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,**,3
*
Departments of Nutritional Sciences and
Paediatrics, University of Toronto, Toronto, Canada M5S 3E2;
**
The Research Institute, The Hospital for Sick Children, Toronto, Canada M5G 1X8;
School of Dietetics and Human Nutrition, McGill University, Montreal, Canada H9X 3V9; and
Department of Agricultural, Food & Nutritional Science, University of Alberta, Edmonton, Canada T6G 2P5
3To whom correspondence should be addressed. E-mail: paul.pencharz{at}sickkids.ca.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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19%. Although actual F13CO2 was higher during oral tracer infusion (P < 0.001), the breakpoint was not different from that determined with IV infusion (P = 0.98), with both yielding a mean lysine requirement of 36.6 mg/(kg · d). The upper 95% confidence intervals were 52.5 and 53.3 mg/(kg · d) for the oral and IV isotope infusions, respectively. These results demonstrate that routes of isotope administration using the indicator amino acid oxidation technique do not affect the estimated amino acid requirement. Therefore, the indicator amino acid oxidation method using the oral route, which is less invasive and allows for studies in vulnerable groups such as infants and children, should be the preferred method for studying amino acid requirements.
KEY WORDS: • splanchnic extraction • lysine requirement • indicator amino acid oxidation • humans
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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–4
). Classically, the tracer is infused intravenously, and blood samples are collected for measuring amino acid kinetics. This protocol has since been adapted to make it minimally invasive by using oral instead of intravenous (IV)4
tracer infusion and urine as opposed to blood sampling (5
). These advancements in methodology have allowed a more practical and less invasive approach to the study of amino acid metabolism and requirements. Consequently, it has been possible to conduct studies in vulnerable groups such as infants and children.
When labeled amino acids are given orally, a large proportion of tracer is extracted because of the first-pass utilization through the splanchnic tissue (6
–11
). First-pass splanchnic uptake ranges from 29 to 58% for phenylalanine (6
,9
), 20 to 40% for leucine (8
,9
,12
), and is 30% for lysine (8
). Splanchnic extraction has implications for the appearance of label in plasma and estimates of tracer kinetics. Isotopic enrichments of labeled amino acids have been shown to be significantly lower and flux significantly higher in plasma of subjects receiving oral vs. IV tracers (13
). This is because amino acids undergo metabolism, such as oxidation, in the splanchnic region, with the remainder being transported to the systemic circulation. Currently, it is unclear whether splanchnic metabolism of the tracer affects amino acid requirement estimates.
Although the effect of route of tracer administration on amino acid kinetics has been studied by several investigators in the past (6
–10
,12
), only one study has compared different routes of isotope infusion on amino acid (tryptophan) requirement estimates (14
). That study was conducted in piglets. To our knowledge, no experiments have been published in humans in which amino acid requirement estimates have been compared between oral and IV infusion of tracer. Therefore, the goal of this study was to compare whether routes of tracer administration affect the lysine requirement estimate by using the indicator amino acid oxidation (IAAO) technique, and to determine whether kinetics and metabolism of the tracer (phenylalanine) are altered by its route of administration.
Lysine was chosen as the indispensable amino acid of study because the requirement has been documented using different methods, including nitrogen balance (15
–17
), plasma amino acid concentrations (18
), direct amino acid oxidation (18
), 24-h tracer balance (19
,20
), IAAO method (21
,22
) and indicator amino acid balance (IAAB) (23
). The mean lysine requirement from all of these methods including nitrogen balance (17
) is in the relatively narrow range of 30–40 mg/(kg · d). Lysine, therefore, is a good amino acid to study for validating a less invasive method of estimating amino acid requirements.
