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Lysine requirements of healthy adult Indian subjects, measured by an indicator amino acid balance technique^1,2,3

¹ From the Department of Physiology and Nutrition Research Center, St John's Medical College, Bangalore, India, and the Laboratory of Human Nutrition, Massachusetts Institute of Technology, Cambridge, MA.

² Supported by the Global Cereal Fortification Initiative, Tokyo, and NIH grants RR88, DK42101, and P30-DK40561.

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. Address correspondence to VR Young, Laboratory of Human Nutrition, Massachusetts Institute of Technology, Cambridge, MA 02139. E-mail: vryoung{at}mit.edu.

	ABSTRACT

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Background: In an earlier study, using a modification of the indicator amino acid oxidation approach, we concluded that the 1985 FAO/WHO/UNU–proposed lysine requirement of 12 mg•kg^-1•d^-1 is likely inadequate to maintain body amino acid homeostasis in apparently healthy south Asian subjects and that our proposed requirement of 30 mg•kg^-1•d^-1 is more appropriate.

Objective: We assessed the lysine requirement in a similar population by using 4 test lysine intakes (12, 20, 28, and 36 mg•kg^-1•d^-1) with an indicator amino acid balance approach.

Design: Sixteen healthy male Indians were studied during each of 2 randomly assigned 8-d L-amino acid diets that supplied either 12 and 28 or 20 and 36 mg lysine. At 1800 on day 8, a 24-h intravenous [¹³C]leucine tracer-infusion protocol was conducted to assess leucine oxidation and daily leucine balance at each lysine intake.

Results: Mean 24-h leucine oxidation rates decreased significantly (P = 0.005) across different lysine intakes and were 104.1, 97.8, 87.3, and 87.3 mg•kg^-1•d^-1 at intakes of 12, 20, 28, and 36 mg•kg^-1•d^-1, respectively; mean 24-h leucine balances were 3.3, 9.1, 19.7, and 20.7 mg•kg^-1•d^-1, respectively (P = 0.015, mixed-model analysis of variance). Oxidation and balances differed significantly between the lower and higher lysine intakes but were not significantly different between the 12- and 20-mg and 28- and 36-mg test intakes. Two-phase regression analysis indicated a mean breakpoint at 29 mg lysine•kg^-1•d^-1 in the relation between lysine intake and leucine oxidation or balance.

Conclusion: We propose a mean lysine requirement of 30 mg•kg^-1•d^-1 for healthy Indian adults, which is the same amount we proposed previously for Western populations.

Key Words: Indian men • lysine requirement • indicator amino acid balance • amino acid oxidation • leucine

	INTRODUCTION

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION REFERENCES

 
We previously assessed the lysine requirements of healthy adults from south Asia by using a stable-isotope-tracer protocol (1). The results of that study supported our earlier conclusion (2) that the upper lysine requirement for adult humans proposed in 1985 by the FAO/WHO/UNU (12 mg•kg^-1•d^-1; 3) is too low. In addition to the results of that study, there is an expanding body of evidence supporting the validity of our proposed lysine requirement of 30 mg•kg^-1•d^-1 (4, 5). This value is based on results of short-term tracer studies and the intake necessary to balance the predicted obligatory losses (4, 6) and was supported by an additional set of criteria that we used to judge the requirement for lysine (7) and by results of a more recent series of 24-h [¹³C]lysine tracer studies (8, 9). It is also consistent with results from tracer studies carried out by the Toronto group (10, 11) and more recently by those of Millward (12), and with our (13) reassessment of the earlier nitrogen balance data of James et al (14).

We extended the design of our previous pilot study to assess 4 different lysine intakes by using a modification of the indicator amino acid oxidation (IAAO) technique (15–17) in which we measured the indicator amino acid balance (IAAB) as an important indicator of the adequacy of amino acid intakes. The IAAO technique was used previously (10, 11) in studies of the lysine requirements of adult humans and was modified by us to include an assessment of IAAB in our initial study in Indian subjects (1). The IAAB approach that we used in the present study involved 3 major modifications to the previously used IAAO method. First, [¹³C]leucine was used as the indicator amino acid rather than labeled phenylalanine (10, 11). Second, [¹³C]leucine was given over an entire 24-h period because our previous studies of amino acid oxidation at inadequate intakes of leucine (18), phenylalanine (19–21), and, more recently, lysine (1), showed a complex temporal pattern of amino acid utilization within a 24-h period. We found that we could determine, with an acceptable degree of accuracy, the quantitative status of whole-body leucine oxidation and, presumably, balance, by following the present paradigm, at least when leucine intakes were sufficient to meet or exceed the leucine requirement (22). Third, we evaluated the relation between leucine intake and leucine balance as a basis for judging minimum physiologic requirements for lysine.

