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Department of Physiology and Division of Nutrition, St. John’s Medical College, Bangalore, India and * Laboratory of Human Nutrition, Massachusetts Institute of Technology, Cambridge, MA 02139
2To whom correspondence should be addressed. E-mail: vryoung{at}mit.edu.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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2.5–5% of the leucine flux rate) in well-nourished Indian men. Healthy subjects (n = 10) in a fasting state were studied during three randomly administered infusions of different, known amounts of leucine, supplying 4.1, 6.6 or 8.3 mg/(kg · 12 h) during the 12-h fast. Mean 12-h leucine oxidation rate and leucine flux for the different levels of leucine infused did not change significantly (P > 0.1) for the three leucine infusion rates. The plasma leucine concentrations increased significantly after 12 h of leucine infusion, rising from between 20 and 50 µmol/L by the end of the infusions over the range of tracer input. We conclude that tracer leucine infused in the fasting state does not measurably increase leucine oxidation at the doses studied. Thus, tracer intake during the 12-h fast contributes to the effective leucine intake in 24-h DAAB studies.
KEY WORDS: • Indian adults • leucine • oxidation • balance
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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,2
) have been suggested to be 2–3 times higher than the current international recommendations (3
). In the case of leucine, our proposed new requirement value [40 mg/(kg · d)] was based initially on results of short-term tracer studies (4
,5
) and predicted obligatory losses (1
). It is now supported by more recent estimates of 24-h direct amino acid balance (24-h DAAB)3
at different levels of leucine intake (6
–8
). These studies showed that the requirement for body leucine equilibrium was far greater than the 1985 FAO/WHO/UNU (3
) value of 14 mg/(kg · d).
The 24-h DAAB approach for measurement of amino acid requirements has a potential model problem in terms of the fate of the tracer that is infused during the fasting state. In this model, with leucine as the test amino acid, the leucine intake found to be just sufficient to obtain equilibrium in 24-h leucine balance is judged to be the requirement level (6
). The intake of leucine that is taken into consideration for the calculation of balance is the sum of the dietary (given in the fed state) and tracer (administered in the fed and fasting state) leucine. The 24-h DAAB protocol is divided into a 12-h fasting and a 12-h fed state; thus, half of the tracer-based leucine intake is infused in the fasting state. Because no other nutrients are administered during the 12-h fast, it is possible that the tracer leucine is oxidized during this period, perhaps due to a mass action of the free leucine or an effect on the activity of branched-chain keto-acid dehydrogenase complex (9
,10
). If this were true, then it might be argued that the tracer leucine intake in the fasting state should not be considered to be nutritionally important; hence, it should not included in either the input side or its equivalent in the output side of the 24-h leucine balance calculation. In the 24-h DAAB experiment alluded to above (6
), this reasoning would effectively result in achievement of a neutral leucine balance at a leucine intake lower than the calculated intake of 40 mg/(kg · d) by the amount of leucine infused during fasting.
On the other hand, if the tracer leucine was retained in protein and perhaps more likely used to expand the body free leucine pool during the fasting state, then this tracer input would be available for net protein synthesis during the subsequent 12-h fed state, and, hence, be nutritionally significant. Therefore, we designed this study to assess the fate of the tracer 13C-leucine intake during the 12-h fast, so that an appropriate calculation could be made in estimating the 12-h fasting state leucine balance.
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SUBJECTS AND METHODS |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Young adult Indian men (n = 10) participated in this experiment. Their characteristics (mean height, 1.7 ± 0.1 m; weight, 60.0 ± 7.9 kg; body mass index, 21.4 ± 2.2 kg/m2; body fat, 17.1 ± 5.9%; arm circumference, 27.2 ± 2.3 cm) and the recruitment criteria were similar to those of the well-nourished subjects we have studied before. Anthropometric and skinfold measurements were carried out on the subjects on d 0 of the experiment, as described earlier (6
). The purpose of the study and the potential risks involved were explained to each subject, in his language. Signed consent was obtained from each subject, and the Human Ethical Approval Committee of St. John’s Medical College approved the research protocol.
