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Nutrient Requirements and Optimal Nutrition

Leucine Is Not a Good Choice as an Indicator Amino Acid for Determining Amino Acid Requirements in Men^1,2

Jean W-C. Hsu^*,, Wantanee Kriengsinyos, Linda J. Wykes^**, Mahroukh Rafii, Laksiri A. Goonewardene, Ronald O. Ball^*,, and Paul B. Pencharz^*,,,,3

^* Department of Nutritional Sciences, University of Toronto, Toronto, ON, Canada M5S 3E2; The Research Institute, The Hospital for Sick Children, Toronto, ON, Canada M5G 1X8; ^** School of Dietetics and Human Nutrition, McGill University, Ste Anne de Bellevue, QC, Canada H9X 3V9; Department of Agricultural, Food and Nutritional Sciences, University of Alberta, Edmonton, AB, Canada T6G 2P5; and Department of Paediatrics, University of Toronto, ON, Canada

³ To whom all correspondence and reprint requests should be addressed. E-mail: paul.pencharz{at}sickkids.ca.

	ABSTRACT

TOP ABSTRACT SUBJECTS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Leucine tracer has been widely used for examining whole-body protein turnover in humans, but has not been evaluated as an indicator to be used in the indicator amino acid oxidation (IAAO) method. The goal of this study was to determine whether the L-[1-¹³C]leucine isotope is an acceptable indicator by comparing it with an established tracer, L-[1-¹³C]lysine. Healthy men (n = 7; 29.9 ± 4.8 y old) were fed in random order a diet with 7 graded intakes of phenylalanine without tyrosine. In the first study (n = 5), subjects were administered an excess leucine intake of 65 mg/(kg·d), and in the second study (n = 5), they were given the mean requirement of 45 mg/(kg·d) to determine whether leucine intake affected the pattern of response. Previous IAAO studies using lysine and phenylalanine demonstrated a clear pattern in ¹³CO₂ production, i.e., increasing test amino acid intake resulted in a linear decrease to plateau, with a readily discernable breakpoint indicating the requirement. This pattern of production of ¹³CO₂, indicates clear partitioning of the indicator amino acid between oxidation and protein synthesis. This was not observed with leucine at an intake of 65 mg/(kg·d). Conversely, at the lower leucine intake of 45 mg/(kg·d), a breakpoint was seen and a total aromatic amino acid requirement estimate that did not differ from that obtain using lysine as the indicator was obtained. In conclusion, leucine may be used as the indicator in the IAAO technique only when the daily intake leucine is given at its mean requirement level and the potential metabolic effects of other variables are taken into consideration.

KEY WORDS: • indicator amino acid oxidation • leucine • aromatic amino acids • phenylalanine • tyrosine

Indicator amino acid oxidation (IAAO),⁴ first developed in piglets (1,2), is a method based on the concept that the partition of any indispensable amino acid between oxidation and protein synthesis is sensitive to the level of the most limiting amino acid in the diet (3–5). It can be applied to determine the requirements of any indispensable amino acid or conditionally indispensable amino acid by measuring carbon oxidation from carbon oxidation production produced by another nonlimiting indispensable amino acid (called the indicator amino acid) (4,6). When the test amino acid is fed at insufficient intake and limits protein synthesis, all other amino acids are in relative excess and will be oxidized. As the dietary intake of the limiting amino acid is increased in graded amounts, the oxidation of the indicator amino acid will decrease in a linear manner until the requirement is reached, after which there is no change.

Criteria for choosing an appropriate indicator amino acid are as follows (5): 1) the indicator amino acid must be indispensable; 2) labeled carboxyl carbon must be irreversibly oxidized and its oxidation calculated quantitatively by the appearance of label in the breath; and 3) the indicator amino acid should have a small well-regulated pool and must undergo no significant pathways other than oxidation to CO₂ and incorporation into protein. Criteria 1) and 2) restrict the choices to a few amino acids. Among indispensable amino acids, only phenylalanine, lysine, and branched-chain amino acids (BCAA) meet criterion (ii). Phenylalanine has a small well-regulated pool. However, to ensure that phenylalanine is channeled to oxidation plus carbon dioxide production, an excess of tyrosine must be used (7). Lysine has no metabolic pathways other than oxidation and protein synthesis but has a larger pool than phenylalanine. The piglet studies that established the IAAO model validated the use of phenylalanine (plus tyrosine) as an ideal indicator (1,2). They further validated the use of lysine as an indicator by comparing the tryptophan requirement using both indicators; the tryptophan requirement did not differ as estimated using the 2 indicators (8).