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SUBJECTS AND METHODS |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Healthy adult men (n = 5) participated in the study on an outpatient basis at the Clinical Investigation Unit at The Hospital for Sick Children, Toronto, Canada. Subject characteristics are described in Table 1
. None of the subjects had a history of unusual dietary practices, recent weight loss, or endocrine disorders, or medication use before or during the study. The purpose of the study and the potential risks associated with the protocol were fully explained to each subject and informed written consent was obtained. Participants were encouraged to maintain their usual physical activities throughout the study period. The study protocol was approved by the Research Ethics Board of The Hospital for Sick Children. All volunteers were remunerated for their participation.
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The study was a completely randomized, crossover design. Each subject was studied using both oral and IV tracer infusions at each level of lysine intake. The study was carried out for six periods of 6 d each. Each subject was allocated to receive each of the six different lysine intakes (10, 20, 30, 40, 50, and 60 mg/(kg · d) in random order. On d 1–2 and 4–5 of each period (before the IAAO study day), subjects were given a controlled milkshake formula that provided adequate energy and protein [1 g/(kg · d)]. This milkshake diet was given for controlling the protein intake level because previous protein intake may affect phenylalanine flux (24
). On study days (d 3 and 6), phenylalanine kinetics were measured at one of the six dietary lysine intakes. The subjects were assigned in random order on those 2 d to receive either the IV or oral tracer administration. A 2-d washout period between the IV and oral study was provided to avoid the effect of isotope recycling. Study periods were separated by 
1 wk and all studies were carried out within 3 mo.
Subjects were weighed on each IAAO study day to ensure accurate prescription of diets and isotopes, and to confirm weight maintenance throughout the study. Lean body mass was calculated from reactance and resistance (25
) measured by bioelectrical impedance analysis (model 101A; RJL Systems, Detroit, MI). Fat-free mass was calculated from density derived from the four skinfold thicknesses (triceps, biceps, subscapular and suprailiac crest) (26
) measured by a skinfold caliper (British Indicators, St. Albans, UK).
Dietary intake and experimental diet.
Dietary intakes during the adaptation period (d 1–2 and 4–5) were provided in the form of milk shakes (Scandishake; Scandipharm, Birmingham, AL), with added carbohydrate (Caloreen; Nestle Clinical Nutrition, North York, Canada), protein (Promod, Ross Laboratories, Columbus, OH) and 3.25% homogenized milk to tailor the formula to the exact energy and protein intakes required. The diet was given as three meals and snacks spread throughout the day as normally consumed by each subject. Energy intakes were based on each subject’s resting metabolic rate after a 12-h overnight fast, as determined by continuous, open-circuit indirect calorimetry (2900 Computerized Energy Measurement System-Paramagnetic; Sensormedics, Yorba Linda, CA) and multiplied by an activity factor of 1.7. No other food or beverages were consumed except water, clear tea, or clear coffee. Subjects also consumed a daily multivitamin supplement (Centrum, Whitehall-Robins, Mississauga, Canada) throughout the study.
The experimental diet used during each IAAO study (d 3 and 6) was a liquid formula and protein-free cookies as previously developed for amino acid kinetic studies (27
). The study protocol for each IAAO is depicted in Figure 1
. The diet was divided into nine isocaloric, isonitrogenous meals representing 1/12th of the subject’s daily requirements. Hourly meals were consumed to ensure metabolic steady state in the fed condition (27
,28
). The diet provided 37% of energy as fat, 53% as carbohydrate, and 10% as protein. The main source of energy came from a nonprotein liquid formula diet (Protein-Free Powder, Product 80056, Mead Johnson, Evansville, IN; Tang and Koolaid, Kraft Foods, Toronto, Canada). The protein content was provided at a level of 1 g/(kg · d), supplied as a crystalline L-amino acid mixture based on the amino acid composition of egg protein. The only amino acids that diverged from this profile were phenylalanine, lysine, and alanine. The intake of the indicator amino acid, phenylalanine, was fixed at 15 mg/(kg · d) (which included the amount of L[1-13C]phenylalanine administered during the tracer) with an excess tyrosine intake [40 mg/(kg · d)]. This level of phenylalanine intake was previously determined to meet the requirements of 95% of adult men when tyrosine was in excess (29
). Lysine was provided at six graded levels as earlier mentioned. Alanine intake was adjusted to maintain a constant nitrogen intake.
