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A tracer investigation of obligatory oxidative amino acid losses in healthy, young adults^1,2,3

¹ From the Laboratory of Human Nutrition, School of Science and Clinical Research Center, Massachusetts Institute of Technology, Cambridge.

² Supported by NIH grants DK 15856 and DK 42101 and CRC core grant RR 88.

³ Address reprint requests to CA Raguso, Massachusetts Institute of Technology, Room E17-434, 77 Massachusetts Avenue, Cambridge, MA 02139. E-mail: cosetta{at}mit.edu.

	ABSTRACT

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Background: Estimation of the minimum requirement for indispensable amino acids (IAAs) has been attempted by assuming that obligatory oxidative losses (OOLs) of IAAs can be approximated from nitrogen losses and that the efficiency of utilization of IAAs at requirement intakes is 70%.

Objective: We wished to determine the rates of OOLs in healthy adults, using L-[1-¹³C]leucine and L-[1-¹³C, methyl-²H₃]methio-nine as tracers, after adjustment to a protein-free diet and how these rates compare with those when either sulfur amino acids (SAAs: methionine and cyst(e)ine) or leucine were removed from an otherwise adequate diet.

Design: Eleven subjects were randomly assigned to a 5-d protein-free diet or a 5-d diet providing adequate nitrogen and amino acids except for the SAAs or leucine. A 24-h constant intravenous infusion of [¹⁵N,¹⁵N]urea and L-[1-¹³C]leucine (Leu group; n = 5) or L-[1-¹³C, methyl-²H₃]methionine (Met group; n = 6 ) began at 1800 on day 5 and rates of amino acid oxidation were determined.

Results: Mean (±SD) oxidation rates (mg•kg^-1•d^-1) of methionine and leucine were 6.4 ± 1.4 and 24.7 ± 3.6, respectively, with the protein-free diet; rates were significantly lower (3.9 ± 2.2 and 7.2 ± 3.4, respectively) after the SAA- and leucine-free diets. Urea production was significantly lower (P < 0.01) with the protein-free than with the SAA- or leucine-free diet.

Conclusions: Isotopically determined OOLs for methionine and leucine are consistent with losses predicted from nitrogen excretion, and consistent with our previous measurements of cysteine oxidation as an index of total SAA losses. The data further support our earlier conclusions regarding methionine sparing by cysteine and tentative recommended SAA requirements in adults.

Key Words: Methionine metabolism • leucine metabolism • obligatory oxidative losses • indispensable amino acid minimum requirement • sulfur amino acids • cyst(e)ine • leucine • humans

	INTRODUCTION

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Irreversible oxidative losses of endogenous amino acids serve as a basis for determining and evaluating the nutritional requirements of indispensable amino acids (1–3). Millward et al (3–5) estimated the obligatory amino acid losses (OAALs) from obligatory nitrogen losses to develop a metabolic model with which the biological basis of protein and amino acid requirements might be better understood and assessed.

We used predictions of so-called OAALs to establish a tentative amino acid requirement pattern for healthy adults (1, 2). The OAALs, as defined earlier (2), are predicted from total obligatory nitrogen losses, which are measured after a relatively constant urinary nitrogen output is achieved in healthy adults receiving a protein-free but otherwise adequate diet (6, 7). In practice, this has been found to occur within 4–10 d of beginning a protein-free diet (6, 7). Assuming a relatively constant amino acid composition of mixed body proteins over the time frame of interest, obligatory losses of the individual indispensable amino acids would occur in proportion to their composition in mixed body proteins. Although a portion of this loss might be in the form of intact amino acids, as shown by the studies of Fuller et al (8) in which losses of amino acids via the intestine were estimated, it is clear that, for the most part, the quantitatively significant loss of amino acids from the body occurs via oxidative metabolism. We were interested in testing this hypothesis by measuring the rates of leucine and methionine oxidation, using ¹³C-labeled tracers and a 24-h tracer protocol (9, 10), to quantify the entire daily rate of oxidation when a protein-free diet is consumed.