Leucine tracer has been widely used for examining whole-body protein turnover in humans, but has not previously been used as an indicator in the short-term indicator amino acid oxidation (IAAO) method. Leucine has a larger and more variable pool size than phenylalanine or lysine, thus potentially making it a less sensitive indicator. Leucine also plays significant roles in the regulation of protein synthesis (9,10) and hormonal secretion (11,12); therefore, it has a direct influence on the measurement parameter: the partitioning between oxidation and protein synthesis. Recently leucine was used as a tracer in the 24-h indicator amino acid balance (IAAB) method, which is modification of the IAAO technique (13–20). However, leucine has not yet been confirmed as a valid indicator amino acid in the literature. Further, leucine intake levels given in the 24-h IAAB varied from 40 to 93 mg/(kg·d) (13,14,16), but this level was fixed at 40 mg/(kg·d) in the later studies (15,17–20). Finally, a normal and expected pattern in ¹³CO₂ production, a linear decrease to plateau, with a readily discernable breakpoint indicating the requirement was not always observed in these studies. For example, a sigmoid response was observed in the study of determination of methionine requirements (20). Thus, the suitability of leucine as the indicator amino acid remains to be determined.

The goal of this study was to determine whether the L-[1-¹³C]leucine isotope can be used as a suitable indicator in the IAAO technique by comparison with an established tracer, lysine. We recently reported an estimate of total aromatic amino acid requirements for healthy men determined using lysine as an indicator (21). In the present study, we attempted to determine aromatic amino acid requirements using leucine as an indicator with the intention of comparing the requirements with those obtained using lysine. Two different daily leucine intake levels were used to examine whether the level of leucine intake affected the pattern of leucine indicator oxidation.

	SUBJECTS AND METHODS

TOP ABSTRACT SUBJECTS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
    Subjects. Healthy men (n = 7) participated in the study on an outpatient basis in the Clinical Investigation Unit at the Hospital for Sick Children, Toronto, Canada. Five of the subjects in the present study participated in the previous lysine indicator study (21). Subject characteristics are described in Table 1. Subjects were excluded from the study if they had a history of an inborn error of metabolism, a chronic disease, an endocrine disorder, unusual dietary practice, recent weight loss, pharmacological therapy, or hormonal treatment. The purpose of the study and the potential risks involved were explained in detail to each subject and written consent was obtained from all subjects. All procedures used in the study were approved by the Research Ethics Board of the Hospital for Sick Children. All men received financial compensation for their participation.

    Tracer protocol. The isotopic labeled tracers used in the studies were NaH¹³CO₃ with 99% enrichment and L-[1-¹³C]leucine with 99% enrichment (Cambridge Isotope Laboratories). The isotopic and optical purity of L-[1-¹³C]leucine was verified by the manufacturer of the isotopes using chemical ionization GC-MS and NMR. The enrichment of the L-[1-¹³C]leucine tracer was reconfirmed by LC/MS of the butanol derivative. The measured fractional molar abundance of L-[1-¹³C]leucine was 98.8%. This value was used in the calculation of leucine flux. The oral tracer study started after a 4-h enteral intake adaptation period. Priming doses of NaH¹³CO₃ (2.023 µmol/kg) and L-[1-¹³C]leucine (18.158 µmol/kg) were given with the fifth meal. In addition, the L -[1-¹³C]leucine isotope (9.079 µmol/kg) was given with hourly meals, starting with the fifth meal, for the next 4 h.