	SUBJECTS AND METHODS

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Subjects
Sixteen young men participated in this experiment. All 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. All subjects were in good health as determined by medical history, physical examination, blood cell count, routine blood biochemical profile, and urinalysis. The subjects' habitual intake of lysine was estimated to be <60 mg•kg^-1•d^-1 on the basis of a 3-d weighed dietary intake record. Subjects who smoked cigarettes, consumed 5 alcoholic drinks/wk, or drank 6 cups of caffeinated beverages/d were excluded from participation. The purpose of the study and the potential risks involved were explained to each subject. Written consent was obtained from each subject and the research protocol was approved by the Human Ethical Approval Committee of St John's Medical College.

Diet and experimental design
The tracer experiment began after an 8-d run-in period. Subjects were studied during 2 separate diet periods, during which time they consumed a weight-maintaining diet based on an L-amino acid mixture providing different daily lysine intakes (Table 2). Daily energy intakes were designed to maintain body weight, and the energy requirement was calculated to be 1.6 x basal metabolic rate (BMR) from days 1 to 8 and 1.35 x BMR on day 9 (tracer study day). The subjects were encouraged to maintain their customary physical activity levels but were asked to refrain from excessive or competitive exercise. The major source of energy was a sugar-oil formula and protein-free wheat-starch cookies (Table 3). Nonprotein energy was provided as fat (43% of energy) and carbohydrate (56% of energy). The main source of carbohydrate was beet sugar and wheat starch, to attain a low ¹³C content in the diet and a relatively steady background in breath ¹³CO₂ enrichment over the 24-h period. 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.

All other nutrients were provided in adequate amounts during the run-in period (Table 3). A choline supplement of 500 mg was given daily and dietary fiber was provided as 20 g psyllium husk (Sat-Isabgol; Charak Pharmaceuticals Ltd, Gujarat, India) when requested by a subject. The total daily food intake was consumed as 3 isoenergetic, isonitrogenous meals (at 0800, 1300, and 2000). Each morning, body weight was measured and vital signs were monitored. All of the subjects' meals were consumed at the kitchen of the Nutrition Research Center, under supervision of the dietary staff.

24-h Tracer-infusion protocol
The primed tracer-infusion protocol was conducted in all subjects according to a standard design (Figure 1). After the subjects consumed their last meal at 1500 on day 8, the tracer administration began at 1800 and ended at 1800 on day 9. Subjects received 10 small isoenergetic, isonitrogenous meals at hourly intervals beginning at 0600 and ending at 1500; together these meals provided the complete 24-h dietary intake. Indirect calorimetry was performed hourly and blood was withdrawn half-hourly for measurement of [¹³C]-ketoisocaproic acid (KIC) enrichment. Throughout the 24-h study, the subjects remained in bed, in a reclining position, except during sleep when they lay supine. Thus, the 24-h study was divided into two 12-h metabolic periods (fasted and fed).

View larger version (16K): [in this window] [in a new window]   FIGURE 1. . Schematic diagram of the 24-h L-[1-13C]leucine infusion study, which began at 1800 on day 8 of the experimental diet period. Breath and blood sampling was performed every 30 min and calorimetry every alternate hour. Ten isonitrogenous and isoenergetic meals were provided every hour for 10 h in the fed period. The numbers on the y axis represent the following: 1, feeding; 2, continuous intravenous infusion; 3, blood samples; 4, breath samples; and 5, calorimetry.

Recovery of ¹³CO₂ and the contribution of dietary ¹³C to breath ¹³CO₂
Because the diets we used 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 previously described (1). The recovery of breath ¹³CO₂ was calculated for every 30-min interval as previously described (1). Values at each time point were used to correct each 30-min estimate of ¹³CO₂ production from [1-¹³C]leucine oxidation (see below).

