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Nutrient Requirements

The Total Branched-Chain Amino Acid Requirement in Young Healthy Adult Men Determined by Indicator Amino Acid Oxidation by Use of L-[1-¹³C]Phenylalanine

Roya Riazi^*,, Linda J. Wykes^**, Ronald O. Ball^*, and Paul B. Pencharz^*,,,,3

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

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

	ABSTRACT

TOP ABSTRACT SUBJECTS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Previous recommendations for branched-chain amino acids (BCAA), based on nitrogen balance studies, were found to be low in a series of stable isotope–labeled amino acid studies. The BCAA requirement was increased in the new dietary reference intake (DRI) report on the basis of a series of stable isotope studies examining the requirement of leucine and valine individually, but not isoleucine. To reduce the possibility of interactions among these amino acids and imbalances in the mixture affecting the estimate of requirements, we decided to determine the requirement for the total BCAA of young healthy adult men, receiving a mixture of BCAA based on the proportion of these amino acids in egg protein, by use of indicator amino acid oxidation. Seven men were assigned to receive nine graded intakes of a BCAA mixture in random order: 34, 50, 66, 80, 100, 120, 140, 160 and 180 mg/(kg · d). The rate of release of ¹³CO₂ from the oxidation of L-[1-¹³C]phenylalanine (F ¹³CO₂) was measured and a two-phase linear regression crossover model was applied to determine total BCAA requirement. The mean requirement and population-safe level (upper limit of 95% confidence interval) of the total BCAA were 144 and 210 mg/(kg · d), respectively. Based on the balance of BCAA in egg protein, our estimate for the mean leucine requirement is 55 mg/(kg · d), which is substantially higher than the 34 mg/(kg · d) recommended by the DRI.

KEY WORDS: • amino acid requirements • branched-chain amino acids • indicator amino acid oxidation

The indispensable amino acid requirement values for human adults have been established from nitrogen balance studies by Rose in males (1 ) and by Leverton in females (2 ), and are the basis of the current FAO/WHO/UNU 1985 (3 ) recommendation for amino acid intake. However, these values have been shown to be underestimated for most amino acids by a series of stable isotope–labeled amino acid studies (4 –8 ), and in particular the requirements for leucine (9 ,10 ) and valine (11 ) were too low. Based on these studies the dietary reference intake (DRI) committee on protein and amino acid requirements increased the estimated average requirement (EAR) for leucine and valine (12 ).

The branched-chain amino acids (BCAA) leucine, isoleucine and valine are among the nine dietary indispensable amino acids for humans. BCAA accounts for 35–40% of the dietary indispensable amino acids in body protein and 14% of the total amino acids in skeletal muscle (13 ). They share a common membrane transport system and enzymes for their transamination and irreversible oxidation (14 –16 ). Administrations of individual BCAA to humans, either orally or intravenously, cause differential responses among the three BCAA (14 ). Interactions among BCAA can cause changes in plasma and tissue BCAA pools [e.g., high intakes of leucine decreased valine and isoleucine concentration in blood and muscle (14 ,17 ,18 ) and elevated rates of valine oxidation (19 )]. In contrast, Pelletier et al. (20 ,21 ) examined the effect of different leucine intakes on the oxidation of valine in adult men, and whether the oxidation of leucine and its requirement were affected by different dietary intakes of valine and isoleucine. They concluded that within the range of amino acid intakes that were studied, changes in either valine and/or isoleucine intake do not affect the leucine requirement, and changes in leucine intakes at the physiological range do not affect valine oxidation.

A concern about the investigation of amino acid requirements is that, by changing the intake of a single test amino acid, the dietary mixture may become imbalanced (22 ); this appears to be particularly important in the case of BCAA. Therefore, we designed a study to reduce the possibility of interactions among these amino acids and imbalances in the mixture affecting the estimate of requirements. We decided to study total BCAA requirement in healthy adult men, with a BCAA pattern based on the proportion of these amino acids in a reference protein (egg) and by use of the indicator amino acid oxidation (IAAO) technique, in response to graded intakes of the total BCAA.

	SUBJECTS AND METHODS

TOP ABSTRACT SUBJECTS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
IAAO is a functional method based on the concept that the amount of the limiting amino acid governs the partition of any indispensable amino acids between retention for protein synthesis and oxidation. In the current study the indicator amino acid used was L-[1-¹³C]phenylalanine and our test amino acid was a mixture of the three BCAA.