Studies in rodents (11) and pigs (12) showed that when one indispensable amino acid is omitted from the diet, body weight loss or rate of nitrogen loss differs depending on which amino acid is removed from the diet. When SAAs are removed, the loss of body weight in adult female rats or the nitrogen excretion rate in young pigs are similar to those seen with a protein-free diet. On the other hand, with removal of only leucine from the diet, rats lose little body protein over a 6-wk period (11) and nitrogen output in the young pig is only 20% of that occurring during a protein-free diet (12). This finding clearly indicates a differential sparing of endogenous amino acids under specific dietary conditions. Thus, for leucine, the oxidation rate during the leucine-free diet would presumably be substantially below the so-called OAAL level. Hence, another aim of this study was to evaluate, in adult humans, the response of whole-body methionine and leucine oxidation when these amino acids were omitted from a diet that was otherwise adequate in indispensable amino acids, nitrogen, and essential nutrients. It was thought that this information would be useful for enhancing our understanding of the efficiency of dietary amino acid utilization and its relation to minimum physiologic requirements.

This study presents and discusses the results of 2 studies: 1 of methionine oxidation and 1 of leucine oxidation in healthy, young adults given a protein-free diet and a diet devoid of either SAAs (methionine and cyst(e)ine; Met group) or leucine (Leu group) but otherwise adequate in indispensable amino acids, nitrogen, and essential nutrients.

	SUBJECTS AND METHODS

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Subjects
A total of 11 young adults participated in this investigation. Six subjects were randomly assigned to the Met group (age: 23 ± 2 y; weight: 63 ± 10 kg; and height: 169 ± 7 cm) and 5 to the Leu group (age: 27 ± 6 y; weight: 71 ± 14 kg; and height: 178 ± 5 cm).

Subjects were screened by conducting a medical history and a physical examination. In addition, blood and urine samples were collected for routine biochemical and clinical screening at the Clinical Research Center (CRC) laboratories. The study and the consent form were approved by the Massachusetts Institute of Technology (MIT) Committee on the Use of Humans as Experimental Subjects and the Advisory Committee of the MIT CRC. Informed consent was obtained from each volunteer and they were paid for their participation in the study.

Protocol design
During the first 5 d of each experimental dietary period, the subjects received either a protein-free diet or a diet based on an L-amino acid mixture that was either devoid of the SAAs or leucine; total nitrogen and other indispensable amino acid intakes were sufficient to meet or exceed minimum physiologic requirements. A break period lasting from 1 to 4 wk was allowed between the 2 dietary periods and their sequence—protein-free diet and leucine- or SAA-free diet—was randomized. The compositions of the diets are given in Table 1, and subjects were given 3 meals each day at 0800, 1200, and 1800. Daily energy intake was constant for each subject, ranging from 172 to 188 kJ/kg (41 to 45 kcal/kg). No significant or persistent body weight changes occurred during the experimental periods. Nonprotein energy was provided as 40% fat (safflower oil and butter) and 60% carbohydrate (beet sugar and wheat starch). Nitrogen, 160 mg•kg^-1•d^-1, was supplied via the L-amino acid mixture (Table 2). Flavoring agents (Vivonex flavor packets; Norwich Eaton Pharmaceuticals, Norwich, NY) were offered to the subjects to improve the taste of the amino acid mixture. Vitamins, minerals, choline, and fiber were supplied as daily supplements (Table 1).

Three meals, each providing one-third of the daily intake, were given at 2000, 0600, and 1200. The constant infusion was terminated at 1800 on day 6. Blood and breath samples were collected every 30 min for determination of ¹³CO₂, plasma [¹³C]-ketoisocaproic acid (KIC), [¹⁵N,¹⁵N]urea, L-[1-¹³C, methyl-²H₃]methionine, and [1-¹³C]methionine enrichments and plasma leucine and methionine concentrations as described previously (9, 13). Blood was collected in chilled, heparin-containing tubes and centrifuged immediately; plasma was stored at -20°C until analyzed.