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Two tracers, NaH13CO3 (99%) andL[1-13C]phenylalanine (99%) (Cambridge Isotope Laboratories, Woburn, MA), were used in this study. Isotopic and optical purity of L[1-13C]phenylalanine was verified by the manufacturer of the isotopes using chemical ionization gas chromatography-mass spectrometer (GC-MS) and nuclear magnetic resonance. The enrichment and enantiomeric purity of the L[1-13C]phenylalanine were reconfirmed by GC-MS of the n-propyl, heptofluorobutyramide derivative (30
) using a chiral column (Chirasil-Val, Alltech, Deerfield, IL). The measured fractional molar abundance of L[1-13C]phenylalanine was 97.5%. This value was used in the calculation of phenylalanine turnover. For the IV tracer infusion, all tracers were prepared in normal saline and passed through a 0.22-µm filter (Millipore, Bedford, MA) under a laminar flow hood and then dispensed into single-dose vials. Each batch of infusates was sterile and pyrogen free by the limulus amebocyte lysate test (31
). The tracers for oral administration were prepared in deionized water and stored at -20°C until used.
Doses of tracers for both oral and IV administrations were equal. Priming doses of NaH13CO3 (2.071 µmol/kg) and L[1-13C]phenylalanine (3.995 µmol/kg) were started at the fifth meal. Four-hour adaptation to the experimental diet was required to ensure sufficient time for 13CO2 background equilibration in expired air (5
). A continuous dose of L[1-13C]phenylalanine [7.991 µmol/(kg · h)] was commenced simultaneously and continued throughout the remaining 5 h of the study.
Sample collection.
Three baseline urine and breath samples were collected 15, 30, and 45 min before the isotope protocol began. Five plateau urine and breath samples were collected at isotopic steady state every 30 min during the period 150–270 min after initiation of the isotope protocol. This isotope protocol had been shown to achieve a satisfactory isotopic steady state 2 h after start of L[1-13C]phenylalanine isotope (5
). The same pattern also was seen in the present study, as shown in Figure 2
. The slope of the plateau enrichment in breath and urine samples on each study day was not significantly different from zero. All urine samples were stored at -20°C until analyzed for L[1-13C]phenylalanine enrichment. Breath samples were collected in disposable Haldane-Priestley tubes (Venoject; Terumo Medical, Elkton, MD) using a collection mechanism that permits the removal of dead-space air. Breath samples were stored at room temperature pending analysis. Expired CO2 production rate was measured using an indirect calorimeter (2900 Computerized Energy Measurement System; Sensormedics) 4 h after consuming the experimental diet on each study day.
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Expired 13CO2 enrichment was measured by a continuous-flow isotope ratio MS (CF-IRMS20/20, PDZ Europa, Cheshire, UK) and was expressed as atom percent excess (APE) relative to a reference standard of compressed CO2 gas. After isolation by cation exchange (Dowex 50W-X8, 100–200 mesh H+, Bio-Rad Laboratories, Richmond, CA), urinary phenylalanine was derivatized to its n-propyl, heptofluorobutyramide derivative (30
). Because the L[1-13C]phenylalanine we used had 1.5% D-isomer, enrichment of urinary L-phenylalanine was measured on a chiral column (Chirasil-Val, Alltech) to separate the D- and L-isomers, and obtain accurate urinary L[1-13C]phenylalanine enrichment. As we noted in our earlier paper (32
), some of the D-phenylalanine can be inverted to L-phenylalanine and then oxidized. However, under this circumstance, the inversion of D- to L-isomers would have a small and probably negligible effect on an estimate of phenylalanine oxidation. Using methane-negative chemical ionization GC-MS (Hewlett-Packard 5890 series GC; Hewlett-Packard 5988A MS system, Mississauga, Canada), selected-ion chromatograms were obtained by monitoring [m + HF-] ions at m/z 383 for L-phenylalanine and 384 for L[1-13C]phenylalanine. Isotopic enrichment in molecules percent excess was calculated from peak area ratios at isotopic steady state and baseline.