Subjects.

Seven young healthy adult male volunteers (mean age ± SD = 26.1 ± 6.6) were assigned to receive nine intakes of the BCAA mixture in random order. Characteristics of the men who participated in the study are summarized in Table 1. There was no history of weight loss, unusual dietary habits, endocrine disorders or use of any kind of medication or hormonal treatment. Each subject was told the purpose of the study and the possible risks involved, and written consent was obtained. All men received financial compensation for their participation in the study. All procedures used in the study were approved by the University of Toronto Human Experimental Committee and the Human Subjects Review Committee of The Hospital for Sick Children (HSC).

The study design was based on the noninvasive IAAO model of Bross et al. (23 ). Each study consisted of a 2-d adaptation period to a prescribed diet (24 ) which provided 1 g protein/(kg · d). Our initial design was for the men to receive dietary intake levels of a BCAA mixture of 26, 34, 50, 66, 80, 100 and 120 mg/(kg · d). However, analysis of ¹³CO₂ enrichment in breath samples obtained from our first adult man (Subject 1) suggested that we might need higher intakes. Thus the design was adjusted so that the test intake of 26 mg/(kg · d) was removed and test intakes of 140 and 160 mg/(kg · d) of the BCAA mixture were added, therefore ensuring sufficient BCAA intakes for estimate of requirement. We also tested 180 mg/(kg · d) BCAA intake on three men. The distribution of all men across the levels of intake is presented in Table 3. All men received different BCAA intakes in random order.

The energy intake for men was determined by measuring their resting metabolic rate (RMR) after a 12-h overnight fast by use of open-circuit indirect calorimetry (2900 Computerized Energy Measurement System; Sensormedics, Yorba Linda, CA). The RMR was multiplied by an activity factor of 1.7 to ensure weight maintenance for individuals during the short-term amino acid oxidation studies (Table 1) (7 ,25 ). All men maintained their ordinary level of activity throughout the study periods.

Dietary intake during the 2-d adaptation period was a milkshake diet (Scandishake; Scandipharm, Birmingham, AL) supplemented with additional protein (Promod; Ross Laboratories, Columbus, OH) and calories (Caloreen; Nestle Clinical Nutrition, North York, Canada) to meet each subject’s requirements. All men were told to add a predetermined volume of homogenized milk containing 3.25% fat to their daily portions and drink the milkshake at regular meal times during the day. No other food or beverages, including products containing artificial sweeteners, were consumed during the adaptation period.

Our BCAA mixture was based on the proportion of these amino acids in egg protein: 38.5, 32.5 and 29% for leucine, valine and isoleucine, respectively. The lowest level studied was 34 mg/(kg · d), representing the FAO/WHO/UNU recommendation for total BCAA, which was based on nitrogen balance studies (1 ,26 –28 ); the next levels, 50 and 66 mg/(kg · d), were based on the Millward’s recalculation of nitrogen balance studies were by taking into account the miscellaneous losses (29 ). The midpoints, 80 and 100 mg/(kg · d), were based on direct amino acid oxidation (DAAO) studies for leucine and valine (9 –11 ), a 24-h tracer balance for leucine (30 ) and the Young et al. tentative set of requirements for BCAA (4 ,5 ). The higher levels were 120, 140, 160 and 180 mg/(kg · d) to ensure that we reached a plateau above the requirement breakpoint. The experimental diet was based on an amino acid mixture developed for amino acid kinetic studies (31 ). The nitrogen content of the diet was supplied at the level of 1 g protein/(kg · d) and was provided as an L-amino acid mixture based on the amino acid profile of egg protein. The experimental diet included 15 mg/(kg · d) phenylalanine, to ensure adequate dietary phenylalanine, as previously determined by amino acid oxidation studies when tyrosine was present in relative excess at about 40 mg/(kg · d) (8 ). The main source of energy in the experimental diet was a flavored liquid protein-free formula (Protein-Free Powder, product 80056, Mead Johnson, Evansville, IN; Tang and Koolaid, Kraft Foods, Toronto, Canada). The remainder of the energy requirement was provided by protein-free cookies (31 ). The experimental diet provided 53% of total energy from carbohydrate, 10% of total energy from protein and 37% of total energy from fat. The protein-free formula provided 65% and the cookies about 25% of total daily energy intake. All the diets were prepared and weighed (scale model PE2000, Mettler, Nanikon, Switzerland) in the HSC research kitchen and consisted of nine hourly isonitrogenous, isocaloric meals that differed in total BCAA content on each study day. Serine and glycine were given to keep the meals isonitrogenous at different total BCAA intakes. Each meal contained a bottle of flavored protein-free formula to which the amino acid mixture was added, plus two protein-free cookies. Although the protein-free formula had a complete mixture of vitamins and minerals, an additional multivitamin supplement (Centrum; Whitehall-Robins Inc., Mississauga, Canada) was given to all men for the entire period of all the studies to ensure adequate vitamin B intake.