To allow the subjects to sleep between 0000 and 0600, blood but not breath samples were drawn for determination of ¹³CO₂ enrichment during this time interval. Breath and blood samples for ¹³CO₂ enrichment were collected as described previously (9) and stored at room temperature until analyzed by isotope ratio mass spectrometry (MAT Delta E; Finnigan, Bremen, Germany). Total carbon dioxide production was measured by indirect calorimetry (Deltatrak; SensorMedics, Yorba Linda, CA) for 25–30 min every hour.

Complete 24-h urine output was collected during the 6-d experimental period. Plasma and urinary urea nitrogen concentrations were determined as described previously (9) and urinary urea excretion was corrected for changes in the body urea pool (see below). Total urinary nitrogen was determined by micro-Kjeldahl analysis.

Sample analysis for isotopic content
We described previously, in detail, treatment of blood and expired air samples for determination of ¹³CO₂ isotopic enrichment (9) and analysis of plasma free methionine (13, 14) and urea and KIC (9) enrichments. Briefly, N-methyl-N-(tert-butyl-dimethylsilyl) trifluoracetamide (MTBSTFA; Pierce Chemical Co, Rockford, IL) was used to form the tert-butyl-dimethylsilyl (t-BDMS) derivative of methionine. For KIC, the equinoxalinol-t-BDMS derivative was formed by adding to the extracted KIC a solution of MTBSTFA and pyridine (1:1). A t-BDMS derivative of urea was also obtained by adding MTBSTFA to the dry, methanolic extract of urea. Isotopic enrichments were measured by using gas chromatography–mass spectrometry (HP 5890 Series II and HP 5988A; Hewlett-Packard, Palo Alto, CA). Methionine, [1-¹³C]methionine, and [1-¹³C, methyl-²H₃]methionine were monitored, respectively, at a mass-to-charge ratio (m/z) of 320, 321, and 324, respectively. KIC and [1-¹³C]KIC were monitored at m/z 259 and 260, respectively. Urea and [¹⁵N,¹⁵N]urea were monitored at m/z 231 and 233, respectively. The isotopic enrichment of the experimental samples was determined by multivariate spectral deconvolution (15) by using the observed abundances of known tracer-tracee combinations, with molar ratios from 0 to 0.1 as standards. The validation standards were analyzed before and after each set of unknowns to adjust for variations in instrument response. In this study, the t-BDMS derivatization approach afforded an average accuracy error and intersample precision of <7% each. All plasma enrichment values reported here are expressed as a molar ratio (%) above baseline (MPE).

Amino acid oxidation
The transsulfuration (TS) rate (methionine oxidation) was calculated as follows:

(1)

where CO₂ is the rate of carbon dioxide production (in µmol•kg^-1•30 min^-1), E¹³CO₂ is the enrichment of ¹³C in expired air, and E₁ and E₄ are the plateau plasma enrichments of [1-¹³C]- and [1-¹³C, methyl-²H₃]methionine, respectively.

The rate of leucine oxidation (µmol•kg^-1•30 min^-1) was calculated, as for methionine transsulfuration, as follows:

(2)

where E_KIC is the plateau plasma enrichment of KIC, assumed to be the intracellular metabolite of leucine. A plateau in plasma methionine and KIC enrichment was reached within 2 h from the beginning of the infusion; therefore, the oxidation rates of methionine and leucine from 0 to 120 min (first 4 half-hourly intervals) were assumed to be equal to the first half-hourly value of the observed plateau period. Whole-body, 24-h oxidation rates of methionine and leucine were then computed as the sum of 48 half-hourly intervals.

Some of the ¹³C liberated as ¹³CO₂ during oxidation of methionine or leucine is retained by the body and it is necessary to correct for this retention. Because the experimental conditions were similar to those in our previous studies (10), we used the [¹³C]bicarbonate recovery factors determined previously (10) to correct for ¹³C retention. Oxidation estimates of methionine and leucine were also corrected for ¹³CO₂ background enrichment (10). As in previous studies of methionine (13, 14), a correction factor was used to account for a likely plasma-intracellular gradient in the methionine tracer enrichment. In the past, we assumed that the intracellular enrichment of tracer methionine was 80% of the measured plasma enrichment of the relevant labeled species. When we applied this correction factor in our earlier studies of methionine kinetics (16), we found that it permitted a determination of methionine oxidation that was consistent with the rate anticipated for methionine intakes at which an equilibrium could be expected for body methionine balance.