Indirect calorimetry
Minute-to-minute total carbon dioxide production (CO₂) and oxygen consumption (O₂) were determined with an open-circuit indirect calorimeter with a ventilated hood, as previously described (1, 26). Whole-system calibration was verified by combustion of pure ethanol; the observed difference between measured and predicted total CO₂ 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 30, 15, and 5 min before the 24-h tracer infusion started and then at consecutive half-hourly intervals throughout the 24-h study. Breath gas was collected in a specially designed bag that permitted the removal of dead space air and was transferred into three 10-mL nonsilicon-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. When the breath-sample collection coincided with hourly meals, the breath sample was collected first. The samples were stored at room temperature until isotope ratio mass spectrometry (Europa Scientific, Crewe, United Kingdom) was used to analyze the ratio of ¹³CO₂ to ¹²CO₂ as previously described (1). The increase in breath enrichment after the isotope administration was expressed as atom percent excess (APE). The APE was calculated by taking the arithmetic difference between the enrichment of each breath sample and the predose basal breath sample.

Collection and analysis of blood and urine samples
Blood samples were collected at 30-min intervals between 0000 and 2400 of the tracer infusion period. Three baseline samples at -30, -5, and -5 min were taken 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 withdrawal while the hand was in a custom-made warming box that was 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 measuring hemoglobin saturation; saturation was >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). The plasma was removed and the samples were stored at -80°C until shipped from Bangalore, India, in dry ice for determination of [¹³C]KIC enrichment in our laboratories at the Massachusetts Institute of Technology (MIT) according to procedures described previously (18, 22). The isotopic abundance of plasma [¹³C]KIC was considered to represent enrichment of the intracellular leucine pool (27) that was undergoing leucine oxidation.

Leucine oxidation
Leucine oxidation (µmol•kg^{- 1}•30 min^-1) 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:

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

where R is the recovery of ¹³CO₂ computed for each time interval as previously described (1).

In addition, within each metabolic state, CO₂ over the time interval when it was not directly measured was derived as the arithmetic average of CO₂ measured just before and after this interval. Leucine oxidation values were converted to µg•kg^-1•d^-1 by multiplying by 131.17 (molecular weight of leucine) and by 48 (30-min time intervals in a day).

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

Statistical methods and data evaluation
Data are presented as means ± SDs. The metabolic variables were analyzed by using mixed-models analysis of variance (PROC MIXED, version 6.12; SAS Institute Inc, Cary, NC). The models for 12-h leucine oxidation and flux included diet period, metabolic phase (fasting compared with fed), and lysine intake—which was a within- or between-subject factor. If the intake by phase interaction was significant, then model contrasts were used to make pairwise comparisons of interest and comparisons of 24-h oxidation between different lysine intakes. If the interaction was not significant and the main effect of lysine was, contrasts were used for comparisons between intakes without regard to metabolic phase; these comparisons applied to 24-h oxidation levels. The model for 24-h IAAB (leucine) included lysine intake—which was a within- or between-subject factor—and the diet period. A P value of 0.05 indicated significance for all tests of interaction and main effects; P values of post hoc comparisons were adjusted by using Tukey's test. Because our working hypothesis was that leucine oxidation would be higher at each of the lower intakes than at the higher lysine intakes and that the IAAB (leucine) would be more negative or less positive at the same lower intakes of lysine, pairwise comparisons of interest were made with one-sided tests.

We also estimated a breakpoint for the relations between lysine intake and leucine oxidation and balance; ie, a two-phase linear regression model was fit to the 24-h oxidation data to estimate at what lysine intake (mg•kg^-1•d^-1) the oxidation no longer decreased with increasing dietary lysine. The least-squares regression model estimated the intercept and slope of one line segment and the intercept of the second line segment; the slope of the second line segment was restricted to zero. The breakpoint was estimated as -1 times the ratio of the difference between intercepts divided by the difference between slopes. The 95% CI for the breakpoint was calculated by using Fieller's theorem (28). The analysis was repeated by using the 12-h fed oxidation data and then by using the daily IAAB (leucine) data to determine when balance no longer increased with increasing dietary lysine.

	RESULTS

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Anthropometry
Mean anthropometric indexes of the subjects are summarized in Table 1. BMI ranged from 18.7 to 20.6 and body fat was relatively low (15%). There were no significant weight changes in the groups of subjects during the 8-d diet periods. All subjects remained in apparent good health throughout.

