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Threonine requirements of healthy Indian men, measured by a 24-h indicator amino acid oxidation and balance technique^1,2,3

¹ From the Department of Physiology and the Division of Nutrition (AVK, TR, JV, BC, and DN) and the Department of Biochemistry (JG), St John’s Medical College, Bangalore, India, and the Laboratory of Human Nutrition, Massachusetts Institute of Technology, Cambridge, MA (MMR and VRY).

² Supported by the Nestlé Foundation, Switzerland, and by NIH grants RR88, DK 42101, and P-30-DK-40561.

³ Address reprint requests to AV Kurpad, Department of Physiology and Nutrition Research Center, St John’s Medical College, Bangalore, India. E-mail: a.kurpad{at}divnut.net.

	ABSTRACT

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Background: We previously questioned the validity of the 1985 FAO/WHO/UNU upper requirement value for threonine (7 mg · kg^-1 · d^-1) and proposed a tentative mean requirement of 15 mg · kg^-1 · d^-1.

Objective: In this study we used a 24-h indicator amino acid oxidation and balance technique, with [1-¹³C]leucine as the indicator amino acid, to assess threonine adequacy at 6 test intakes (7, 11, 15, 19, 22, and 27 mg · kg^-1 · d^-1) with a 6-d dietary adaptation phase in healthy, well-nourished Indian men.

Design: Sixteen men were randomly allocated to 3 of 6 test intakes and were studied after 6 d of adaptation to the experimental diets. Diets were based on an L-amino acid mixture in which the threonine content was varied. At 1800 on day 6, a 24-h intravenous [¹³C]leucine tracer infusion protocol was conducted to assess 24-h leucine oxidation and daily leucine balances.

Results: Leucine balances differed significantly (P = 0.02) between the different intakes of threonine. Two-phase linear regression analysis from 12-h and 24-h leucine oxidation and 24-h leucine balance gave a breakpoint at a threonine intake of 15 mg · kg^-1 · d^-1, with 95% CIs ranging from 11 to 27 mg · kg^-1 · d^-1. There was no significant effect of threonine intake on 24-h leucine flux.

Conclusion: The results of the 24-h indicator amino acid oxidation and balance experiments indicate that the current FAO/WHO/UNU threonine recommendation of 7 mg · kg^-1 · d^-1 is inadequate. A mean threonine intake of 15 mg · kg^-1 · d^-1 is sufficient to achieve the indicator (leucine) amino acid balance in healthy Indian men.

Key Words: Threonine requirements • indicator amino acid oxidation • indicator amino acid balance • leucine kinetics • healthy Indian men

	INTRODUCTION

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION REFERENCES

 
The 1985 FAO/WHO/UNU Expert Consultation set the upper requirement for threonine at 7 mg · kg^-1 · d^-1 for healthy men and women (1). This value was derived from nitrogen balance studies in men and women (2–4). We judged this value to be too low (5, 6) and proposed a mean threonine requirement of 15 mg · kg^-1 · d^-1 on the basis of estimates of the intakes of amino acids necessary to balance the minimum obligatory losses of amino acids as predicted from the composition of mixed body proteins. Furthermore, a study that used [¹³C]threonine as a tracer, followed by estimations of threonine oxidation and balance at different threonine intakes (7), also suggested that the mean requirement for threonine was in the range of 10–20 mg · kg^-1 · d^-1.

On the basis of a short-term (4-h) fed-state tracer study that used phenylalanine as the indicator amino acid in subjects who had not received a period of dietary adaptation to threonine intake before the study began, Wilson et al (8) proposed a mean threonine requirement value in adults of 19 mg · kg^-1 · d^-1. However, there is a complex diet-dependent, temporal pattern to the rate of amino acid oxidation and, therefore, also in amino acid use within a 24-h period (9–11). Hence we also conducted a study in adult US subjects (12). We used 24-h leucine oxidation and balance as indicators of threonine use and equilibrium, and our results supported the proposition that the mean threonine requirement was similar to that suggested by our previous studies, or 15 mg · kg^-1 · d^-1.