Estimation of isotope kinetics.
The model used to study phenylalanine metabolism was a simplified single-pool model (33
):
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where Q is phenylalanine flux [µmol/(kg · h)]; S is the rate of phenylalanine nonoxidative disposal, a measure of the rate of phenylalanine incorporation into body protein; O is the rate of phenylalanine oxidation; B is the rate of phenylalanine released from body protein; and I is the rate of exogenous phenylalanine intake. Phenylalanine flux (Q) used in the simplified single pool model was calculated from the dilution of the L-[1-13C]phenylalanine infused into the body amino acid pool at isotopic steady state by using the following equation (34
):
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where i is the rate of [1-13C]phenylalanine infused [µmol/(kg · h)] and Ei and Eu are the isotopic enrichments as mole fractions of the infusate and urinary phenylalanine at isotopic plateau. The -1 removes the contribution of the isotope infusion to the flux.
The rate of phenylalanine oxidation (O) was calculated as follows:
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where F13CO2 represents the rate of 13CO2 released by phenylalanine tracer oxidation [µmol 13CO2/(kg · h)] calculated by the following equation:
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where FCO2 is the CO2 production rate (mL/min), ECO2 is the 13CO2 enrichment in expired breath at isotope steady state (APE), and W is the weight of the subject (kg). The constants 44.6 µmol/mL and 60 min/h convert FCO2 to µmol/h and the factor 100 changes APE to a fraction. The factor 0.82 accounts for 13CO2 retained in the body because of bicarbonate fixation (35
).
The fraction of oral tracer not extracted on the first pass by the splanchnic bed was determined by comparing the urinary enrichment after oral administration of tracer [Eu(oral)] with that during the IV infused tracer [Eu(IV)]. The fraction of oral tracer extracted on the first pass by the splanchnic bed (f) was then calculated by difference. Because the tracer infusion rate during both routes in this study was equal, the splanchnic extraction was calculated as follows:
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where Eu(oral) and Eu(IV) are the urinary enrichments of the tracer infused by the oral and IV routes, respectively.
Statistical analysis.
Results are expressed as means ± SD. Multifactorial ANOVA with repeated measures was used to determine the effects of individual, orders of study, routes of isotope administration and levels of lysine intake on F13CO2, phenylalanine flux and oxidation. When warranted, post-hoc analysis was performed with Duncan’s multiple range test. Body weight and body composition during the study period were compared by repeated-measures ANOVA. Estimates of the mean and population-safe lysine intakes for adult men were derived by breakpoint analysis of the F13CO2 data using a two-phase linear regression crossover model similar to that described previously (21
). The 95% confidence interval (CI) for the mean lysine requirement was calculated using Fieller’s theorem; the upper 95% CI was used to represent the population-safe intake. The statistical difference between the two breakpoints was assessed using the comparison of the two-sample t procedure (36
). The comparisons for all parameters between the oral and IV tracer infusions were tested by paired two-tailed t tests. All statistical analyses were performed using the SAS program (version 8.0, SAS Institute, Cary, NC); differences were considered significant at P < 0.05.
In addition, the mean lysine requirement obtained from this study when isotope was infused IV was compared with that from our earlier IAAO studies in which the tracer was also given IV using the comparison of the two-sample t-procedure and significantly different after adjusted for Bonferroni correction (P < 0.017) (21
,22
).