Leucine oxidation and balance
Data for the primary variables measured, including CO₂, breath ¹³CO₂ enrichment, ¹³CO₂ production, and plasma [¹³C]KIC enrichment at each test lysine intake, were similar to those published previously (1) and are not shown here. The temporal pattern of leucine oxidation over the 24-h period indicated that the rates of leucine oxidation were significantly lower during the fasting than during the fed period at all 4 lysine intakes (P < 0.0001; Figure 2). For the entire group of subjects, the mean oxidation rate was equivalent to 31.4 mg leucine•kg^-1•12 h^-1 during the fasting period (Table 4).

View larger version (30K): [in this window] [in a new window]   FIGURE 2. . Mean (±SD) leucine oxidation measured for each 30-min interval throughout a 24-h intravenous infusion of 12 (A), 20 (B), 28 (C), and 36 (D) mg L-[1-13C]leucine•kg-1•d-1. Feeding began with small meals at 0600 to provide 93 mg dietary leucine•kg-1•d-1 and ended with the last meal at 1500.

Breakpoint analysis
The results of fitting a two-phase linear regression model to the data are summarized in Table 5. The breakpoint estimated from each of the 3 variables approximated a lysine intake of 29 mg•kg^-1•d^-1; however, the 95% CIs were wide.

	DISCUSSION

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION REFERENCES

Using [¹³C]lysine as a tracer, we more recently conducted a series of 24-h tracer studies to estimate the lysine requirement of healthy adults (8, 9). On the basis of these findings, we concluded that our previously proposed lysine requirement of 30 mg•kg^-1•d^-1—derived from limited direct experimental data (6) as well as from a predictive approach (2, 5)—was well supported. We recommended that this value be used in considerations of lysine nutrition in healthy young and adult humans. This proposed requirement is considerably higher than the upper requirement of 12 mg•kg^-1•d^-1 proposed in 1985 by the FAO/WHO/UNU (3), but is also supported by our analysis of earlier nitrogen balance data (13) and is intermediate between values of 23–27 mg•kg^-1•d^-1 as suggested by Millward (12) and estimates of 37 and 45 mg lysine•kg^-1•d^-1 proposed by the Toronto group (10, 11). In all of these studies, [¹³C]leucine oxidation, retention, or both were used as indicators. The lower of the 2 estimates now reported in detail by Millward et al (36) may be underestimates of the true, minimum lysine requirement. This may be true because if Millward et al's subjects had been adapted to a low lysine intake before the short-term [1-¹³C]leucine tracer studies were conducted, the amount of free lysine in the intracellular pool for recycling into tissue protein would have been lower when a meal containing wheat protein was consumed. This would have resulted in a lower efficiency of protein utilization from the meal than observed in their study (36). This, together with our present observation that leucine oxidation in the fasted state remained the same at all 4 lysine intakes, would have resulted in a higher estimate of the lysine requirement because Millward et al based their proposed requirement on the lysine content of the estimated average requirement of wheat protein; the estimated protein requirement was calculated as 24 x the hourly postabsorptive (fasting) loss of leucine. This view is supported by the fact that the relative protein value of wheat protein reported by Millward et al, based on short-term tracer studies in subjects who were adapted to generous protein and lysine intakes, was much higher than that reported by us (37) when based on nitrogen balance slope assays.

The global applicability of this earlier estimate of the lysine requirement (30 mg•kg^-1•d^-1) should be questioned because it was obtained in a study using healthy well-nourished subjects whose habitual lysine intakes would have been expected to exceed 100 mg•kg^-1•d^-1. In populations in developing regions whose diet is based predominantly on cereals, especially wheat, the mean habitual lysine intake might be expected to be in the range of 30–40 mg•kg^-1•d^-1. Hence, the possibility exists that such populations can achieve body amino acid balance with a greater metabolic efficiency, relative to the balance obtained by populations in developed regions, because of an adaptive response. Therefore, we considered it necessary to conduct appropriate tracer studies of amino acid oxidation in healthy subjects in less-developed countries to resolve the question of whether lower intakes than our proposed, tentative new requirements for the indispensable amino acids (2, 5) would be sufficient to meet minimum physiologic requirements in these populations. In addition, it seemed important to conduct such studies initially in "adequately nourished" Third World subjects, so that the requirement for health maintenance would be measured for these subjects. The subjects in the present study were healthy by all usual anthropometric, biochemical, and clinical indexes and their mean habitual lysine intake was 52.6 ± 11.9 mg•kg^-1•d^-1. This is considerably lower than the usual intake of our MIT subjects and presumably that of the Canadian (10, 11) and UK (38) subjects, although it still exceeds our tentative minimum requirement.