    Sample collection. Breath and blood samples were collected on each IAAO study day; 3 baseline breath and 2 baseline blood samples were collected between 45 and 15 min before the tracer protocol began. Because isotopic plateau in CO₂ and plasma can be reached within 2 h after initiating isotope infusion (30), expired breath ¹³CO₂ and blood samples were collected every 30 min, beginning 2.5 h after the tracer protocol began. Breath samples were collected in disposable Haldane-Priestley tubes (Venoject; Terumo Medical) using a collection mechanism that allows the removal of dead-space air. The breath samples were stored at room temperature until ¹³C enrichment analysis. The arterialized blood samples were collected in heparinized tubes (31); then plasma was separated by centrifuging at 1500 x g for 10 min at 4°C. The plasma samples were stored at –20°C until ¹³C enrichment and concentration analyses.

    Analytical procedures. Expired ¹³CO₂ enrichment was measured by a continuous-flow isotope ratio MS (CF-IRMS20/20, PDZ Europa) and was expressed as atom percent excess (APE) against a reference standard of compressed CO₂ gas.

Plasma L-[1-¹³C]leucine enrichment was measured by a triple quadrupole mass spectrometer MS API 4000 (Applied Biosystems/MDS SCIEX) operated in positive ionization mode with a TurbolonSpray ionization probe source (operated at 5800 V and 600°C), which was coupled to an Agilent 1100 HPLC system (Agilent Technologies Canada). The butylated plasma samples were reconstituted in 0.1% formic acid. The individual components were separated using a Hypercarb^® column from Hypersil 5 µm, 4.6 x 50 mm column (Thermo Electron) at 50°C and eluted with a binary LC gradient (10–50% aqueous acetonitrile containing 0.025% formic acid and 0.05% trifluoroacetic acid in 5 min). The retention time was 1.2 min. Selected-ion chromatograms were obtained by monitoring the fragmentation of the protonated [M+H]⁺ molecule at m/z = 188 (leucine) and 189 ([¹³C]leucine) for precursor (parent) ion and at m/z 86 for product (daughter) ion.

Plasma [¹³C]- ketoisocaproic (-KIC) acid enrichment was measured by methane-negative chemical ionization GC-MS (Hewlett Packard 5890, GC; Hewlett Packard 5988A MS system). Plasma -KIC, an index of intracellular leucine enrichment, was derivatized to its pentafluorobenzyl ester based on an extractive derivatization process (32). A highly polar column [SP-2380 (0.20 µm x 0.25 mm x 30 m); Supelco] was necessary to separate KIC from the keto acid of isoleucine -keto-ß-methylvalerate, which has the same molecular weight as KIC. Selected-ion chromatograms were obtained by monitoring [M-H-PFB]^– ions at m/z = 129 for -KIC and 130 for [¹³C]-KIC. Because of technical difficulties, only the plasma samples from Part B were analyzed for [¹³C]-KIC enrichment.

Plasma free amino acid concentrations were determined by HPLC analysis. The plasma samples and norleucine (as the internal standard) were extracted using a cation exchange column (Dowex 50 W-X8, 100–200 mesh H⁺ form; Bio-Rad Laboratories). Plasma phenylalanine and tyrosine were derivatized with phenylisothiocyanate (PITC; adapted from PicoTag; Waters) (33–35) and their PITC derivatives were separated and analyzed against an amino acid standard mix (Sigma) by reversed-phase (C18, 2.9 mm x 300 mm Pico Tag column, Waters) column using HPLC (Dionex Summit HPLC System, Dionex, operated under HPLC pump model P580A LPG and UV/VIS 170S). The areas under the peaks were integrated using Chromeleon software (version 6.2; Dionex).

    Estimation of isotope kinetics. Whole-body leucine or KIC flux was calculated from the dilution of isotope in the body amino acid pool at isotopic steady state (36):

where Q is the rate of leucine or KIC flux [µmol/(kg·h)], i is the isotope infusion rate [µmol/(kg·h)], and E_i and E_p are the enrichments as mole fractions of the infused isotope (APE) and plasma leucine or KIC at isotopic plateau (APE).