Diet and experimental design.
During the 10-d experimental period, the subjects received a weight-maintaining diet based on an L-amino acid mixture containing 40 mg leucine/(kg · d). Daily energy intakes were designed to maintain body weight, and energy requirement was calculated to be 1.6 x basal metabolic rate on all days of the study. The major energy supply was given in the form of a sugar-oil formula and as protein-free, wheat starch cookies as described earlier (6
,11
).
The 12-h fasting 13C-leucine tracer studies were begun after the subjects had received the experimental diet for 4 d. These tracer experiments were carried out at one of three rates of infusion, i.e., 4.1, 6.6 and 8.3 mg leucine/(kg · 12 h) during the entire overnight fast. The tracer 13C-leucine infusion rate was constant in all three experiments, and leucine increments were delivered by co-infusing 2H3-leucine in differing amounts (see below). The subjects were randomly assigned to the order in which they would undergo the three tracer experiments. The first tracer experiment was performed on the night of d 4 of the experimental feeding period; the following two tracer experiments were performed on the nights of d 7 and 9, respectively. On the day before the tracer study, i.e., d 4, 7 and 9, the subjects consumed their last meal of the day at 1500 h.
Tracer-infusion protocol
The primed 12-h tracer-infusion began at 1800 h on d 4, 7 or 9 and lasted until 0600 h on d 5, 8 or 10, respectively. The primed, intravenous administration of 1-13C-leucine (99.3 atom %; MassTrace, Woburn, MA) was given at a known rate of 2.5 µmol/(kg · h); the prime was 4.2 µmol/kg, and was administered as a bolus at the start of the experiment. 2H3-leucine was co-infused in two of the experiments, at a rate of either 1.5 or 2.5 µmol/(kg · h), such that the confirmed total amount of leucine given in the entire 12-h fasting state was 4.1, 6.6 and 8.3 mg/(kg · 12 h). The bicarbonate pool was primed intravenously with 0.8 µmol/kg of 13C-sodium bicarbonate (99.9 atom%; MassTrace). The tracers were prepared in physiologic saline, under sterile conditions.
Indirect calorimetry and analysis of breath and blood samples.
Total carbon dioxide production (VCO2) and oxygen consumption (VO2) were determined hourly, with the aid of an open circuit indirect calorimeter, as previously described (6
,12
). Breath and blood samples were collected every hour throughout the 12-h protocol. The breath samples were analyzed for their 13CO2/12CO2 ratio by isotope ratio mass spectrometry (Europa Scientific, Crewe, UK), as described earlier (6
,11
). A correction was made for the small contribution of dietary and endogenous 13C-substrate oxidation over the 24-h study period, as described earlier, and the estimates of 13CO2 corrected for recovery, also as described earlier (11
). Plasma was analyzed for leucine concentrations, and 13C-enrichments of plasma 
-keto-isocaproate were as described previously; these values were used to calculate leucine oxidation and flux (6
,13
).
Statistical methods and data evaluation.
Data are presented as means ± SD. The metabolic variables were regressed as a linear function of total leucine tracer infused using random effects regression models, and the 95% confidence interval (95% CI) of the slope was calculated. Plasma leucine concentration was regressed as a linear function of total leucine tracer infused, which was interacted with time (i.e., pre- or postinfusion). A two-sided P-value of <0.10 indicated a significant interaction; when the interaction was significant, then pre- and postinfusion slopes and 95% CI were calculated. Each model also assessed the effect of the study day. The data analysis used SAS version 8.2 (SAS Institute, Cary, NC).
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RESULTS |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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and Fig. 1
for individual data) [slope = 0.33 mg/(kg · 12 h); 95% CI: -0.18, +0.83]. Fasting leucine balance increased (P = 0.012) with increasing total tracer infused.
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. About 13% of the leucine flux was associated with oxidation and this did not differ (P = 0.21) with increasing tracer infusion rate.