Therefore, we previously conducted a preliminary study in healthy adult Indian subjects who were given diets providing lysine intakes of 12 and 28 mg•kg^-1•d^-1 by using a modification of the IAAO technique (1). The 24-h [¹³C]leucine balance (IAAB) strengthened the view that the FAO/WHO/UNU (3) upper requirement of 12 mg•kg^-1•d^-1 was also too low for Indian subjects and that 28 mg•kg^-1•d^-1 was an approximate minimum mean lysine requirement. The present study was an extension of this earlier study (1) and involved 24-h [¹³C]leucine tracer studies at 4 test lysine intakes: 12, 20, 28, and 36 mg•kg^-1•d^-1. Subjects were given 1 of 2 experimental diets during 8-d periods before the tracer study began on day 9. This design was chosen because of the complexity of the investigation and our anticipation that the Indian subjects would not be able to successfully complete all 4 diet and tracer periods.

The 24-h leucine oxidation rates were clearly higher at the lowest 2 lysine intakes but they were not significantly different between the 2 highest lysine intakes. These rates, in turn, only tended to be higher than the rate at the 20-mg lysine intake and nonsignificantly so. Using a two-phase regression model, we estimated a mean breakpoint of 29 mg•kg^-1•d^-1. These findings strengthen the view that the mean lysine requirement for these Indian subjects is 30 mg•kg^-1•d^-1 and that it applies to healthy populations worldwide. We concluded earlier that there was no evidence of an adaptive reduction in the quantitative requirements for protein to maintain an adequate state of protein nutriture by populations whose habitual intake is lower than that characteristic of affluent, Western societies (7, 39).

The findings of a distinct positive daily balance at the higher lysine intakes were unexpected on the basis of our earlier studies conducted at MIT. The explanation for these apparent high leucine balances is not straightforward. They may have been due to a true biological phenomenon; these Indian subjects may have been showing a repletion-type response to a complete, adequate experimental diet. We do not consider this to be likely because the subjects were all healthy and normal and their intakes of protein and energy were all apparently adequate. The possibility exists, however, that they were responding to one or more micronutrients present in the experimental diet that were perhaps marginal or inadequate in their free-choice diets. Again, this seems unlikely because there were no trends toward increased body weights during the study period in any of the groups.

Another possible explanation is that leucine balances were overestimated for model or technical reasons. Thus, it is conceivable that there was some ileal or fecal loss of leucine but, even if this was corrected for by a factor of 3% of total intake to account for such a loss [approximated from data of Fuller et al (40)], it would not entirely explain the magnitude of the apparent positive balance. None of the subjects reported any episodes of diarrhea or other intestinal problems. It is possible that there was a relatively large extraction of dietary leucine within the splanchnic area in these Indian subjects and that all of the absorbed dietary leucine may not have equilibrated with the tracer before its oxidation. Boirie et al (41) also investigated this problem and found that leucine oxidation was higher within this region when both the tracer and meal were given orally than when given intravenously. In that study, the increment in leucine oxidation increased by a factor of 1.6. Applying a similar factor to the increment in oxidation during the fed state in the present study would have yielded a leucine balance of -19 mg•kg^-1•d^-1 at the lowest lysine intake and a leucine balance of -2 mg•kg^-1•d^-1 at the highest intake. These approximations represent an equivalent increment in the observed 24-h leucine oxidation of 20%. We recently completed the human phase of a study in healthy Indian adults in which the ¹³C tracer was administered orally (unpublished observations, 1999). Other than this difference, the same protocol was used in the present study and in similar subjects provided similar lysine intakes. Preliminary results indicate that the 24-h leucine oxidation rate increased, ranging from 5% at the lowest lysine intake to 15% at the 2 highest lysine intakes. The average increment in daily leucine oxidation across all lysine intakes was 10%. This finding, in part, explains the positive leucine balances observed in the present study, but perhaps differs from our earlier findings in subjects studied at MIT, for whom estimates of whole-body leucine oxidation rates were similar when the [¹³C]leucine tracer was given intravenously and orally (42, 43). Therefore, it might be that our Indian subjects process dietary leucine in a way that differs from that of our US subjects, who were largely white. Recent research (44–48) has further elucidated the major, metabolic significance of the intestine in the utilization and catabolism of endogenous amino acids and the role it plays in channeling dietary amino acids to the portal-drained viscera and peripheral tissues. It is likely, therefore, that the quantitative fate of dietary amino acids in subjects at St John's Medical College, Bangalore, differs from that of the subjects studied at MIT. We intend to explore this hypothesis. Nevertheless, the relation between leucine intake and balance indicated that whole-body leucine metabolism was less favorable at the lower lysine intakes than at the higher lysine intakes. We conclude that the 2 lower lysine intakes were not adequate.