The rate of leucine or KIC oxidation was calculated with the following equation (36):

where O represents leucine or KIC oxidation [µmol/(kg·h)] and F¹³CO₂ represents the rate of ¹³CO₂ released by leucine tracer oxidation [µmol/(kg·h)] calculated by the following equation:

where FCO₂ is the CO₂ production rate (mL/min), ECO₂ is the enrichment in expired breath at isotopic steady state (APE), the constants 44.6 µmol/mL and 60 min/h converted FCO₂ to µmol/h, W is the weight (kg) of the subject, the factor 0.82 is the correction for CO₂ retained in the body due to the bicarbonate fixation (37), and the factor 100 changes APE to a fraction.

    Statistical analysis. Estimates of the mean aromatic amino acid requirement intake were derived by breakpoint analysis of the rate of release of ¹³CO₂ (F¹³CO₂) data using the mixed procedure of SAS (version 8.2, SAS Institute) (38) (subject as a random effect) followed by a 2-phase linear regression crossover model as described previously (39). PROC MIXED was used to determine the effects of phenylalanine intake level on F¹³CO₂ (Tables 3 and 4), leucine flux, KIC flux, leucine oxidation, KIC oxidation, and plasma phenylalanine and tyrosine concentrations. The subject was included as a random effect in the model and the P-value was adjusted to the Tukey test for multiple comparison. Because plasma concentration of a test amino acid responds to the intake level of that test amino acid and the test amino acid in our previous study (21) was also phenylalanine, the data on plasma concentrations of phenylalanine and tyrosine in both parts of the present study and the previous study (21) were pooled. The statistical difference between the breakpoints determined in the previous study (21) and the present study was calculated using the comparison of a 2-sample t procedure (40). Differences were considered significant at P < 0.05.

	RESULTS

TOP ABSTRACT SUBJECTS AND METHODS RESULTS DISCUSSION LITERATURE CITED

View larger version (17K): [in this window] [in a new window]   FIGURE 1  Plasma phenylalanine and tyrosine concentrations in healthy men after 8 h of feeding. Data were combined from the 2 parts of the study and the study of Hsu et al. (21). Values are means ± SD, n = 15 for phenylalanine intake = 5, 15, 25, 35, and 45 mg/(kg·d); n = 10 for phenylalanine intake = 10 and 60 mg/(kg·d); n = 5 for phenylalanine intake = 55 and 65 mg/(kg·d); n = 4 for phenylalanine intake = 70 mg/(kg·d). Means for an amino acid without a common letter differ, P < 0.05.

    Data evaluation. The ideal IAAO pattern of the partition of any indispensable amino acid between oxidation and protein synthesis did not occur with the higher daily leucine intake in Part A (Fig. 2), but was apparent at the lower daily leucine intake in Part B (Fig. 3), except at the lowest phenylalanine intake, 5 mg/(kg·d). In Part A, the F¹³CO₂ remained steady and was reduced only when the phenylalanine intake was >35 mg/(kg·d). In Part B, when the data for 5 mg/(kg·d) of phenylalanine intake were omitted, phenylalanine intake still tended to affect F¹³CO₂ (P = 0.06) and did not significantly affect leucine flux, KIC flux, leucine oxidation, and KIC oxidation. The mean total aromatic amino acid requirement determined by the 2-phase linear regression model at the lower leucine intake was estimated to be 41.9 mg/(kg·d); it was estimated to be 39.6 mg/(kg·d) when the data of the lowest phenylalanine intake [5 mg/(kg·d)] were omitted. When comparing the slope ratio analyzed with and without the data of 5 mg/(kg·d) phenylalanine intake, the breakpoints were very similar, even though dropping the 5 mg/(kg·d) data improved the P-value from 0.048 to 0.027, R² from 0.17 to 0.24, and the SE of the breakpoint from 16.0 to 8.7. Thus, the data of 5 mg/(kg·d) phenylalanine intake were kept and the mean total aromatic amino acid requirement was estimated to be 41.9 mg/(kg·d).

View larger version (14K): [in this window] [in a new window]   FIGURE 2  Effect of phenylalanine intake on production of 13CO2 from leucine oxidation in men at a leucine intake of 65 mg/(kg·d) in Part A. Values are all observations, n = 35, and means (–), n = 5.