Because there are also concerns that the determination of amino acid requirements in white and US subjects may not be representative of global human amino acid requirement patterns, the aim of the present study was to determine threonine requirements in young, well-nourished Indian men, with the use of the 24-h indicator amino acid oxidation (IAAO) and balance (IAAB) approaches. The indicator amino acid used was [¹³C]leucine, and the tracer study was conducted over an entire 24-h period to include an examination of the response of the IAAO to feeding as well as the estimation of the daily IAAB.

	SUBJECTS AND METHODS

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Subjects
Sixteen young men participated in this experiment. All the subjects were recruited from the student population of St John’s Medical College, Bangalore, India. The physical characteristics of the subjects are given in Table 1 and were similar to subjects studied previously at this site (13, 14). All were in good health as determined by medical history, physical examination, analysis for blood cell count, routine blood biochemical profile, and urinalysis. Subjects who smoked cigarettes, consumed 5 alcoholic drinks/wk, or drank > 6 cups caffeinated beverages/d were excluded from participation in this study. The purpose of the study and the potential risks involved were explained to each subject. Written, informed consent was obtained from each subject. The Human Ethical Approval Committee of St John’s Medical College and the Massachusetts Institute of Technology Committee on the Use of Humans as Experimental Subjects approved the research protocol.

Diet and experimental design
All subjects were studied during 3 separate 7-day diet periods, during which they received a weight-maintaining diet based on an L-amino acid mixture (Table 2). The tracer experiments were carried out after a 6-d period of adjustment to the experimental diet. Daily energy intakes were designed to maintain body weight, and energy requirement was calculated to be 1.6 times the basal metabolic rate during the days of feeding and 1.35 times the basal metabolic rate on day 7 (tracer study day). The subjects were encouraged to maintain their customary levels of physical activity but were asked to refrain from excessive, or competitive, exercise. The major energy supply was given in the form of a sugar-oil formula and as protein-free wheat-starch cookies (Table 3). Nonprotein energy was provided as fat (43%) and carbohydrate (56%). The main source of carbohydrate was beet sugar and wheat starch, to attain a low ¹³C content in the diet so that a relatively steady background in breath ¹³CO₂ enrichment over the 24-h period could be obtained. Breath ¹³CO₂ enrichments obtained during the leucine-tracer studies were corrected to account for the small changes in background ¹³CO₂ output that would have occurred without the [¹³C]leucine tracer.

During the run-in experimental period, all other nutrients were provided in adequate amounts (Table 3). A choline supplement of 500 mg was given daily, and dietary fiber was provided in the form of 20 g isapgul (Charak Piramal Healthcare Ltd, Mumbai, India) when requested by the subject. The total daily food intake was consumed as 3 isoenergetic, isonitrogenous meals (at 0800, 1300, and 2000). Every morning, body weight and vital signs were monitored. All of the subjects’ meals were consumed at the kitchen of the Division of Nutrition, under the supervision of the dietary staff.

Twenty-four–hour tracer-infusion protocol
The primed tracer-infusion approach was used in this study, following a standard design in all subjects. The tracer administration began at 1800 on day 6 of each 7-d diet period, with the subjects having consumed their last meal of that day at 1500, and lasted until 1800 on day 7. The subjects received, at hourly intervals, 10 isoenergetic, isonitrogenous small meals from 0600 through 1500 (which together were equivalent to the 24-h dietary intake for that day). Indirect calorimetry was performed every other hour, with half-hourly withdrawals of blood samples for [¹³C]-ketoisocaproic acid (KIC) enrichment measurements. Throughout the 24-h study the subjects remained in bed in a reclining position, except during sleep, when they lay supine. This tracer design therefore divided the 24-h study into two 12-h metabolic periods, fasted and fed.

The primed, constant intravenous infusion of [1-¹³C]leucine (99.3 atom%; MassTrace, Woburn, MA) was given through a 20-gauge, 5-cm catheter placed into an antecubital vein on the nondominant side. Leucine was infused at a known rate of 2.8 µmol · kg^-1 · h^-1; the prime was 4.2 µmol/kg, and was administered over 1 min. The bicarbonate pool was primed with 0.8 µmol sodium [¹³C]bicarbonate/kg (99.9 atom%; MassTrace). The tracers were prepared in physiologic saline, under sterile conditions, and infused at 8 mL/h with the aid of a screw-driven pump (Model 919; Harvard Apparatus, Millis, MA).