Body-composition measurements.

Men were weighed each morning on a balance scale (model 2020; Toledo Scale, Windsor, Canada) to the nearest 0.1 kg after voiding. Standing heights were measured to the nearest 0.1 cm with the wall-mounted stadiometer on the prestudy day. Multiple skinfold thicknesses (triceps, biceps, subscapular and suprailiac) were measured before the study day to the nearest 1 mm with a Harpenden caliper (British Indicators Ltd., St. Albans, UK) to estimate fat mass and fat-free mass, by subtraction from body weight (32 ). Bioelectrical impedance analysis (33 ,34 ) was performed on fasting subjects on the prestudy day by use of a fixed-frequency analyzer (50 KHz) (BIA, model 101A; RJL Systems, Detroit, MI). Resistance (R) and reactance (X_C) measurements were made by use of a four-terminal bioelectrical impedance analyzer. Three readings of both R and X_C (in ) were taken for each subject and equations were used to determine the lean body mass (34 ).

Isotope infusion studies.

The stable isotope tracers used in these studies were as follows: NaH¹³CO₃ (Cambridge Isotope Laboratories, Woburn, MA) and L-[1-¹³C]phenylalanine (Mass Trace, Woburn, MA) with a 99 atom %. Isotopic and optical purity of L-[1-¹³C]phenylalanine was verified by the manufacturer through the use of chemical ionization gas chromatography–mass spectrometry (GC-MS) and nuclear magnetic resonance. The enrichment and enantiomeric purity of the L-[1-¹³C]phenylalanine tracer were reconfirmed by GC-MS of the N-heptafluorobutyryl (HFB) n-propyl ester derivative (35 ) by use of a chiral column (ChirasilVal; Alltech Associates, 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. Tracer solutions were prepared in deionized water and stored at -20°C. The subjects consumed four hourly meals in the morning of the study day; with the 5th meal, the subjects were given a priming oral dose of NaH¹³CO₂ (2.07 µmol/kg) and a priming oral dose of L-[1-¹³C]phenylalanine (3.99 µmol/kg), with a constant infusion of L-[¹³C]phenylalanine (7.22 µmol/kg) thereafter with each hourly meal until the end of the study. The amount of dietary phenylalanine in the last five meals was reduced by an amount that corresponded to the amount of L-[1-¹³C]phenylalanine given orally during the tracer infusion so that the phenylalanine intake remained unchanged.

Sample collection.

It was previously shown in our lab that plasma amino acid enrichments can be determined from urine (23 ,36 ,37 ). Three baseline samples of breath and urine were collected at 60, 45 and 30 min before the isotope was given orally. A background isotopic steady state was achieved in breath ¹³CO₂ within 4 h of the start of the feeding. Isotopic steady state was achieved in both breath and urine by 120 min after the initiation of the isotope protocol and was maintained to the end of the study at 270 min. Five plateau breath and urine samples were collected by sampling every 30 min during a period of 150–270 min after the start of the isotope protocol.

A 5-mL blood sample was also taken from each subject at the end of each study day to determine the profile of the amino acids in the plasma. The blood samples were kept on ice until centrifugation at 1200 x g for 10 min at 4°C. Plasma was stored at -20°C until analysis by HPLC (Dionex Summit HPLC System; Oakville, ON, Canada). Urine samples stored at -20°C, and breath samples were collected in disposable Halden-Priestly tubes (Venoject; Terumo Medical Corp., Elkton, MD) with use of a collection mechanism that allows the removal of dead-air space (38 ). Samples were stored at room temperature until analysis. In addition to collecting breath samples, indirect calorimetry (2900 Computerized Energy Measurement System) was carried out to determine the carbon dioxide production rate for 30 min, after 4 h of consuming the experimental diet on each study day.