View larger version (16K): [in this window] [in a new window]   FIGURE 3  Effect of phenylalanine intake on the production of 13CO2 from leucine oxidation in men at a leucine intake of 45 mg/(kg·d) in Part B. Values are all observations, n = 30, and means (–), n = 5. The phenylalanine intake between 2 regression lines represents the mean total aromatic amino acid requirement of 41.9 mg/(kg·d).

	DISCUSSION

TOP ABSTRACT SUBJECTS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The BCAA, leucine, isoleucine, and valine, have a common membrane transport system and a common enzyme for their transamination and oxidative decarboxylation steps in the catabolic pathways (42). Altering any one of BCAA in the diet influences the metabolism of the other 2 due to antagonistic effects among them (42). In the earlier IAAB studies by Kurpad et al. (13,14,16), the authors attempted using mean [40 or 50 mg/(kg·d)] and generous [93 mg/(kg·d)] leucine intakes in the studies to determine lysine requirements, but because only 2–4 test amino acid intake levels were studied, the effect of leucine intake in response to the test amino acid levels could not be statistically determined from the studies. From Figures 2 and 3, it is clear that the level of leucine intake affected the pattern of response. In Part A, at 65 mg/(kg·d) leucine intake, the expected and ideal pattern for the IAAO method, linear change in partitioning between oxidation and protein synthesis with increasing intake of the test amino acid was not observed. Intake of leucine in excess of requirement appears to have made leucine oxidation insensitive as an indicator. Valine was shown to be the most limiting BCAA amino acid in the proportion of egg protein in humans (43), and BCAA intake was given at its egg protein ratio in the first part of the study; thus, valine may also have influenced the leucine response to the test amino acid.

The daily leucine intake in Part B was close to the mean requirement, 45 mg/(kg·d), and isoleucine and valine intakes were given at their ratio in egg protein to avoid their potential for interference with leucine metabolism. In Figure 3, the ideal IAAO pattern could be observed except at the lowest phenylalanine intake level, 5 mg/(kg·d). Leucine oxidation at a phenylalanine intake of 5 mg/(kg·d) was less than leucine oxidation at 10 mg/(kg·d). We reason that the plateau in leucine oxidation at a phenylalanine intake of 5 mg/(kg·d) was due to an inability to oxidize the large excess of leucine (44). Although dropping the data of 5 mg/(kg·d) phenylalanine intake improves the best-fit model for breakpoint analysis, the estimated breakpoint was similar to that from the data that included all of the data points. Hence we chose the model that included the data of 5 mg/(kg·d) phenylalanine intake. At a leucine intake of 45 mg/(kg·d), the mean requirement of the total aromatic amino acids for healthy men was estimated to be 42 mg/(kg·d), which did not differ from the requirement determined from our previous study (21) using lysine as the indicator, 44 mg/(kg·d) (P = 0.30). In our previous study using lysine tracer as indicator (21), the clear ideal IAAO pattern was observed. In comparing the slope ratio analyzed between the studies using lysine (21) and leucine as the indicator, the best-fit model in the study using lysine as the indicator gave a better statistical outcome; for example, R² and the P-value were 0.29 and 0.002, respectively, compared with 0.17 and 0.048, respectively, in the present study.

The F¹³CO₂ (¹³C-lable oxidation) is the direct measurement of the end product (CO₂) in leucine catabolism, and leucine oxidation was calculated from plasma [1-¹³C]leucine or KIC enrichment in addition to F¹³CO₂. Although the leucine oxidation data follow a pattern similar to that of the F¹³CO₂ data, variance in the oxidation data was large enough that a breakpoint could not be defined by 2-phase linear crossover analysis. However, we were able to define a breakpoint in the F¹³CO₂ data. Similar observations were also made in the other IAAO studies of lysine requirement in men (45), tryptophan requirement in women (46), phenylalanine requirement in children with classical phenylketonuria (47), and aromatic amino acid requirements in men (21). Plasma may not always represent the intracellular pool from which amino acid oxidation takes place (47); thus, a breakpoint may not be detected in the oxidation of the indicator amino acid, despite finding one with F¹³CO₂.

