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
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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, open circle } 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, open circle ) 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, open circle ) 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, open circle ) 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, open circle ) 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, open circle ) 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, open circle ) protein restriction. *Significantly different from postabsorptive concentration (P < 0.05).

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).

                              
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Table 1.   Postabsorptive and 2-h postmeal serum urea and amino acid concentrations


                              
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Table 2.   Postmeal 15N transfer into urea


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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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