In summary, this investigation of 24-h [¹³C]leucine tracer kinetics in healthy Indian subjects, in whom the effects of 4 lysine intakes were studied, indicates that the lysine requirement proposed by the FAO/WHO/UNU in 1985 (3) of 12 mg•kg^-1•d^-1 is not adequate for the healthy Indian population. We further conclude that the proposed tentative lysine requirement of 30 mg•kg^-1•d^-1, based on [¹³C]lysine tracer studies in US subjects (8, 9) and now generally supported by those of other investigators (10–12), similarly applies to healthy adults in south Asia.

	REFERENCES

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION REFERENCES

 

1. Kurpad AV, El-Khoury AE, Beaumier L, et al. An initial assessment, using 24-h [¹³C]leucine kinetics, of the lysine requirement of healthy adult Indian subjects. Am J Clin Nutr 1998;67:58–66.[Abstract]
2. Young VR, Bier DM, Pellett PL. A theoretical basis for increasing current estimates of the amino acid requirements in adult man, with experimental support. Am J Clin Nutr 1989;50:80–92.[Abstract/Free Full Text]
3. World Health Organization. FAO/WHO/UNU Expert Consultation. Energy and protein requirements. World Health Organ Tech Rep Ser 1985;724:1–264.[Medline]
4. Young VR, Pellett PL. Current concepts concerning indispensable amino acid needs in adults and their implications for international nutrition planning. Food Nutr Bull 1990;12:289–300.
5. Young VR, El-Khoury AE. Can amino acid requirements for nutritional maintenance in adult humans be approximated from the amino acid composition of body mixed proteins? Proc Natl Acad Sci U S A 1995;92:300–4.[Abstract/Free Full Text]
6. Meredith CN, Wen Z-M, Bier DM, Matthews DE, Young VR. Lysine kinetics at graded intakes in young men. Am J Clin Nutr 1986;43: 787–94.[Abstract/Free Full Text]
7. Young VR, El Khoury AE. Human amino acid requirements: a re-evaluation. Food Nutr Bull 1996;17:191–203.
8. El-Khoury AE, Basile A, Beaumier L, et al. Twenty-four–hour intravenous and oral tracer studies with L-[1-¹³C]-2-aminoadipic acid and L-[1-¹³C]lysine as tracers at generous nitrogen and lysine intakes in healthy adults. Am J Clin Nutr 1998;68:827–39.[Abstract]
9. El-Khoury AE, Pereira PCM, Borgonha S, et al. Twenty-four–hour oral tracer studies with L-[1-¹³C]lysine at a low (15 mg•kg^-1•d^-1) and intermediate (29 mg•kg^-1•d^-1) lysine intake in healthy adults. Am J Clin Nutr 2000;72:122–30.[Abstract/Free Full Text]
10. Zello GA, Pencharz PB, Ball RO. Dietary lysine requirement of young adult males determined by the oxidation of L-[1-¹³C]-phenylalanine. Am J Physiol 1993;264:E677–85.[Abstract/Free Full Text]
11. Duncan AM, Ball RO, Pencharz PB. Lysine requirement of adult males is not affected by decreasing protein intake. Am J Clin Nutr 1996;64:718–25.[Abstract/Free Full Text]
12. Millward DJ. Post-prandial protein utilization: implications for clinical nutrition. In: Fürst P, Young VR, eds. Proteins, peptides and amino acids in enteral nutrition. Vol 3. Basel, Switzerland: Nestlé/Karger, 2000:135–55. (Nestlé Nutrition Workshop Series Clinical and Performance Programme.)
13. Rand WM, Young VR. Statistical analysis of nitrogen balance data with reference to the lysine requirement in adults. J Nutr 1999;129: 1920–6.[Abstract/Free Full Text]
14. Jones EM, Baumann CA, Reynold MS. Nitrogen balance of women maintained on various levels of lysine. J Nutr 1956;60:549–59.
15. Kim KI, McMillan I, Bayley HS. Determination of amino acid requirements of young pigs using an indicator amino acid. Br J Nutr 1983;50:369–82.[Medline]
