the single-dose end-product model, which calculates flux as the
15N dose divided by the 15N enrichment in urea
produced over the subsequent 12 h (12). The flux
obtained when the dose is administered as an intrinsically labeled
protein is approximately twice that obtained using
[15N]Ala (13). We presume that very rapid
mixing of 15N from [15N]Ala in the urea
precursor pool is the reason why our main outcome measure, fractional
change in tracer appearance in urea, was closely similar for
[15N]Ala and [15N]SPI.
The notion that splanchnic first-pass amino acid conservation or
catabolism is important in nutritional adaptation is supported by
several studies that have used sophisticated tracer approaches in
humans and piglets (4, 8, 23, 29, 35, 36). Notwithstanding the complex nature of splanchnic amino acid metabolism indicated by
these studies and the important effects on model parameters when
individual tracer amino acids or intrinsically labeled protein tracers
are used (6, 29), it may be useful, in certain situations, to bypass the details of how the body achieves its homeostatic aims
and, as in the present study, simply measure the system's regulated
output: short-term amino acid catabolism after a metabolic or
nutritional challenge.
The present results support such a conceptual approach. They suggest
that, despite serum amino acid 15N labeling that differed
greatly from what was produced by an intrinsically labeled protein,
[15N]Ala gave a similar system output, i.e., an
equivalent reduction in 15N tracer incorporation into urea.
The advantages of this method are simplicity and robustness. These
render it amenable to use in a variety of clinical settings, with the
potential for insights that can be coupled with the results of more
sophisticated tracer methodologies.
We found no indication of excess postmeal transfer of 15N
from [15N]Ala into urea in persons with mildly
hyperglycemic IDDM. This observation is consistent with their normal
postmeal sulfate and total urea production (20) and with
our earlier finding in conventionally treated IDDM by using a
high-protein test meal (22). Insulin withdrawal increases
urea production (1, 14) and leucine plasma concentration,
oxidation, and turnover in IDDM (31, 32). Conventional
IDDM therapy is also commonly associated with increased circulating
branced-chain amino acid concentrations (11, 28, 37, 41),
but, as in the present study, this is not always the case (7, 42,
43). Where and how does insulin therapy of IDDM regulate amino
acid incorporation into body proteins? At one level, it restrains
muscle proteolysis, a restraint that is released when insulin provision
is grossly inadequate (31, 32). Tracer studies of postmeal
(40) or fed-state (5) amino acid kinetics in
insulin-deprived IDDM indicate defective suppression of whole body
proteolysis before the meal, with persistence to a varying extent into
the fed state. Milder insulin-deficient states may yet suffice to
restrain proteolysis, while exerting anabolic and regulatory effects
when injected insulin, together with dietary amino acids, reaches the
liver (10) and the periphery to stimulate muscle protein
synthesis (15). We suggest that the low serum leucine
concentrations measured in our IDDM subjects are evidence that they had
sufficient exogenous insulin in their tissues to prevent a
protein-catabolic state despite their hyperglycemia. The results
would presumably have been different had their insulin deficiency been
more severe.
In conclusion, we have found that inclusion of [15N]Ala
in a test meal provided information about adaptive changes in dietary amino acid catabolism equivalent to that provided by the use of an
intrinsically labeled protein tracer. Under conditions of adaptation to
a high protein intake, persons with conventionally treated IDDM
demonstrated normal first-pass dietary amino acid retention.
 |
ACKNOWLEDGEMENTS |
We thank Line Robitaille for technical assistance, Chantal
Bellerose for dietetic expertise and assistance, and Alicia Schiffrin for assistance in the study involving the patients with diabetes. Ross
Laboratories provided the product used in the test meals.
 |
FOOTNOTES |
M. J. Hamadeh was a recipient of the McGill University
1999-2000 Standard Life Dissertation Fellowship. The Clinical
Research Unit is supported by the Fonds de la Recherche en Santé
du Québec. This study was supported by Grants MT8725 and MME6712
from the Canadian Institutes for Health Research.