Analytical procedure.

Expired ¹³CO₂ enrichment was measured by a continuous flow isotope ratio mass spectrometer (model 20/20, PDZ Europa Ltd., Cheshire, U.K.) and was expressed as atom percent excess against a reference standard of compressed CO₂ gas. Urinary L-[1-¹³C]phenylalanine enrichment was measured by the modified method of Patterson et al. (35 ) by use of negative chemical ionization GC-MS with a chirasil (Val-D) fused-silica capillary column (Alltech Associates). Briefly, a 1-mL urine sample was deproteinized and acidified with 500 µL of 2.5 mol/L trichloroacetic acid and centrifuged at 7000 x g. Amino acids were separated from the supernatant by use of cation-exchange columns (Dowex 50 W-X8, 100–200 mesh H⁺ form; Bio-Rad Laboratories, Hercules, CA). The eluate from the column was freeze-dried (Freezone 12 L; Labconco Corp., Kansas City, MO) before amino acids were derivatized to their HFB n-propyl ester derivatives. By use of methane-negative chemical ionization GC-MS (GC system: Hewlett Packard 5890 series; MS system: Hewlett Packard 5988A, 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. Isotope enrichment in molecule percent excess was calculated from the peak area ratios at isotopic steady state and baseline.

Plasma-free amino acids were separated by use of a cation exchange column, as mentioned earlier, with norleucine as an internal standard. Plasma BCAA, tyrosine and phenylalanine concentrations were determined by reverse-phase HPLC technique (Dionex Summit HPLC system; Dionex, Sunnyvale, CA; operated under HPLC pump model P580A LPG and UV/VIS Detector UVD 170S) by use of a precolumn derivatization with phenylisothiocyanate (adapted from Pico Tag; Waters, Milford, MA) (39 –41 ). The areas under the peaks were integrated by use of Chromeleon software (version 6.2), provided by the Dionex Summit HPLC system (Dionex, Oakville, ON, Canada).

Isotope kinetics.

The model used to evaluate phenylalanine kinetics was described by others (7 ,8 ,42 ) using a constant-infusion approach to study amino acid oxidation. The isotopic steady state in the metabolic pool was represented by plateaus in ¹³CO₂ enrichments in breath and L-[1-¹³C]phenylalanine enrichments in urine. A plateau was defined as a CV < 5% and the absence of a significant slope. The difference between mean breath ¹³CO₂ enrichments of the three baseline and five plateau samples was used to determine atom percent excess above baseline at isotopic steady state.

Phenylalanine flux [µmol/(kg · h)] was calculated from the dilution of L-[1-¹³C]phenylalanine at isotopic steady state, as represented by urinary phenylalanine by use of standard equations (8 ). The rate of ¹³CO₂ production from L-[1-¹³C]phenylalanine oxidation [F ¹³CO₂ in µmol ¹³CO₂/(kg · h)] was calculated according to the model of Matthews et al. (42 ) from ¹³CO₂ expiration, by use of a factor of 0.82 to account for the ¹³CO₂ retained in the body, in the fed state, attributed to bicarbonate fixation (43 ). The rate of L-[1-¹³C]phenylalanine oxidation [µmol/(kg · h)] was calculated from F ¹³CO₂ and urinary phenylalanine enrichment (7 ,42 ).

Statistical analysis.

A three-factor general linear model was performed to assess the relationship of F ¹³CO₂, phenylalanine flux and phenylalanine oxidation to the following variables: total BCAA intakes, order of intakes, subjects and interactions through use of SAS statistical software (SAS, version 8.0; SAS Institute Inc., Cary, NC). In all cases, differences were considered significant at P < 0.05.

Estimation of the mean and safe total BCAA intakes for the healthy adult male population was derived by breakpoint analysis through use of a two-phase linear regression crossover model, similar to the previously described method in animal and human studies using IAAO (7 ,44 ). The 95% confidence interval for the mean total BCAA requirement was determined by use of Fieller’s theorem (45 ). The total BCAA requirements for each individual subject was determined by visual inspection of the breakpoint from the phenylalanine oxidation curves. The effect of total BCAA on plasma BCAA, phenylalanine and tyrosine concentrations was evaluated by a repeated-measures ANOVA, followed by Student–Newman–Keuls post hoc test using SAS software (version 8.0). ANOVA with repeated measures, followed by Student–Newman–Keuls post hoc test, was also used to determine the effect of individual and total BCAA intake on nonoxidative phenylalanine disposal (NOPD), phenylalanine release from proteolysis (B_phe) and phenylalanine balance using SAS software (version 8.0).