16. Ball RO, Bayley HS. Tryptophan requirement of the 2.5 kg piglet determined by the oxidation of an indicator amino acid. J Nutr 1984;114:1741–6.
17. Zello GA, Wykes LJ, Ball RO, Pencharz PB. Recent advances in methods of assessing dietary amino acid requirements for adult humans. J Nutr 1995;125:2907–15.
18. El-Khoury AE, Fukagawa NK, Sánchez M, et al. The 24-h pattern and rate of leucine oxidation, with particular reference to tracer estimates of leucine requirements in healthy adults. Am J Clin Nutr 1994;59:1012–20.[Abstract/Free Full Text]
19. Basile-Filho A, El-Khoury AE, Beaumier L, Wang SY, Young VR. Continuous 24-h L-[1-¹³C]phenylalanine and L-[3,3-²H₂]tyrosine oral-tracer studies at an intermediate phenylalanine intake to estimate requirements in adults. Am J Clin Nutr 1997;65:473–88.[Abstract/Free Full Text]
20. Sanchez M, El-Khoury AE, Castillo L, Chapman TE, Young VR. Phenylalanine and tyrosine kinetics in young men throughout a continuous 24-h period, at a low phenylalanine intake. Am J Clin Nutr 1995;61:555–70.[Abstract/Free Full Text]
21. Sanchez M, El-Khoury AE, Castillo L, et al. Twenty-four–hour intravenous and oral tracer studies with L-[1-¹³C]phenylalanine and L-[3,3-²H₂]tyrosine, at a tyrosine free, generous phenylalanine intake. Am J Clin Nutr 1996;63:532–45.[Abstract/Free Full Text]
22. El-Khoury AE, Fukagawa NK, Sanchez M, et al. Validation of the tracer-balance concept with reference to leucine: 24-h intravenous tracer studies with L-[1-¹³C]leucine and [¹⁵N-¹⁵N]urea. Am J Clin Nutr 1994;59:1000–11.[Abstract/Free Full Text]
23. Lohman GT, Roche AF, Martorell R. Skinfold thicknesses and measurement technique. In: Anthropometric standardization reference manual. Champaign, IL: Human Kinetics Books, 1988:55–70.
24. Durnin JVGA, Womersley J. Body fat assessed by total body density and its estimation from skinfold thickness measurements on 481 men and women aged from 16 to 72 years. Br J Nutr 1974;32:77–97.[Medline]
25. Siri WE. Body composition from the fluid spaces and density: analysis of methods. In: Brozek J, Henschel A, ed. Techniques for measuring body composition. Washington, DC: National Academy of Sciences, 1961:223–44.
26. Shetty PS, Sheela ML, Murgatroyd PR, Kurpad AV. An open circuit indirect whole body calorimeter for the continuous measurement of energy expenditure of man in the Tropics. Indian J Med Res 1987; 85:453–60.[Medline]
27. Matthews DE, Schwarz HP, Yang RD, Motil KJ, Young VR, Bier DM. Relationship of plasma leucine and -ketoisocaproate during a L-[1-¹³C]leucine infusion in man: a method for measuring human intracellular tracer enrichment. Metabolism 1982;31:1105–12.[Medline]
28. Seber GAF. Linear regression analysis. New York: John Wiley and Sons, 1977.
29. Young VR. 1987 McCollum award lecture. Kinetics of human amino acid metabolism: nutritional implications and some lessons. Am J Clin Nutr 1987;46:709–25.[Abstract/Free Full Text]
30. Young VR, El-Khoury AE. The notion of nutritional essentiality of amino acids revisited, with a note on the indispensable amino acid requirements in adults. In: Cynober L, ed. Amino acid metabolism and theory in health and nutritional disease. Boca Raton, FL: CRC Press, 1995:191–232.
31. Young VR, Marchini JS. Mechanisms and nutritional significance of metabolic responses to altered intakes of protein and amino acids, with reference to nutritional adaptation in humans. Am J Clin Nutr 1990;51:270–89.[Abstract/Free Full Text]