Address for reprint requests and other correspondence: L. J. Hoffer, Lady Davis Inst. for Medical Research, Jewish General Hospital, 3755 Cote-Ste-Catherine Rd., Montreal, QC H3T 1E2, Canada (E-mail:mi90{at}musica.mcgill.ca).
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. Section 1734 solely to indicate this fact.
Received 1 September 2000; accepted in final form 21 March 2001.
 |
REFERENCES |
1.
Almdal, TP,
Jensen T,
and
Vilstrup H.
Increased hepatic efficacy of urea synthesis from alanine in insulin-dependent diabetes mellitus.
Eur J Clin Invest
20:
29-34,
1990[ISI][Medline].
2.
Battezzati, A,
Haisch M,
Brillon DJ,
and
Matthews DE.
Splanchnic utilization of enteral alanine in humans.
Metabolism
48:
915-921,
1999[ISI][Medline].
3.
Berthold, HK,
Hachey DL,
Reeds PJ,
Thomas OP,
Hoeksema S,
and
Klein PD.
Uniformly 13C-labeled algal protein used to determine amino acid essentiality in vivo.
Proc Natl Acad Sci USA
88:
8091-8095,
1991[Abstract].
4.
Berthold, HK,
Jahoor F,
Klein PD,
and
Reeds PJ.
Estimates of the effect of feeding on whole-body protein degradation in women vary with the amino acid used as tracer.
J Nutr
125:
2516-2527,
1995[ISI][Medline].
5.
Biolo, G,
Inchiostro S,
Tiengo A,
and
Tessari P.
Regulation of postprandial whole-body proteolysis in insulin-deprived IDDM.
Diabetes
44:
203-209,
1995[Abstract].
6.
Boirie, Y,
Gachon P,
Corny S,
Fauquant J,
Maubois JL,
and
Beaufrère B.
Acute postprandial changes in leucine metabolism as assessed with an intrinsically labeled milk protein.
Am J Physiol Endocrinol Metab
271:
E1083-E1091,
1996[Abstract/Free Full Text].
7.
Borghi, L,
Lugari R,
Montanari A,
Dall'Argine P,
Elia GF,
Nicolotti V,
Simoni I,
Parmeggiani A,
Novarini A,
and
Gnudi A.
Plasma and skeletal muscle free amino acids in type I, insulin-treated diabetic subjects.
Diabetes
34:
812-815,
1985[Abstract].
8.
Cayol, M,
Boirie Y,
Rambourdin F,
Prugnaud J,
Gachon P,
Beaufrère B,
and
Obled C.
Influence of protein intake on whole body and splanchnic leucine kinetics in humans.
Am J Physiol Endocrinol Metab
272:
E584-E591,
1997[Abstract/Free Full Text].
9.
DCCT Research Group.
The diabetes control and complications trial (DCCT): design and methodologic considerations for the feasibility phase.
Diabetes
35:
530-545,
1986[Abstract].
10.
De Feo, P.
Fed state protein metabolism in diabetes mellitus.
J Nutr
128:
328S-332S,
1998[ISI][Medline].
11.
Devlin, JT,
Scrimgeour A,
Brodsky I,
and
Fuller S.
Decreased protein catabolism after exercise in subjects with IDDM.
Diabetologia
37:
358-364,
1994[ISI][Medline].
12.
Fern, EB,
Garlick PJ,
Sheppard HG,
and
Fern M.
The precision of measuring the rate of whole-body nitrogen flux and protein synthesis in man with a single dose of [15N]-glycine.
Human Nutr Clin Nutr
38C:
63-73,
1984[Medline].
13.
Fern, EB,
Garlick PJ,
and
Waterlow JC.
Apparent compartmentation of body nitrogen in one human subject: its consequences in measuring the rate of whole-body protein synthesis with 15N.
Clin Sci (Colch)
68:
271-282,
1985[ISI][Medline].
14.
Freyse, EJ,
Rebrin K,
Schneider T,
Petrzika M,
and
Fischer U.
Increased urea synthesis in insulin-dependent diabetic dogs maintained normoglycemic: effect of portal insulin administration and food protein content.
Diabetes
45:
667-674,
1996[Abstract].