Kurpad et al. (13–20) used extensively leucine as an indicator amino acid in the 24-h IAAB method. In that method, only 2–4 test amino acid levels were studied initially (13–16) raising questions regarding the statistical confidence in the requirement estimates. In later experiments (17–19), the test amino acid intakes increased to 7 levels; however, each subject did not participate in all of the intake levels. Thus, the important issue of subject variability could not be taken fully into account. The ideal IAAO pattern was not always observed in these studies. For example, in the study of determining daily methionine requirements of healthy Indian men (20), a sigmoid pattern was observed instead of the ideal pattern of the IAAO. The ¹³CO₂ responses to phenylalanine intakes at the higher leucine intake in the present study (Part A, Fig. 2) had a similar sigmoid pattern. When the test amino acid was limiting for protein synthesis and leucine was in excess of requirement, leucine was not sensitive enough to respond to increasing phenylalanine intake.

Plasma amino acid response is another one of the approaches used to determine the requirement for individual amino acids. In our previous study (21), a 2-phase response was not observed in either phenylalanine or tyrosine plasma concentrations with increasing phenylalanine intake. When the data from the present study and the data from the previous study (21) were pooled, a two-phase response was still not observed. This is further evidence that the plasma phenylalanine or tyrosine concentrations cannot be used to determine their requirements in adult humans.

Leucine is not only incorporated into protein, but also plays a role in the regulation of protein metabolism (9,10) and hormonal secretion (11,12). Leucine stimulates protein synthesis in the skeletal muscles by increasing availability of eukaryotic initiation factor (eIF) 4E, which is associated with enhanced phosphorylation of 4E binding protein 1 (4E-BP1) and activation of ribosomal protein S6 kinase (S6K1) (48,49). The mechanism of hyperphosphorylation of 4E-BP1 and S6K1 by leucine is via a signal pathway involving mammalian target of rapamycin protein kinase (49–51). In addition, dietary leucine enhances protein metabolism by reducing muscle protein breakdown (9). BCAA, especially leucine, were shown to stimulate insulin secretion (11,12,52) and may also inhibit glucagon secretion (12). Garlick and Grant (52) found that leucine enhances the sensitivity of insulin during feeding in the rat model, which then stimulates muscle protein synthesis. Leucine, however, has significant effects on intermediate metabolism other than oxidation to CO₂ and incorporation into protein (53–55). Whether the results of the metabolic effects of leucine during in vitro and animal studies can be applied directly to humans in vivo is not clear, but it is clear that leucine is a much less suitable choice as an indicator than phenylalanine or lysine. Thus, leucine does not fulfill the third criteria, mentioned previously, for choosing an appropriate indicator amino acid.

In summary, the total aromatic amino acid requirements for healthy men using leucine as the indicator amino acid were estimated to be 42 mg/(kg·d), which was not significantly different from the requirements determined previously [44 mg/(kg·d)] by using lysine as the indicator amino acid. For the reasons discussed above, we conclude that leucine is not an ideal indicator amino acid and is less suitable than either phenylalanine or lysine. However, leucine may be a usable indicator when it is fed at the mean requirement and due consideration is given to its other potential metabolic effects.

	ACKNOWLEDGMENTS

 
We thank Karen Chapman for coordinating the activity in the Clinical Investigation Unit of the Hospital for Sick Children and Linda Chow for preparing the protein-free cookies. We are also grateful to Mead Johnson Nutritionals (Canada) for providing the protein-free powder for the experimental diet.

	FOOTNOTES

 
¹ Presented in part in abstract form [Hsu JWC, Rafii M, Kriengsinyos W, Ball RO, Pencharz PB. Is leucine a suitable indicator amino acid (abstract)? FASEB J. 2004;18: 370.6]. Experimental Biology: 04; Washington DC.

² Supported by the Canadian Institute of Health Research (grant MOP-10321).

⁴ Abbreviations used: -KIC, -ketoisocaproic; BCCA, branched-chain amino acid; 4E-BP1, 4E binding protein 1; eIF, eukaryotic initiation factor; IAAO, indicator amino acid oxidation; IAAB, indicator amino acid balance; S6K1, S6 kinase 1.

Manuscript received 8 November 2005. Initial review completed 6 December 2005. Revision accepted 28 December 2005.

	LITERATURE CITED

TOP ABSTRACT SUBJECTS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

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