Recovery of ¹³CO₂ and the contribution of dietary ¹³C to breath ¹³CO₂
Because the present study was conducted with diets that contained low amounts of ¹³C-enriched carbohydrate, the contribution to breath ¹³CO₂ from the experimental diet was expected to be low, although a correction was made for this small contribution of endogenous ¹³C-substrate oxidation over the 24-h study period, as described previously (13, 14).

The recovery of ¹³CO₂ in the breath was calculated for every 30-min interval, as described previously (13, 14), and values for each time point were used to correct each 30-min estimate of ¹³CO₂ production from oxidation of [1-¹³C]leucine (see the equations below).

Indirect calorimetry
Total carbon dioxide production (^·VCO₂) and oxygen consumption (^·VO₂) were determined with the aid of an open-circuit indirect calorimeter with a ventilated hood, as previously described (18). Minute-to-minute O₂ consumption and CO₂ production were thus obtained for each subject. Whole-system calibration was verified by combustion of pure ethanol, where the observed difference between measured and predicted total CO₂ production was < 3% and the average respiratory quotient was between 0.64 and 0.68. Measurements of respiratory exchange were made during alternate hours throughout the entire 24-h period.

Collection and analysis of breath samples
Three baseline breath samples were collected at -30, -15, and -5 min before the 24-h tracer infusion started, after which samples were collected at consecutive half-hourly intervals throughout the 24-h study except between 0600 and 1200, when hourly samples were collected, because this period corresponded to the time when the subjects were sleeping. Breath gas was collected in a specially designed bag, with a mechanism that permitted the removal of dead-space air, and was transferred into three 10-mL non-silicon-coated glass tubes (Vacutainer, Becton & Dickinson, Franklin Lakes, NJ) with a thin needle (PrecisionGlide, 24G; Becton & Dickinson) that was attached to the bag by means of a 3-way tap. At time intervals when the breath sample collection coincided with hourly meals, the breath sample was collected first. The samples were stored at room temperature until analyzed for their ¹³CO₂-¹²CO₂ ratio by isotope ratio mass spectrometry (Europa Scientific, Crewe, United Kingdom), as described previously (13, 14). The increase in breath enrichment after the administration of isotope was expressed as atom percent excess (APE). The APE was calculated by taking the arithmetic difference between enrichment of each breath sample and the predose basal breath sample.

Collection and analysis of blood samples
Blood samples were collected at 30-min intervals between 0000 and 2400 of the tracer-infusion period except between 0600 and 1200, when hourly samples were collected, because this period corresponded to the time when the subjects were sleeping. Three baseline samples were taken at -30, -15 and -5 min before administration of the [¹³C]leucine tracer. Blood sampling (5 mL/sample) was performed through a 20-gauge, 5-cm catheter placed into a superficial vein of the dorsal hand or wrist on the nondominant side. The catheter was introduced in an antiflow position to facilitate blood drawing while the hand was placed into a custom-made warming box, maintained at 65 °C, for 15 min before withdrawal of each sample to achieve arterialization of venous blood. The arterialization of the blood sample was checked earlier by measurement of hemoglobin saturation in the withdrawn blood; the saturation was found to be > 90%. The patency of the vein was maintained by slow infusion of normal saline. Blood samples were drawn into 5-mL syringes and transferred into anticoagulant tubes and centrifuged for 15 min at 1200 x g in a refrigerated centrifuge (4 °C). Plasma was removed, and samples were stored at -80 °C. They were later analyzed for plasma leucine and threonine concentrations by HPLC, with the use of a binary gradient on a 5-µm Luna C18 column (Phenomenex, St Torence, CA), and fluorescence detection (Shimadzu, Kyoto, Japan) with the o-phthalaldehyde and 2-mercaptoethanol derivative method (19). Homoserine was used as the internal standard. The linearity of the analysis was determined at threonine and leucine concentrations of 50–200 µmol/L, and the assay was found to be linear (r = 0.99, P < 0.05). The precision of the assay was evaluated by replicating analyses of a standard solution; CVs of 2.3% and 2.9% for threonine and leucine, respectively, were obtained. The plasma samples were also analyzed for isotopic enrichment of KIC according to procedures described previously (9, 20). The isotopic abundance of plasma [¹³C]KIC was considered, for the present purpose, to be representative of the enrichment of the intracellular leucine pool (21) that was undergoing leucine oxidation.