Urea metabolism
Twenty-four–hour urea production (mg•kg^-1•30 min^-1) was computed as the sum of 48 half-hourly measurements obtained as follows:

(3)

where IR[¹⁵N₂]urea and E_tracer are the infusion rate and the enrichment of the tracer infusate, respectively, and E[¹⁵N₂]urea is plasma [¹⁵N₂]urea enrichment.

Urea nitrogen concentration was measured in the 24-h urine specimens. The urea nitrogen excretion rate was corrected for changes in the size of the body urea pool as described previously by Fern et al (17), with total body water (TBW) estimated according to the method of Watson et al (18). Hence, the urea nitrogen excretion rate (mg•kg^-1•d^-1) was computed as follows:

(4)

where 0–1440 min is the difference in plasma urea concentration from time 0 to 1440 min.

Urea nitrogen recycling was estimated as urea production/2.156 (to convert urea production to urea nitrogen production) and urea nitrogen excretion.

Total nitrogen excretion (mg•kg^-1•d^-1) was computed as follows:

(5)

where 8 mg•kg^-1•d^-1 accounts for unmeasured and miscellaneous nitrogen losses (19).

Statistical methods
Data are presented as means ± SDs unless otherwise specified. A paired t test was used for comparisons between the protein-free and SAA- or leucine-free diets. An unpaired t test was used for comparisons between the Leu and Met groups. A P value <0.05 was considered statistically significant.

	RESULTS

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION REFERENCES

 
The level and pattern of expired ¹³CO₂ and of either plasma KIC or methionine enrichment, respectively, during the 24-h period while subjects consumed the protein-free or SAA- or leucine-free diets are shown in Figures 1 and 2. From these data, estimates were made of methionine and leucine oxidation rates. Thus, results for the measured rates of methionine and leucine oxidation together with those for urea metabolism and nitrogen excretion are summarized in Table 3.

View larger version (31K): [in this window] [in a new window]   FIGURE 1. Mean (±SE) 13CO2 enrichment of the leucine (A; n = 5) and methionine (B; n = 6) groups given the protein-free diet () or sulfur amino acid (SAA: methionine and cyst(e)ine)–free or leucine-free () diet. The filled circles indicate when the 3 meals were given during the 24-h tracer study. APE, atom percent excess.

View larger version (32K): [in this window] [in a new window]   FIGURE 2. Mean (±SE) plasma 13C enrichment of plasma -ketoisocaproic acid (KIC) in subjects fed the protein-free (; n = 5) or leucine-free (; n = 5) diet, plasma enrichment of [1-13C, methyl-2H3]methionine in subjects fed the protein-free (; n = 6) or sulfur amino acid (SAA: methio-nine and cyst(e)ine)–free (; n = 6) diet, and plasma enrichment of [1-13C]methionine in subjects fed the protein-free (; n = 6) or SAA-free (; n = 6) diet. Filled circles indicate when the 3 meals were given during the 24-h tracer study. MPE, mole percent excess.

View larger version (29K): [in this window] [in a new window]   FIGURE 3. Mean (±SE) rates and patterns of leucine (n = 5) and methionine (n = 6) oxidation in subjects fed the protein-free () or sulfur amino acid– or leucine-free () diet. Filled circles indicate when the 3 meals were given during the 24-h tracer study.

The lower rates of irreversible oxidative losses of methionine and leucine during the SAA- or leucine-free diet than during the protein-free diet were reflected by differences in circulating plasma free methionine and leucine concentrations. As shown in Figure 4, leucine and methionine concentrations, respectively, were lower throughout the entire 24-h period of the leucine- and SAA-free diet periods than during the protein-free diet period.

View larger version (25K): [in this window] [in a new window]   FIGURE 4. Mean (±SD) plasma leucine (n = 5) and methionine (n = 6) concentrations in subjects fed the protein-free () or sulfur amino acid (methionine and cyst(e)ine)– or leucine-free () diet. Filled circles indicate when the 3 meals were given during the 24-h tracer study.