Plasma leucine concentrations were determined on 6 of the 10 subjects. The interaction between total tracer infused and timing (pre- vs. postinfusion; n = 6) was significant at the 0.1 level (P = 0.0970); plasma leucine concentration did not differ between leucine infusion levels preinfusion (P = 0.73), whereas plasma leucine increased postinfusion (P = 0.01) within increasing total tracer infused (Table 1)
.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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40 mg/(kg · d) be used for the adult requirement of this amino acid (14
). However, because the oxidation of the tracer in the fasting state could theoretically range from none to complete oxidation, this could contribute to a variable effective 24-h leucine intake in our 24-h DAAB experiments (6
–8
). Because the amount of leucine tracer that is infused in the fasting state is 5 mg/(kg · 12 h), an assumption of complete oxidation of the fasting tracer intake would result in a lowering of our estimate of the daily leucine requirement from 40 to 35 mg/(kg · d). It was therefore considered important to provide a direct, quantitative estimate of the fate of the infused 13C-tracer during a 12-h fast. An elegant way to determine whether the 13C-tracer contributes to the oxidation rate would be to compare separate estimates of oxidation using the finite amount of 13C-tracer vs. that obtained with a massless 14C-tracer. However, in the absence of ethical approval to use a radiolabeled tracer, we explored the question by giving different doses of leucine within a range of tracer infusion rates that is common in experiments of this kind.
Thus, we observed that the 12-h leucine oxidation rates at the tracer input rates of 4.1, 6.6 and 8.3 mg/(kg · 12 h) intakes did not differ. There were also no differences in the endogenous leucine flux rate or in the oxidation:flux ratio. This confirms that when assigning effective leucine intake values for tracer-based estimates of leucine balance (i.e., intake - oxidation = balance), the entire leucine intake (diet plus tracer) should be used. The retained leucine tracer presumably expands the body free leucine pool in the absence of a change in net protein synthesis. In the present instance, the total amount of leucine infused over the 12 h ranged from 1.8 to 3.6 mmol for the subjects with mean weight of 60 kg. If we assume a total body water of 60% of body weight and even distribution of the leucine in this space, this would mean an increase in leucine concentration of 50–100 µmol/L. However, the plasma increase was less than this, indicating that the leucine was probably concentrated at a somewhat higher level in the free leucine pools in muscle and liver. Nevertheless, the change in concentration of leucine in the intracellular water of muscle that would be expected with the retention of this range of tracer input appears to be well within the range of transient changes observed after ingestion of protein-free or protein-rich meals (15
). Thus, the rates of 13C-leucine tracer infusion that we used during the 12-h fast appear to be well accommodated by expansion of the intracellular leucine pools.
Earlier experiments also showed no significant effects on leucine oxidation or flux, but small and significant changes in plasma leucine concentration when leucine was infused at a dose of up to 10% of the leucine flux in humans (16
). In contrast, leucine oxidation was stimulated only when leucine was infused at much higher doses of 28–56 µmol/(kg · h) in humans, in which the slope of the relationship between leucine oxidation and infusion rate suggested that between 50 and 75% of the infused dose was oxidized (17
). In comparison, the present study assessed doses of leucine that are typically employed in our 24-h tracer experiments, with doses of 2.5–5 µmol/(kg · h), which is 2.5–5% of leucine flux. This is in support of our view that no reduction in total leucine intake should be made for tracer input during the fasting state when assigning an intake value that corresponds to a zero leucine balance. Hence, we conclude, on the basis of this and our earlier studies (6
–8
), that 40 mg leucine/(kg · d) is the best approximation of the leucine requirement as determined by the 24-h DAAB approach.
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FOOTNOTES |
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3 Abbreviations used: DAAB, direct amino acid balance.
Manuscript received 12 January 2002. Initial review completed 1 March 2002. Revision accepted 21 March 2002.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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1. Young, V. R., Bier, D. M. & Pellett, 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.
2. Young, V. R. & Borgonha, S. (2000) Nitrogen and amino acid requirements; the Massachusetts Institute of Technology amino acid requirement pattern. J. Nutr. 130:1841S-1849S.
3. World Health Organization (1985) FAO/WHO/UNU Expert Consultation. Energy and Protein Requirements. WHO Technical Report no. 724 1985 WHO Geneva, Switzerland. .