32. World Health Organization. FAO/WHO Consultation. Protein quality evaluation. Rome: Food and Agriculture Organization, 1991:1–66.
33. Young VR, Borgonha S. Nitrogen and amino acid requirements: the Massachusetts Institute of Technology amino acid requirement pattern. J Nutr 2000;130:1841–9S.
34. Hoshiai K. World balance of dietary essential amino acids related to the 1989 FAO/WHO protein scoring pattern. Food Nutr Bull 1995; 16:166–77.
35. Young VR, Scrimshaw NS, Pellett PL. Significance of dietary protein source in human nutrition. Animal or plant proteins? In: Waterlow JC, Armstrong DG, Fowder L, Riley R, eds. Feeding a world population of more than eight billion people. A challenge to science. Oxford, United Kingdom: Oxford University Press, 1998:205–22.
36. Millward DJ, Fereday A, Gibson NR, Pacy PJ. Human adult amino acid requirements: [1-¹³C]leucine balance evaluation of the efficiency of utilization and apparent requirements for wheat protein and lysine compared with those for milk protein in healthy adults. Am J Clin Nutr 2000;72:112–21.[Abstract/Free Full Text]
37. Young VR, Fajaroda L, Murray E, Rand WM, Scrimshaw NS. Protein requirements of man: comparative nitrogen balance response within the submaintenance-to-maintenance range of intakes of wheat and beef proteins. J Nutr 1975;105:534–44.
38. Millward DJ. The nutritional value of plant-based diets in relation to human amino acid and protein requirements. Proc Nutr Soc 1999; 58:249–69.[Medline]
39. Young VR, Borgonha S. Nutritional adaptation (genetic, physiological and behavioral): implications for requirements. In: Fitzpatrick DW, Anderson JE, L'Abbe' ML, eds. From nutritional science to nutrition process for better global health. Ottawa: Canadian Federation of Biological Societies, 1998:57–160.
40. Fuller MF, Milne A, Harris CI, Reid TMS, Keenan R. Amino acid losses in ileostomy fluid on a protein-free diet. Am J Clin Nutr 1994; 59:70–3.[Abstract/Free Full Text]
41. Boirie Y, Gachon P, Beaufrere B. Splanchnic and whole-body leucine kinetics in young and elderly men. Am J Clin Nutr 1997;65:489–95.[Abstract/Free Full Text]
42. Hoerr RA, Mathews DE, Bier DM, et al. Effects of protein restriction and acute refeeding on leucine and lysine kinetics in young men. Am J Physiol 1993;264:E567–75.[Abstract/Free Full Text]
43. Raguso CA, El-Khoury AE, Young VR. Leucine kinetics in reference to the feeding mode as three discrete meals. Metabolism 1999;48:1–10.[Medline]
44. Stoll B, Burrin DG, Henry JF, Jahoor F, Reeds PJ. Dietary and systemic phenylalanine utilization for mucosal and hepatic constitutive protein synthesis in pigs. Am J Physiol 1999;276:G49–57.[Abstract/Free Full Text]
45. Stoll B, Burrin DG, Henry J, Yu H, Jahoor F, Reeds PJ. Substrate oxidation by the portal drained viscera of fed piglets. Am J Physiol 1999;277:E168–75.[Abstract/Free Full Text]
46. Stoll B, Henry J, Reeds PJ, Yu H, Jahoor F, Burrin DG. Catabolism dominates the first-pass intestinal metabolism of dietary essential amino acids in milk protein-fed piglets. J Nutr 1998;128:606–14.[Abstract/Free Full Text]
47. Cepaldo B, Castaldelli A, Antoniello S, et al. Splanchnic and leg substrate exchange after ingestion of a natural mixed meal in humans. Diabetes 1999;48:958–66.[Abstract]
48. Wu G. Intestinal mucosal amino acid metabolism. J Nutr 1998;128: 1249–52.[Abstract/Free Full Text]

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