Rates of urea production, based on the [¹⁵N,¹⁵N]urea tracer method, showed trends that were in the direction of the total nitrogen excretion values. However, there were no significant differences between the Met and Leu groups during either the protein- or SAA- or leucine-free diet periods (Table 3).

	DISCUSSION

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION REFERENCES

 
This investigation was carried out to examine the relations between daily rates of methionine or leucine oxidation and nitrogen metabolism under conditions of a protein-free or specific amino acid–free diet. The reason for this interest was that these relations have been assumed for purposes of estimating OAALs and, in turn, the tentative requirements for some of the indispensable amino acids (1, 2). Furthermore, studies in rats (11) and young pigs (12) have shown that the irreversible OAALs can be reduced below the OAAL level, as defined previously (1), when diets otherwise adequate but devoid in single, specific indispensable amino acids are consumed. These previous studies suggest that this sparing of OAALs is greater for some indispensable amino acids than for others. For example, in growing pigs, Fuller et al (12) reported that rates of body nitrogen loss were greatest when sulfur amino acids were removed from the diet and least with omission of the individual branched-chain amino acids. In adult female rats, Said and Hegsted (11) observed that body weight loss was similar to that occurring with a protein-free diet when they fed diets free of either threonine, isoleucine, or methionine-cysteine, whereas those fed lysine- and leucine-free diets lost little weight. These results imply a greater sparing of OAALs for leucine and lysine than for the sulfur amino acids when otherwise adequate diets that are devoid in each of these particular amino acids are consumed. Hence, we were interested in determining the extent to which the measured rates of methionine or leucine oxidation might similarly be reduced in healthy human adults fed diets devoid in either the SAAs or in leucine compared with these rates when subjects are adjusted to a protein-free diet.

There was good agreement between the daily rates of both methionine and leucine oxidation and rates predicted from the measured rate of nitrogen excretion when the subjects were fed a protein-free diet for 5 d. This finding supports the notion that the major route of OAALs is via oxidative metabolism, with minor but measurable losses via sweat (21) and the gastrointestinal tract (8). In addition, these results imply that our 24-h tracer technique provided a reliable estimate of the daily rate of body methionine and leucine losses under these experimental conditions. Previously, we confirmed that our estimates of daily leucine oxidation in subjects consuming a generous amount of leucine are consistent with the predicted rates (nitrogen intake - nitrogen output) and when the dietary leucine-to-protein ratio is similar to that for mixed body proteins (9).

We found that there was a significant difference in urinary nitrogen output, after 5 d of the protein-free diet, between the Met and Leu groups, with that for the Met group being higher. It is likely that this was due to differences in the time needed for a relatively steady state of nitrogen output to be achieved after ingestion of the protein-free diet. In earlier studies we found that on average it took 4–6 d for subjects to achieve their likely obligatory urinary nitrogen output (6, 7), but for some individuals this could take as long as 10 d. In addition, there are variations (by up to 40%) in reported obligatory urinary nitrogen losses among groups of apparently similar, healthy, young adults (6). Nevertheless, because all subjects had received the protein-free diet for 5 d before the 24-h tracer study, it is clear that the oxidation rates reflected those for amino acids of endogenous origin.

We used OAAL estimates to predict the minimum requirements per kilogram per day for the indispensable amino acids (1, 2). OAALs were calculated to be 13 mg for the SAAs (methionine and cysteine) and 27 mg for leucine, assuming a total obligatory nitrogen loss of 54 mg•kg^-1•d^-1 (1). A 70% efficiency of utilization of dietary amino acids at minimum requirement intakes was also assumed from whole-body balance studies in humans (1), and so the minimum requirement for leucine was predicted to be 40 mg•kg^-1•d^-1. This value was used to develop our tentative MIT amino acid requirement pattern (MIT-AARP) for healthy adults (2). On the basis of the mean oxidative loss of leucine in the present study (22.2 mg•kg^-1•d^-1), based on nitrogen excretion, the leucine requirement would be 32 mg•kg^-1•d^-1. This value is slightly lower than our MIT-AARP value because subjects in the present study had obligatory nitrogen losses that were lower than the mean value generally accepted as indicative of good protein nutrition in healthy adults (19). Furthermore, if we included in this estimate an additional 3 mg•kg^-1•d^-1 to account for possible gastrointestinal amino acid losses (8) and 1 mg•kg^-1•d^-1 for the loss of free leucine via sweat (21), our tentative recommendation of 40 mg•kg^-1•d^-1 as the minimum leucine requirement of healthy adults would appear to be well supported. This value is 3 times greater than the 1985 FAO/WHO/UNU requirement for leucine (19), namely 14 mg•kg^-1•d^-1.