Evaluation of primary data
Leucine oxidation
Leucine oxidation was computed for consecutive half-hourly intervals to improve the accuracy of the 24-h leucine oxidation value, because there was a variable rate of leucine oxidation throughout the 24-h period. For each half-hourly interval, leucine oxidation was computed as follows:

(1)

where [¹³C]KIC enrichment is the average of the 2 enrichments determining the specific half-hour interval and where

(2)

where R is the recovery of ¹³CO₂ computed for each time interval, as described previously (13, 14).

In addition, within each metabolic period, ^·VCO₂ over the time interval when it was not directly measured was derived as the arithmetic average of ^·VCO₂ measured just before and after this interval.

Leucine balance
The 24-h leucine balance (input - measured output) was computed as follows:

(3)

(4)

Statistical analysis
Data are presented as means ± SDs. The primary analysis estimated a breakpoint for the relations between threonine intake and leucine oxidation and balance. A two-phase linear regression model was fit to the 24-h oxidation data (IAAO) to estimate at what threonine intake (mg · kg^-1 · d^-1) the oxidation no longer decreased with increasing dietary threonine. A mixed-models analysis of variance regression model estimated the intercept and slope of one line segment and the intercept of the second line segment, and the slope of the second line segment was restricted to zero. The model was constrained such that the 2 line segments intersect at the unknown breakpoint. The breakpoint variable was estimated as -1 times the ratio of the difference between intercepts divided by the difference between slopes (22). The 95% CI for the breakpoint was calculated by using Fieller’s theorem. The analysis was repeated by using the 12-h fed-state oxidation data and by using daily IAAB (leucine) data to determine when the balance no longer increased with increasing dietary threonine. We also examined the plasma threonine data to determine at what dietary threonine intake the plasma threonine concentrations began increasing with further increases in threonine intake.

In the initial review of this paper, a question was raised as to whether the design with unequal allocation of subjects to intakes may have affected the resulting estimate. Therefore, a simulation study was undertaken to provide insight into what would be expected if the same experiment were conducted many times. The simulation study used intakes of 7, 11, 15, 19, 22, and 27 mg threonine · kg^-1 · d^-1 and assumed that the two-phase model for 24-h leucine balance estimated from our experiment was the true model. One thousand Monte Carlo simulations of the experiment were run under each of 2 design assumptions, both resulting in a total of 48 infusions being undertaken: 1) assuming 48 subjects were randomized equally to the 6 intakes (8 subjects/intake) and 2) assuming 48 subjects were randomized as in the present experiment. A simplifying assumption of independent subjects was made to eliminate additional assumptions about the correlation of repeated measures on a subject. To generate the 48 observations for each experiment, we used the SAS RANNOR (SAS Institute Inc, Cary, NC) function to generate normally distributed mean 0 and SD 1 random errors, then multiplied the error by the assumed SD of 5 mg · kg^-1 · d^-1 (a value estimated from our data) and added the intake-specific mean 24-h balance value. The data analysis of each experiment was conducted exactly as for our experimental values.

The metabolic variables were also analyzed by using mixed-models analysis of variance. The models for 12-h leucine oxidation and flux included diet period, metabolic period (fasted compared with fed), threonine intake, and the interaction between intake and metabolic period. If the interaction between intake and phase was significant, then model contrasts were used to make pairwise comparisons of interest and comparisons of 24-h values between different threonine intakes. If the interaction was not significant and the main effect of threonine intake was, then model contrasts were used for comparisons between intakes without regard to metabolic period. The model for 24-h IAAB (leucine) included diet period and threonine intake; comparisons with zero balance were made by using the model, and model contrasts were used for comparisons between intakes if the main effect was significant. A two-sided P value of 0.05 indicated significance for all tests of interaction and main effects; P values of pairwise comparisons were adjusted by using Tukey’s method. To correspond with the breakpoint analyses of leucine oxidation (24-h and 12-h fed phase) and 24-h IAAB, an additional set of comparisons between intakes was made in the corresponding mixed-models analysis of variance: the value of the variable at each intake below the breakpoint was compared with the value averaged across the intakes above the breakpoint. Data analysis used SAS PROC MIXED, version 8.2 (SAS Institute Inc).