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RESULTS |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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. When the tracer was given orally, the enrichment of urinary phenylalanine was consistently below that achieved when the tracer was given IV, at all lysine test intakes (P < 0.0001). Regardless of tracer infusion route, phenylalanine enrichment, and flux, nonoxidative phenylalanine disposal, a measure of the rate of phenylalanine incorporation into body protein, and phenylalanine release from proteolysis were not affected by lysine intake. Lysine intake, however, had a significant effect on phenylalanine oxidation (P = 0.006 and 0.04 for IV and oral tracer infusions, respectively). Oxidation rates of phenylalanine showed a linear decline at lysine intakes between 10 and 40 mg/(kg · d), but were not different at lysine intakes between 40 and 60 mg/(kg · d).
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and 3)
. Splanchnic extraction, calculated from the urinary [13C]phenylalanine enrichments, during the fed state was 18.8% of the orally infused phenylalanine tracer. There were no differences in splanchnic extraction of [13C]phenylalanine among lysine test intakes (repeated-measures ANOVA, P = 0.29)
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illustrates the mean breakpoint in the F13CO2 data (representative of the mean requirement), as analyzed by two-phase linear regression crossover. This breakpoint occurred at a dietary lysine intake of 36.6 (derived from 36.64) and 36.6 (derived from 36.59) mg/(kg · d) for the IV and oral isotope infusions, respectively. There was no difference between these two breakpoints [(P = 0.98), two-sample t-procedure (35
)]. The upper 95% CI, which were calculated from variation about the estimate and represented the safe population requirement, were estimated to be 52.5 and 53.3 mg lysine/(kg · d) for the IV and oral isotope infusion, respectively. Because all subjects were studied at all six dietary lysine intakes, individual requirements, estimated from visual inspection of the F13CO2 curve, varied from 30 to 50 mg/(kg · d). There was no difference between breakpoints obtained from F13CO2 or phenylalanine oxidation data (P = 0.31 and 0.57 for IV and oral isotope infusion, respectively). Less variation was observed with the F13CO2 data than with the phenylalanine oxidation data.
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,22
) when subjects received 1.0 g/(kg · d) protein (equal to protein intake in the present study) and 0.8 mg/(kg · d) protein. The estimates of the lysine requirement as defined by the breakpoint of 36.6 mg lysine/(kg · d) in the present study and 39.3 mg/(kg · d) in the subjects receiving the same amount of total protein intake (1.0 g/(kg · d) in the study of Zello et al. (21
) were not different (P = 0.11). However, the estimate of the lysine requirement in this study was lower than that [45.0 mg/(kg · d)] obtained from subjects with the lower [0.8 g/(kg · d)] protein intake (22
) (P = 0.004). |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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. This result is consistent with the IAAO study of Cvitkovic who found that the tryptophan requirement estimates of piglets were not different whether tracer ([1-14C]phenylalanine) was given IV or intragastrically (14
). As intakes of the limiting amino acid increase from deficient to adequate, the indicator amino acid is partitioned between incorporation into proteins and oxidation (4
,37
). The breakpoint is the level at which maximal protein anabolism first occurs and further intake of the test amino acid has no effect on oxidation of the indicator. The breakpoint is an estimate of the mean lysine requirement. The measured phenylalanine oxidation (Table 2)
and labeled carbon dioxide rate of production differed between the two routes of isotope administration. The breakpoint requirement, however, remained the same. In the present study, we found that F13CO2 increased by 7–14% when tracer was infused orally compared with IV, and the proportional difference (IV vs. oral) did not change at any level of lysine intake (P = 0.24). For each route of isotope administration, changes in lysine intake influenced indicator amino acid ([1-13C]phenylalanine) oxidation but did not affect #phenylalanine flux (Table 2)
. This means that the phenylalanine pool did not change with differences in lysine intake within route of tracer administration. This is important because IAAO assumes that the flux of the indicator is constant as it is partitioned between oxidation and incorporation into protein (37
).