In addition to consideration of methionine losses and tentative SAA (methionine and cyst(e)ine) requirements, account should be made of the fact that methionine serves as a precursor of cysteine; therefore, methionine oxidation (transsulfuration) acts as a source of cysteine in addition to that supplied, preformed, via the diet. Hence, to predict the methionine requirement (in the absence of dietary cysteine) from the present value of 6.4 mg•kg^-1•d^-1 for methionine oxidation during a protein-free intake, we propose the following. To balance the loss of 6.4 mg (43 µmol) methionine•kg^-1•d^-1 alone, with an assumed retention efficiency for dietary methionine of 70%, the intake (and, therefore, at balance this would be the oxidation rate) is estimated to be 61 µmol•kg^-1•d^-1. However, this would not balance the total SAA loss because the cysteine concentration in the mixed proteins of the body is 1.7 times that of methionine, as noted above. Therefore, the total obligatory loss of sulfur from methionine and cysteine would be 43 and 74 µmol•kg^-1•d^-1, respectively, with a total obligatory oxidative loss from methionine and cysteine of 117 µmol S•kg^-1•d^-1. This value is similar to the daily rate of cysteine oxidation because we measured it directly with [1-¹³C]cysteine in healthy subjects given a protein-free diet (22). Hence, if 61 µmol methionine•kg^-1•d^-1 is given initially to balance the theoretical total methionine output of 61 µmol•kg^-1•d^-1, then 61 µmol cysteine•kg^-1•d^-1 will be made available, assuming a conversion efficiency of 100%, to partially meet the 74 µmol S•kg^-1•d^-1 loss from cysteine that occurs in the absence of an intake of methionine and cyst(e)ine. Thus, an additional intake of either methionine or cyst(e)ine would be needed to achieve a total SAA amino acid balance. Again, assuming a 70% retention efficiency for dietary methionine, an additional 19 µmol (2.8 mg) S•kg^-1•d^-1 as methionine would be required, for a total requirement in the absence of dietary cysteine of 12 mg•kg^-1•d^-1. This finding is also similar to our experimental observations, suggesting a mean requirement of 13 mg•kg^-1•d^-1 for methionine in the absence of cysteine (14, 23, 24). Alternatively, if dietary cysteine were to provide for the additional intake of SAA needed to balance obligatory SAA losses, it would require about an additional 2.2 mg•kg^-1•d^-1 as cysteine or the combined SAA intake would again be 12 mg•kg^-1•d^-1.

In summary, therefore, our findings with the protein-free diet support both our direct isotopic tracer balance estimates of the requirements for SAAs (23, 24) and leucine (25) and further support the use of OAALs to predict tentative amino acid requirements (1, 2). Additionally, our findings also support our recommendation that a greater proportion of the total SAA requirement be supplied as methionine rather than as cysteine (14). The total SAA requirement and proportion of methionine and cysteine needed to support SAA homeostasis effectively will require further careful investigation. Furthermore, in these calculations it was assumed that methionine serves as a highly efficient precursor for meeting the tissues' needs for cysteine. This may not be true (26), in which case the estimated methionine requirement of 13 mg•kg^-1•d^-1, in the absence of dietary cysteine, might be too low.