	RESULTS

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Breakpoint analysis
The results of fitting a two-phase linear regression model to the data are summarized in Table 4. The breakpoint estimated from each of the 3 variables evaluated approximated a threonine intake of 15 mg · kg^-1 · d^-1, with 95% CIs ranging from 11 to 27 mg · kg^-1 · d^-1. This indicates that the mean threonine requirement in these subjects is 15 mg · kg^-1 · d^-1. The daily mean rate of leucine oxidation at threonine intakes at and above the breakpoint was 43 mg · kg^-1 · d^-1, or essentially the daily intake. A summary of the group mean leucine kinetic data is given in Table 5, and the individual data for 24-h leucine oxidation and daily balance are shown in Figures 1 and 2, respectively.

View larger version (14K): [in this window] [in a new window]   FIGURE 1. Twenty-four–hour leucine oxidation at different threonine intakes in adult Indian men. The solid line is based on the regression analysis in Table 4.

View larger version (15K): [in this window] [in a new window]   FIGURE 2. Daily leucine balance at different threonine intakes in adult Indian men. The solid line is based on the regression analysis in Table 4.

The 24-h leucine oxidation rate was significantly lower at the 7 and 11 mg threonine · kg^-1 · d^-1 intakes than at the 15, 19, and 22 mg threonine · kg^-1 · d^-1 intakes (each P < 0.05), which were not different from one another or from the oxidation rate at 27 mg threonine · kg^-1 · d^-1. The absence of significant differences between oxidation at the 7 and 11 mg threonine · kg^-1 · d^-1 intakes and oxidation at the 27 mg threonine · kg^-1 · d^-1 intake may be a result of the small sample size (n = 4) at 27 mg threonine · kg^-1 · d^-1.

Corresponding to the breakpoint analysis, which assumes that oxidation does not differ across intakes at or above the estimated breakpoint of 15 mg threonine · kg^-1 · d^-1, the average oxidation across these intakes was computed for comparison with oxidation at intakes below the breakpoint. The 24-h leucine oxidation rates at the 7 and 11 mg threonine · kg^-1 · d^-1 intakes were each significantly higher than the average oxidation at or above the estimated breakpoint of 15 mg threonine · kg^-1 · d^-1 (each P < 0.01). Similarly, 12-h fed oxidation at the 7 mg threonine · kg^-1 · d^-1 intake was significantly higher than 12-h fed oxidation averaged across intakes at or above the estimated breakpoint (P < 0.01); oxidation at the 11 mg threonine · kg^-1 · d^-1 intake was also higher than the average oxidation at or above the breakpoint, but the difference was not significant (P = 0.10).

Leucine balance
With respect to leucine balance, the results were essentially the same whether expressed as an absolute balance or as a percentage of threonine intake (Table 5). Daily leucine balance was signficantly affected by threonine intake (P = 0.02) and was signficantly lower at the 7 mg threonine · kg^-1 · d^-1 intake than at the 15, 19, and 27 mg threonine · kg^-1 · d^-1 intakes (each P < 0.01). Corresponding to the foregoing breakpoint analysis, leucine balances at the 7 and 11 mg threonine · kg^-1 · d^-1 intakes were each significantly lower than the average balance at intakes at or above the estimated breakpoint of 15 mg threonine · kg^-1 · d^-1 (each P < 0.05). Leucine balances at 7 and 11 mg threonine · kg^-1 · d^-1 were 10 and 5 mg · kg^-1 · d^-1 lower, respectively, than the average balance at threonine intakes above the breakpoint.

Leucine flux
There was no significant effect of metabolic period nor of threonine intake on leucine flux (Table 5). There was however, evidence of an effect of diet period: flux was lower by 8 µmol · kg^-1 · 30 min^-1 in the first diet period than in the other 2 diet periods.

Plasma amino acids
Results for plasma threonine and leucine concentrations for the fasted and 3- and 6-h fed periods are summarized in Table 7. Inspection of the threonine data suggested that for the fed period there may be a breakpoint in the plasma concentration–threonine intake response relation. The results of fitting a two-phase linear regression model to the plasma threonine data are summarized in Table 8. The breakpoint estimated from each of the 3 variables evaluated approximated a threonine intake of 13–15 mg · kg^-1 · d^-1, with 95% CIs ranging from 1 to 19 mg · kg^-1 · d^-1. Although the variability in these data was greater than that observed for the other response variables, such as the leucine balance or oxidation rate, the breakpoint for the mean threonine intake on the plasma threonine–threonine intake response curve approximated that for the 24-h IAAO and 24-h IAAB variables (Table 4).