15.
Fryburg, DA,
and
Barrett EJ.
Insulin, growth hormone, and IGF-I regulation of protein metabolism.
Diabetes Rev
3:
93-112,
1995.
16.
Fuller, MF,
and
Reeds PJ.
Nitrogen cycling in the gut.
Annu Rev Nutr
18:
385-411,
1998[ISI][Medline].
17.
Goldstein, MH,
Lenz PR,
and
Levitt MF.
Effect of urine flow rate on urea reabsorption in man: urea as a "tubular marker."
J Appl Physiol
26:
594-599,
1969[Free Full Text].
18.
Gray-Donald, K,
Jacobs-Starkey L,
and
Johnson-Down L.
Food habits of Canadians: reduction in fat intake over a generation.
Can J Public Health
91:
381-385,
2000[ISI][Medline].
19.
Hamadeh, MJ,
and
Hoffer LJ.
Tracer methods underestimate short-term variations in urea production in humans.
Am J Physiol Endocrinol Metab
274:
E547-E553,
1998[Abstract/Free Full Text].
20.
Hamadeh, MJ,
Schiffrin A,
and
Hoffer LJ.
Sulfate production depicts fed-state adaptation to protein restriction in humans.
Am J Physiol Endocrinol Metab
281:
E341-E349,
2001[Abstract/Free Full Text].
21.
Hoffer, LJ.
Adaptation to protein restriction is impaired in insulin-dependent diabetes mellitus.
J Nutr
128:
333S-336S,
1998[ISI][Medline].
22.
Hoffer, LJ,
Taveroff A,
and
Schiffrin A.
Metabolic adaptation to protein restriction in insulin-dependent diabetes mellitus.
Am J Physiol Endocrinol Metab
272:
E59-E67,
1997[Abstract].
23.
Hunter, KA,
Ballmer PE,
Anderson SE,
Broom J,
Garlick PJ,
and
McNurlan MA.
Acute stimulation of albumin synthesis rate with oral meal feeding in healthy subjects measured with [ring-2H5]phenylalanine.
Clin Sci (Colch)
88:
235-242,
1995[ISI][Medline].
24.
Hutchison, WC,
and
Munro HN.
The determination of nucleic acids in biological materials. A review.
Analyst
86:
768-813,
1961[ISI].
25.
Kay, JDS,
Seakins JWT,
Geiseler D,
and
Hjelm M.
Validation of a method for measuring the short-term rate of urea synthesis after an amino acid load.
Clin Sci (Colch)
70:
31-38,
1986[ISI][Medline].
26.
Lo, S,
Russell JC,
and
Taylor AW.
Determination of glycogen in small tissue samples.
J Appl Physiol
28:
234-236,
1970[Free Full Text].
27.
Long, CL,
Jeevanandam J,
and
Kinney JM.
Metabolism and recycling of urea in man.
Am J Clin Nutr
31:
1367-1382,
1978[Abstract].
28.
Luzi, L,
Castellino P,
Simonson DC,
Petrides AS,
and
DeFronzo RA.
Leucine metabolism in IDDM: role of insulin and substrate availability.
Diabetes
39:
38-48,
1990[Abstract].
29.
Metges, CC,
El-Khoury AE,
Selvaraj AB,
Tsay RH,
Atkinson A,
Regan MM,
Bequette BJ,
and
Young VR.
Kinetics of L-[1-13C]leucine when ingested with free amino acids, unlabeled or intrinsically labeled casein.
Am J Physiol Endocrinol Metab
278:
E1000-E1009,
2000[Abstract/Free Full Text].
30.
Munro, HN,
and
Fleck A.
Analysis of tissues and body fluids for nitrogenous constituents.
In: Mammalian Protein Metabolism, edited by Munro HN. New York: Academic, 1969, vol. III, p. 424-525.
31.
Nair, KS,
Ford GC,
Ekberg K,
Fernqvist-Forbes E,
and
Wahren J.
Protein dynamics in whole body and in splanchnic and leg tissues in type I diabetic patients.