As mentioned earlier, phenylalanine kinetics were affected by the route of isotope infusion. Although the tracer dose was the same for the IV and oral routes, urinary 13C-phenylalanine enrichment was greater during IV than oral infusion at all levels of lysine intake (Table 2)
. This leads to the lower calculated estimates of phenylalanine oxidation and flux when the tracer was given IV (Table 3)
. These findings are in agreement with previous results obtained in both fasting and fed subjects (9
,14
,38
–40
). Splanchnic utilization of phenylalanine is likely the reason for this observed difference. When the label is given orally, it is absorbed by the gastrointestinal tract and undergoes first-pass metabolism in the splanchnic bed. First-pass metabolism could involve incorporation into protein and oxidation. This utilization means that a lower proportion of the isotope enters the systemic circulation; hence, urinary enrichments are lower when the tracer is given orally.
There is still considerable debate about the human lysine requirement. Thus, it is worthwhile to compare the lysine requirement obtained from this study with others in the literature. The lysine requirement of 36.6 mg/(kg · d) obtained in this study is in the upper range [17–36 mg/(kg · d)] of the reanalyzed lysine requirement initially determined by the nitrogen balance method by Jones et al. (17
), but it is higher than the lysine requirement of 23.3 mg/(kg · d) suggested by Millward et al. (41
). It should be noted that the latter study was an indirect experimental model in which the lysine requirement was obtained from consumption of high and low amounts of wheat and milk proteins, and the lysine requirements suggested are relatively lower than those proposed by other investigators discussed below (17
–20
,23
). Further, in the present study the lysine requirement of 36.6 mg/(kg · d) is somewhat higher than that of 30 mg/(kg · d) proposed by the Massachusetts Institute of Technology (MIT) group. The suggested lysine requirement of 30 mg/(kg · d) was initially based on their whole-body lysine oxidation balance studies, which showed a slightly positive balance at a lysine intake of 29 mg/(kg · d) in the study by El-Khoury et al. (20
) and 30 mg/(kg · d) in the study by Meridith et al. (18
). However, in the latter study, the oxidation rates, as measured by direct oxidation, were relatively constant for intakes from 2 to 35 mg/(kg · d); therefore, it can be interpreted that the lysine requirement should not be < 35 mg/(kg · d). This number is in close agreement with the lysine requirement obtained in the present study. A modification of our method (IAAO) was used recently by Kurpad et al. (23
). Those authors used [1-13C]leucine as an indicator amino acid (rather than [1-13C]phenylalanine) and a 24-h IAAB with IV tracer infusion to estimate lysine requirement in healthy Indian adults. Again, they estimated the lysine requirement estimate to be 29 mg/(kg · d). However, only three levels of lysine [15, 29 and 77 mg/(kg · d)] were used in the 24-h lysine balance studies (19
,20
), and only four levels of lysine intake were used in the IAAB study of Kurpad et al. (23
). The number of lysine intake levels studied may not have been adequate for proper statistical derivation of a requirement estimate. Although eight levels of lysine intake were used in the study of Meridith et al. (18
), each subject was studied at only three to five levels, and some levels were studied in only two subjects. We have shown in every one of our requirement studies, using repeated measurements, that significant differences exist among subjects (22
,42
–44
). Because of intraindividual variability and the difficulty in carrying out these kinds of studies, it is important that every subject should be studied at all test levels.