	RESULTS

TOP ABSTRACT SUBJECTS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Characteristics and energy intake of men who participated in the study are outlined in Table 1. Weight, lean body mass and fat-free mass did not change over the experimental period. Plasma BCAA concentration (Table 2)increased with increasing total BCAA intake (P < 0.0001). Conversely, levels of phenylalanine and tyrosine decreased with the increase of total BCAA, presumably because of increased incorporation of these amino acids into protein synthesis as the requirement for BCAA was reached. The distribution of subjects across the intake levels and their individual label oxidation are presented in Table 3. Visual inspection of individual breakpoint from label oxidation (F ¹³CO₂) determined total BCAA requirement to be 100, 100, 80, 140, 120 and 140 mg/(kg · d) for subjects 1, 2, 3, 4, 5 and 6, respectively. Because subject 7 did not complete all the test intakes of total BCAA, we were unable to estimate a requirement. The rate of the release of ¹³CO₂ varied between the subjects (P < 0.05), although the overall response pattern was consistent. The order of total BCAA intake did not affect the ¹³CO₂ production rate. The effect of total BCAA intakes on oxidation of L-[1-¹³C]phenylalanine was determined from the rate of release of ¹³CO₂ (F ¹³CO₂) (Fig. 1 ). The two regression lines represent the partitioning of data points that minimized the total error and provided the best fit. The breakpoint analysis from the production of labeled carbon dioxide in breath (F ¹³CO₂) provided a mean requirement of 144 mg/(kg · d) and a safe-population intake of 210 mg/(kg · d). The mean L-[1-¹³C]phenylalanine flux, phenylalanine oxidation, NOPD, phenylalanine release from proteolysis (B_phe) and phenylalanine balance are given in Table 4. Mean phenylalanine flux was not affected either by total BCAA intake or by order of test total BCAA intake. Total BCAA intake did not have a significant effect on phenylalanine flux (P > 0.28), NOPD (P > 0.67) or phenylalanine release from proteolysis (P > 0.57). Flux was significantly different among individuals (P = 0.0001). Oxidation rates were not different among the individuals (P = 0.059), however, BCAA intake had a significant effect on phenylalanine oxidation and phenylalanine balance (P = 0.01). By use of the two-phase linear regression crossover model, breakpoint analysis provided a mean total BCAA requirement of 125.7 mg/(kg · d), from the oxidation of L-[1-¹³C]phenylalanine and a safe-population intake of total BCAA of 170.7 mg/(kg · d) (Fig. 2 ). The total BCAA requirement was also estimated by use of the two-phase linear regression crossover model, from phenylalanine balance (Table 5).

View larger version (16K): [in this window] [in a new window]   FIGURE 1 Effect of total BCAA intake on oxidation of L-[1-13C]phenylalanine determined from the rate of release of 13CO2 (F 13CO2), at each of the nine levels of total BCAA intakes for all seven men (n = 53 observations). Values are mean oxidation rate ± SEM. The total BCAA intake between two regression lines represents the mean total BCAA requirement of 144 mg/(kg · d).

View larger version (15K): [in this window] [in a new window]   FIGURE 2 Effect of total BCAA intake on oxidation of L-[1-13C]phenylalanine determined from urinary enrichment of 13C-phenylalanine, at each of the nine levels of total BCAA intakes for all seven men (n = 52 observations). Values are mean oxidation rate ± SEM. The total BCAA intake between two regression lines represents the mean total BCAA requirement of 126 mg/(kg · d).