J Clin Invest
95:
2926-2937,
1995[ISI][Medline].
32.
Nair, KS,
Ford GC,
and
Halliday D.
Effect of intravenous insulin treatment on in vivo whole body leucine kinetics and oxygen consumption in insulin-deprived type I diabetic patients.
Metabolism
36:
491-495,
1987[ISI][Medline].
33.
Patterson, BW,
Carraro F,
and
Wolfe RR.
Measurement of 15N enrichment in multiple amino acids and urea in a single analysis by gas chromatography/mass spectrometry.
Biol Mass Spectrom
22:
518-523,
1993[ISI][Medline].
34.
Read, WWC,
Harrison RA,
and
Halliday D.
A resin-based method for the preparation of molecular nitrogen for 15N analysis from urinary and plasma components.
Anal Biochem
123:
249-254,
1982[ISI][Medline].
35.
Stoll, B,
Burrin DG,
Henry J,
Yu H,
Jahoor F,
and
Reeds PJ.
Dietary amino acids are the preferential source of hepatic protein synthesis in piglets.
J Nutr
128:
1517-1524,
1998[Abstract/Free Full Text].
36.
Stoll, B,
Henry J,
Reeds PJ,
Yu H,
Jahoor F,
and
Burrin DG.
Catabolism dominates the first-pass intestinal metabolism of dietary essential amino acids in milk protein-fed piglets.
J Nutr
128:
606-614,
1998[Abstract/Free Full Text].
37.
Tamborlane, WV,
Sherwin RS,
Genel M,
and
Felig P.
Restoration of normal lipid and amino acid metabolism in diabetic patients treated with a portable insulin-infusion pump.
Lancet
1:
1258-1261,
1979[ISI][Medline].
38.
Taveroff, A,
and
Hoffer LJ.
Are leucine turnover measurements valid in the intravenously fed state?
Metabolism
43:
1338-1345,
1993[ISI].
39.
Taveroff, A,
Lapin H,
and
Hoffer LJ.
Mechanism governing short-term fed-state adaptation to dietary protein restriction.
Metabolism
43:
320-327,
1994[ISI][Medline].
40.
Tessari, P,
Pehling G,
Nissen SL,
Gerich JE,
Service FJ,
Rizza RA,
and
Haymond MW.
Regulation of whole-body leucine metabolism with insulin during mixed-meal absorption in normal and diabetic humans.
Diabetes
37:
512-519,
1988[Abstract].
41.
Trevisan, R,
Marescotti C,
Avogaro A,
Tessari P,
Del Prato S,
and
Tiengo A.
Effects of different insulin administrations on plasma amino acid profile in insulin-dependent diabetic patients.
Diabetes Res.
12:
57-62,
1989[ISI][Medline].
42.
Tuttle, KR,
Bruton JL,
Perusek MC,
Lancaster JL,
Kopp DT,
and
DeFronzo RA.
Effect of strict glycemic control on renal hemodynamic response to amino acids and renal enlargement in insulin-dependent diabetes mellitus.
N Engl J Med
324:
1626-1632,
1991[Abstract].
43.
Vannini, P,
Marchesini G,
Forlani G,
Angiolini A,
Ciavarella A,
Zoli M,
and
Pisi E.
Branched-chain amino acids and alanine as indices of the metabolic control in type 1 (insulin-dependent) and type 2 (non-insulin-dependent) diabetic patients.
Diabetologia
22:
217-219,
1982[ISI][Medline].
44.
Walser, M.
Determinants of ureagenesis, with particular reference to renal failure.
Kidney Int
17:
709-721,
1980[ISI][Medline].
45.
Wolfe, RR,
Jahoor F,
and
Shaw JHF
Effect of alanine infusion on glucose and urea production in man.
J Parenter Enteral Nutr
11:
109-111,
1987.
46.
Young, VR,
El-Khoury AE,
Raguso CA,
Forslund AH,
and
Hambraeus L.
Rates of urea production and hydrolysis and leucine oxidation change linearly over widely varying protein intakes in healthy adults.
J Nutr
130:
761-766,
2000[Abstract/Free Full Text].
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