4. Meguid, M. M., Mathews, D. E., Bier, D. M., Meredith, C. N., Soeldner, J. S. & Young, V. R. (1986) Leucine kinetics at graded leucine intakes. Am. J. Clin. Nutr. 43:770-780.
5. Marchini, C. J., Cortiella, J., Hiramatsu, T., Chapman, T. E. & Young, V. R. (1993) Requirements for indispensable amino acids in adult humans: longer term amino acid kinetic study with support for the adequacy of the Massachusetts Institute of Technology amino acid requirement pattern. Am. J. Clin. Nutr. 58:670-683.
6. Kurpad, A. V., Raj, T., El-Khoury, A. E., Kuriyan, R., Maruthy, K., Borgonha, S., Chandukudlu, D., Regan, M. M. & Young, V. R. (2001) The daily requirement for, and splanchnic uptake of, leucine in healthy adult Indian subjects. Am. J. Clin. Nutr. 74:747-755.
7. El- Khoury, A. E., Fukagawa, N. K., Sanchez, M., Tsay, R. H., Gleason, R. E., Chapman, T. E. & Young, V. R (1994) The 24h pattern and rate of leucine oxidation, with particular reference to tracer estimates of leucine requirements in healthy adults. Am. J. Clin. Nutr. 59:1012-1020.
8. El-Khoury, A. E., Fukagawa, N. K., Sanchez, M., Tsay, R. H., Gleason, R. E., Chapman, T. E. & Young, V. R. (1994) Validation of the tracer-balance concept with reference to leucine: 24-h intravenous tracer studies with L-[1-13C]leucine and [15 N-15 N]urea. Am. J. Clin. Nutr. 59:1000-1011.
9. Harris, R. A., Kobayashi, R., Murakami, T. & Shimomura, Y. (2001) Regulation of branched-chain -keto acid dehydrogenase kinase expression in rat liver. J. Nutr. 131:841S-845S.
10. Shimomura, Y., Obayashi, M., Murakami, T. & Harris, R. A. (2001) Regulation of branched-chain amino acid metabolism: nutritional and hormonal regulation of activity and expression of the branched-chain -keto acid dehydrogenase kinase. Curr. Opin. Clin. Nutr. Metab. Care 4:419-423.[Medline]
11. Kurpad, A. V., El-Khoury, A. E., Beaumier, L., Srivatsa, A., Kuriyan, R., Raj, T., Borgonha, S., Ajami, A. M. & Young, V. R. (1998) An initial assessment using 24 hour 13C-leucine kinetics, of lysine requirements of adult man. Am. J. Clin. Nutr. 67:58-66.[Abstract]
12. Shetty, P. S., Sheela, M. L., Murgatroyd, P. R. & Kurpad, A. V. (1987) An open circuit indirect whole body calorimeter for the continuous measurement of energy expenditure of man in the Tropics. Indian J. Med. Res. 85:453-460.[Medline]
13. Kurpad, A. V, Raj, T., Regan, M. M., Vasudevan, J., Caszo, B., Nazareth, D. & Young, V. R. (2002) Threonine requirements of healthy Indian adults, measured by a 24h indicator amino acid oxidation and balance technique. Am. J. Clin. Nutr. :-in press.
14. Upcoming Expert Consultations FAO 2001, http://www.fao.org/es/ESN/require/upcoming.htm [accessed 8 December, 2001].
15. Bergstrom, J., Furst, P. & Vinnars, E. (1990) Effect of a test meal with and without protein on muscle and plasma free amino acids. Clin. Sci. (Lond.) 79:331-337.[Medline]
16. Tessari, P., Tsalikian, E., Schwenk, W. F., Nissen, S. L. & Haymond, M. W. (1985) Effects of [15N] leucine infused at low rates on leucine metabolism in humans. Am. J. Physiol. 249:E121-E130.
17. Schwenk, W. F. & Haymond, M. W. (1987) Effects of leucine, isoleucine, or threonine infusion on leucine metabolism in humans. Am. J. Physiol. 249:E121-E130.
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