	DISCUSSION

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION REFERENCES

Wilson et al (8) used a 4-h protocol with phenylalanine as the indicator amino acid in nonadapted subjects studied in the fed state. These subjects were studied over a range of threonine intakes from 5 to 35 mg · kg^-1 · d^-1, with 5-mg increments. The mean threonine requirement, based on the breakpoint in the [¹³C]phenylalanine oxidation–threonine intake curve, was 19.0 mg · kg^-1 · d^-1, with an upper safe limit of intake of 26 mg · kg^-1 · d^-1. The individual breakpoints (determined by visual inspection) ranged from 10 to 35 mg · kg^-1 · d^-1.

The concordance of the present mean threonine requirement estimate in Indian subjects with that found in Western subjects (7, 8, 12) also provides evidence against the possibility that there may be profound metabolic adaptations, including sparing effects of dietary nonessential nitrogen and urea nitrogen recycling through the gut, in healthy, well-nourished populations from developing countries, who usually have lower amino acid intakes than do Western subjects (25). We earlier explored this possibility, and our recent studies in well-nourished Indian men (11, 13, 14) that used the 24-h IAAO and IAAB indicator amino acid approaches indicated that the requirements for lysine and leucine in these subjects are similar to those in Western subjects. These studies also showed that a 1-wk experimental feeding period before the tracer study is adequate for adaptation to the experimental L-amino acid–based diet (26), as was shown for US subjects previously (12). The present results confirm these previous findings. It must be recognized, however, that the Indian subjects in the present study were well nourished and came from affluent backgrounds; whether the same amino acid requirements exist in chronically undernourished subjects from poor socioeconomic backgrounds is still unknown, and we have begun to address this question.

The similarity of the present threonine requirement estimate and the range proposed by Zhao et al (7), who used the direct amino acid oxidation technique, also validates the latter approach, because doubts have been raised about the accuracy of this approach when there is more than one degradative pathway of the amino acid in question (27, 28). The use of leucine as an indicator of threonine equilibrium obviates the 2 problems associated with direct amino acid oxidation and balance: first, the more general problem of tracer contribution to amino acid intake in the fasted state is removed, because it is the indicator (leucine) that is infused, and threonine intakes in the diet can be manipulated to as low an intake as required. Second and more specifically, the potential problem of the 2 degradation pathways for threonine (27, 28) is no longer present because the indicator amino acid kinetics are independent of the complexities of threonine metabolism. The concordance of our 24-h IAAO and 24-h IAAB estimates of threonine requirement with the earlier direct amino acid oxidation and balance estimates of Zhao et al (7), despite the potential problems related to the complexity of threonine metabolism (27, 28), is probably due to several factors. First, in the latter study, the fed-state threonine oxidation rates decreased with decreased threonine intake, reaching a nadir at a threonine intake of 20 mg · kg^-1 · d^-1 and decreased slightly thereafter as threonine intakes decreased further. Thus, there was an inflection point in the threonine oxidation-intake curve at 20 mg · kg^-1 · d^-1. It is this pattern of the threonine oxidation in response to graded intakes of threonine that was used to arrive at the earlier proposed threonine requirement value. Second, it was recently found that the threonine dehydrogenase pathway is a minor pathway of threonine catabolism in the human adult (29), implying that the threonine dehydratase (EC 4.2.1.16) pathway is the dominant route in the catabolism of threonine in adult humans. This also means that problems of underestimation of oxidation, due to the sequestration of the ¹³C label in glycine, would not be as great.

In conclusion, the results of the present study, considered in association with the tracer-based findings we reported earlier (7, 12), our theoretical prediction model (6), and the findings of others (8, 23), indicate that it is reasonable to propose for practical use a mean requirement for threonine in healthy men of 15 mg · kg^-1 · d^-1. Furthermore, it can be concluded tentatively that this value for men is applicable globally.

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