	DISCUSSION

TOP ABSTRACT SUBJECTS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The total BCAA requirement has not been estimated by DAAO. Estimates of the requirement for BCAA using stable isotopes have been limited to several studies of leucine (9 ,10 ,20 ,30 ,47 ) and one study of valine (11 ). However, to date there is no experimental data for the isoleucine requirement by use of carbon oxidation. Nitrogen balance studies estimated the leucine requirement to be 14 mg/(kg · d) and the valine and isoleucine requirements to be 10 mg/(kg · d) each, thus estimating the total BCAA requirement to be 34 mg/(kg · d) (1 ,26 –28 ). Millward (29 ) estimated the leucine requirement to be 26 mg/(kg · d) based on his recalculation of the Hegsted (48 ) data, using a value of 5 mg N/(kg · d) for miscellaneous losses and a body weight of 60 kg. However, studies by Meguid et al. (9 ) for leucine and for valine (11 ) estimated the leucine requirement to be in the range of 30–40 mg/(kg · d) and valine to be 20 mg/(kg · d). Corteilla et al. (10 ) studied leucine requirement and estimated it to be >30 and 40 mg/(kg · d). El-Khoury et al. (30 ), using 24-h tracer balance studies, also estimated the leucine requirement to be 40 mg/(kg · d). A more recent study by Kurpad et al. (47 ), using a 24-h leucine balance method in an Indian population, estimated the requirement for leucine to be 40 mg/(kg · d). Therefore, all these studies suggest that the current requirements for leucine and valine, based on nitrogen balance studies, are too low. Recently, the DRI committee on protein and amino acid requirements derived an estimated average requirement (EAR) for leucine and valine from stable isotope studies to be 34 and 19 mg/(kg · d), respectively (12 ).

By use of the IAAO technique, we determined the total BCAA requirement to be 144 mg/(kg · d) and the mean leucine requirement to be 55.4 mg/(kg · d), which is higher than the estimate of leucine from previous stable isotope studies. Therefore, it is worthwhile to compare the leucine requirement estimated from our study with the others in the literature. Meguid et al. (9 ) was the first to determine the leucine requirement using carbon oxidation techniques. Although a wide range of leucine intake [eight levels of leucine from 4 to 79 mg/(kg · d)] was studied, each subject received only four to five levels of leucine intake. In addition, the estimation of leucine oxidation was from plasma leucine enrichment rather than from -ketoisocaproate (KIC). Leucine-based oxidation estimates are lower than KIC-based oxidation estimates (46 ); when corrections were made to KIC-based oxidation, the leucine balance at 79 mg/(kg · d) intake (the highest level that was given) would approach equilibrium rather than a marked positive balance value (9 ). Thus their study does not allow a statistically valid estimate of leucine requirement. The study of Cortiella et al. (10 ) was designed primarily to compare intravenous and intragastric tracers. Measurements were made at only four levels of leucine intake, and none was higher than 40 mg/(kg · d). The results for tracer balance showed that at 40 mg/(kg · d), two of five men were in a negative daily leucine balance at 40 mg/(kg · d) and three of five men were in a negative daily leucine balance at 30 mg/(kg · d). Because the study involved very few men and levels of intake (five men and four levels of leucine intake) and the highest intake did not exceed the requirements of all men, a statistically valid estimate of requirement was not possible. El-Khoury et al. (30 ) studied a leucine intake of 38.3 mg/(kg · d), and reported that all subjects were in a negative leucine balance, approximating an amount equivalent to 16% of dietary leucine intake. They (30 ) predicted that leucine balance would occur when the leucine intake was 50–55 mg/(kg · d). In the recent study of Kurpad et al. (47 ) with Indian adult men, each subject was studied at two levels of leucine intake (10 men at two levels of intake) and concluded that the requirement was 40 mg/(kg · d); however, the highest level studied was 40 mg/(kg · d), which does not allow the determination of whether the leucine requirement is any higher than 40 mg/(kg · d).

The minimally invasive IAAO method allowed us to study a wide range of total BCAA, from a very low level to demonstrable excess intakes. Each adult man was studied on at least six levels of intake; the large number of observations enabled us to derive a valid estimate of requirement. Additional strengths of this study were the use of a functional method based on an independent indispensable amino acid’s response to the test levels of total BCAA, the oral administration of the test amino acid and the tracer, and the fact that we used a mixture of total BCAA based on the proportion of these amino acids in a reference protein to reduce the possible interaction between the three.