The lysine requirement [36.6 mg/(kg · d)] obtained in the present IAAO study was not significantly different from that [39.3 mg/(kg · d)] of our earlier study (21
) in which subjects had the same total protein intake[1 g/(kg · d)] and the tracer was infused IV. This suggests the reproducibility of the IAAO method to determine amino acid requirements. However, in the study of Duncan et al. (22
), when the total protein content of the experimental diet was 0.8 g/(kg · d), the lysine requirement estimate was 45 mg/(kg · d), which is significantly higher than that obtained by Zello et al. (21
) and in the present study in which total protein intake was 1 g/(kg · d). On the basis of these results, we suggest that the total protein intake of 0.8 g/(kg · d) may not have been sufficient to meet the protein requirement of every subject in the study of Duncan et al. (22
). In a study by Millward et al. (45
) in which the concept of postprandial protein utilization was used, the results confirmed that the apparent protein requirement of young men is 0.99 ± 0.09 g/(kg · d). In addition, in a leucine balance study, Zello et al. (46
) suggested that a protein intake of 0.8 g/(kg · d) may be limiting for some subjects. In all IAAO studies, the protein was patterned after egg protein, which contains sufficient lysine and other indispensable amino acids so as not to limit protein synthesis at an intake of 0.8 g/(kg · d). Yet, the study of Duncan et al. (22
) suggests that protein synthesis, measured by IAAO, is limited until additional lysine is given. It is possible that total nitrogen intake was inadequate.
This study supports our previous studies and those of the MIT group that the lysine requirement in healthy adults is 2 to 3 times higher than that proposed by the FAO/WHO/UNU in 1985 (47
). Moreover, this study demonstrates that although the route of isotope administration affects tracer kinetics when using the IAAO technique, it has no effect on amino acid requirement estimates. Therefore, the IAAO method using the oral route, which is less invasive and enables studies in vulnerable groups such as infants and children, should be the preferred method for studying amino acid requirements.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Supported by grant MT 10321 of the Canadian Institutes of Health Research. W. K. was supported by the Connaught Scholarship, University of Toronto and RESTRACOMP, The Research Institute, The Hospital for Sick Children, Toronto, Canada.
4 Abbreviation used: APE, atom percent excess; CI, confidence interval; F13CO2, rate of release of 13CO2 from 13C-phenylalanine oxidation; GC-MS, gas chromatography-mass spectrometer; IAAB, indicator amino acid balance; IAAO, indicator amino acid oxidation; IV, intravenous.
Manuscript received 13 February 2002. Initial review completed 7 April 2002. Revision accepted 8 May 2002.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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4. Zello, G. A., Wykes, L. I., Ball, R. O. & Pencharz, P. B. (1995) Recent advances in methods of assessing dietary amino acid requirements for adult humans. J. Nutr. 125:2907-2915.
5. Bross, R., Ball, R. O. & Pencharz, P. B. (1998) Development of minimally invasive protocol for the determination of phenylalanine and lysine kinetics in humans during the fed state. J. Nutr. 128:1913-1919.
6. Biolo, G., Tessari, P., Inchiostro, S., Bruttomesso, D., Frongher, C., Sabadin, L., Fratton, M. G., Valerio, A. & Tiengo, A. (1992) Leucine and phenylalanine kinetics during mixed meal ingestion: a multiple tracer approach. Am. J. Physiol. 262:E455-E463.
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8. Hoerr, R. A., Matthews, D. E., Bier, D. M. & Young, V. R. (1993) Effects of protein restriction and acute refeeding on leucine and lysine kinetics in young men. Am. J. Physiol. 264:E567-E575.
9. Matthews, D. E., Marano, M. A. & Campbell, R. G. (1993) Splanchnic bed utilization of leucine and phenylalanine in humans. Am. J. Physiol. 264:E109-E118.
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14. Cvitkovic, S., Ball, R. O. & Pencharz, P. B. (2000) Comparison of oral and intravenous isotopic tracers in determining tryptophan requirements of piglets. FASEB. J. 14:A745(abs.).
15. Rose, W. C., Borman, A., Coon, M. J. & Lambert, G. F. (1955) The amino acid requirements of man. X. The lysine requirement. J. Biol. Chem. 214:579-587.
16. Jones, E. M., Baumann, C. A. & Reynolds, M. S. (1956) Nitrogen balances of women maintained on various level of lysine. J. Nutr. 60:549-561.
17. Rand, W. M. & Young, V. R. (1999) Statistical analysis of nitrogen balance data with reference to the lysine requirement in adults. J. Nutr. 129:1920-1926.
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