In agreement with studies in experimental animals, we found that when an otherwise adequate diet devoid of leucine was fed, the daily oxidation of this branched-chain amino acid was substantially lower than that measured when subjects were fed a protein-free diet. The apparent daily sparing of the irreversible oxidation of leucine was 70%. This compared with a value of 78% in young pigs in the nitrogen-balance study by Fuller et al (12). These investigators did not observe any substantial sparing of the nitrogen loss, compared with that occurring with a protein-free diet, when all SAAs were removed from a diet supplying nitrogen and other amino acids normally sufficient to approach an approximate nitrogen equilibrium. A similar conclusion was drawn from a study in adult female rats by Said and Hegsted (11).

In the present study of adult humans, we observed some sparing of the OAAL for methionine, but it was equivalent to only about half of that observed for leucine. Therefore, our findings do not agree entirely with those in adult rats and young pigs, although the metabolic basis for the apparent difference is unclear. It may be, in part at least, related to the fact that the animals used in both of these earlier studies had considerable growth potential remaining, possibly resulting in relatively greater metabolic demands for the SAA than would be the case for a grown, healthy adult.

The total urinary nitrogen output by the Met group when given the SAA-free diet was higher than that for the Leu group during the leucine-free diet. However, the increment in nitrogen output above that measured during the protein-free diet period was essentially identical for both groups. These nitrogen excretion results can thus be compared with the rates of methionine and leucine oxidation that occurred when the specific amino acid–free diets were given. Thus, a methionine oxidation rate of 3.9 mg•kg^-1•d^-1 would predict a negative nitrogen balance of 35 mg•kg^-1•d^-1 and the leucine oxidation rate would predict a negative nitrogen balance of 15 mg•kg^-1•d^-1. Although total-body nitrogen balances were not measured in this study, we approximated what they might have been on the basis of the nitrogen intake with each diet (160 mg•kg^-1•d^-1) and on total urinary nitrogen excretion plus assumed unmeasured losses of 8 mg N•kg^-1•d^-1 (19), which may be somewhat low. Mean nitrogen balances with the SAA- and leucine-free diets were approximated to be 41 and 15 mg N•kg^-1•d^-1, respectively. Hence, these reasonable approximations agree very well with those derived from the ¹³C-labeled amino acid tracer estimates.

Finally, rates of urea production were measured under conditions of protein-free and SAA- and leucine-free feeding. Mean urea production rates during the protein-free diet periods were 80 and 71 mg•kg^-1•d^-1 in the Met and Leu groups, respectively (P = 0.35). These rates increased to 330–350 mg• kg^-1•d^-1 with consumption of a diet supplying 160 mg N•kg^-1•d^-1, but devoid of either SAA or leucine. Hence, urea production is sensitive to nitrogen intake, as we showed previously at a higher range of dietary nitrogen intake (27). The percentage of urea produced that was hydrolyzed (difference between production and excretion) differed between the 2 groups, but in both cases the fraction hydrolyzed was no greater for the protein-free than for the nitrogen-containing, amino acid–free diets.

In conclusion, the present findings on the OAALs for methionine and leucine are consistent with those predicted from obligatory nitrogen losses, lending credence to our tracer-based estimates of daily methionine and leucine oxidation. The present data are also consistent with our previous estimates of cysteine oxidation (22), support our conclusions about limited methionine sparing by dietary cysteine (13, 14, 24), and strengthen our recommendations, based on the tracer balance approach, concerning the SAA requirement and the desirable proportions of methionine and cysteine to meet this requirement.

The significant, although differential, sparing of oxidative losses of methionine and leucine in response to a diet devoid of either amino acid but adequate in nitrogen and other indispensable amino acids deserves further study, especially with respect to underlying physiologic and biochemical mechanisms. In addition, the meaning of this response on estimates of amino acid requirements demands additional investigation for 2 reasons. First, Millward (28) used results from studies in which individual amino acids were removed from the diet of experimental animals to support his view that there are marked differences in the pattern of amino acids required for maintenance and growth, a conclusion that we have questioned (2, 29). Second, Millward and Rivers (4) used the observation that FAO/WHO/UNU (19) requirements for adults are significantly lower than OAALs to develop their metabolic model of protein and amino acid requirements.

	REFERENCES

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION REFERENCES

 

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