The reported interactions between the BCAA (14 ,17 –19 ) and the criticism that changing the test amino acid may make the dietary mixture imbalanced (22 ) must be considered in the case of BCAA requirement studies. We believe that it is reasonable to begin by estimating the requirement of total BCAA with proportions that approximate those in a reference protein such as eggs. However, the proportions in our BCAA mixture, which was based on egg protein, may not be optimal. In our next study we plan to estimate which of the BCAA is limiting in this mixture. If the BCAA proportions are not ideal, then the estimated total BCAA requirement and therefore the resulting calculated requirement for leucine may have been overestimated.

We also estimated the total BCAA requirement from different variables (phenylalanine balance, phenylalanine oxidation and the F ¹³CO₂) with and without the data points at the 180 mg/(kg · d) level, by use of the two-phase linear regression crossover model. The averaged total BCAA requirement calculated from all these estimations is 133.6 mg/(kg · d) or 134 mg/(kg · d), which is about 7% different from the total BCAA requirement estimated from the rate of release of ¹³CO₂ from ¹³C-phenylalanine oxidation (F ¹³CO₂) with all the data points.

We conclude that 144 mg/(kg · d) as the mean population requirement and 210 mg/(kg · d) as the population-safe intake, for total BCAA, were reasonable estimates. These are substantially higher than FAO/WHO/UNU (3 ) estimates of the requirement for the average total BCAA, which is 34 mg/(kg · d), and higher than the current DAAO and 24-h direct amino acid oxidation balance estimates for leucine.

	ACKNOWLEDGMENTS

 
We acknowledge the technical assistance of Mahroukh Rafii. We thank Karen Chapman for coordinating the activity in the Clinical Investigation Unit of The Hospital for Sick Children (HSC) and Linda Chow (Department of Nutrition and Food Services, HSC) for preparing the protein-free cookies. Special thanks to the subjects who participated in this study. We are also grateful to Mead Johnson Nutritionals (Canada) for providing the protein-free powder for the experimental diets and to Whitehall Robins Inc. (Canada) for providing the multivitamin supplements.

	FOOTNOTES

 
¹ Supported by the Canadian Institute of Health Research (grant MOP-10321).

² Presented in part in abstract form [Riazi, R., Ball, R. O. & Pencharz, P. B. (2001) Total branched chain amino acid requirement in young healthy adult men determined by indicator amino acid oxidation using L-[1-¹³C]phenylalanine. FASEB J. 15: A266, 234.7 (abs.)].

⁴ Abbreviations used: B_phe, phenylalanine release from proteolysis; DAAO, direct amino acid oxidation; DRI, dietary reference intake; EAR, estimated average requirement; F ¹³CO₂, rate of release of ¹³CO₂ from ¹³C-phenylalanine oxidation; GC-MS, gas chromatography-mass spectrometry; IAAO, indicator amino acid oxidation; KIC, -ketoisocaproate; NOPD, nonoxidative phenylalanine disposal; RMR, resting metabolic rate.

Manuscript received 5 December 2002. Initial review completed 8 January 2003. Revision accepted 28 January 2003.

	LITERATURE CITED

TOP ABSTRACT SUBJECTS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

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2. Leverton, R. M. (1959) Amino acid requirements of young adults. Albanese, A. A. eds. Protein and Amino Acid Nutrition 1959:477-506 Academic Press New York, NY. .

3. FAO/WHO/UNU (1985) Energy and protein requirements. Report of a joint FAO/WHO/UNU Expert Consultation 1985 Geneva, Switzerland. World Health Organization Technical Report Ser. 724.

4. Young, V. R., Bier, D. M. & Pellet, P. L. (1989) A theoretical basis for increasing current estimates of the amino acid requirements in adult man with experimental support. Am. J. Clin. Nutr. 50:80-92.[Abstract/Free Full Text]

5. Young, V. R. & El-Khoury, A. E. (1995) Can amino acid requirements for nutritional maintenance in adult human be approximated from the amino acid composition of body mixed proteins. Proc. Natl. Acad. Sci. U.S.A. 92:300-304.[Abstract/Free Full Text]

6. Young, V. R. & Marchini, S. (1990) Mechanism of nutritional significance of metabolic response to altered intakes of protein and amino acids with reference to nutritional adaptation in humans. Am. J. Clin. Nutr. 51:270-289.[Abstract/Free Full Text]

7. Zello, G. A., Pencharz, P. B. & Ball, R. O. (1993) The dietary lysine requirement of young adult males determined by the oxidation of an indicator amino acid, L-¹³C phenylalanine. Am J. Physiol. 264:E677-E668.

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