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American Journal of Clinical Nutrition, Vol. 72, No. 1, 122-130, July 2000
© 2000 American Society for Clinical Nutrition

Original Research Communications

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 adults1,2,3

Antoine E El-Khoury, Paulo CM Pereira, Sudhir Borgonha, Anibal Basile-Filho, Louis Beaumier, San Y Wang, Cornelia C Metges, Alfred M Ajami and Vernon R Young

1 From the Laboratory of Human Nutrition and Clinical Research Center, Massachusetts Institute of Technology, Cambridge, MA; Shriners Burns Hospital, Boston; and MassTrace Inc, Woburn, MA.

2 Supported by NIH grants RR88, DK 42101, and P-30-DK-40561; grants-in-aid from the Global Cereal Fortification Initiative, Tokyo; and Shriners Hospitals for Children.

3 Reprints not available. Address correspondence to VR Young, Massachusetts Institute of Technology, Room E17–434, 77 Massachusetts Avenue (for express courier: 40 Ames Street), Cambridge, MA 02139. E-mail: vryoung{at}mit.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Background: We proposed previously that the mean lysine requirement value is {approx}30 mg • kg-1 • d-1 rather than the proposed 1985 FAO/WHO/UNU estimate of the upper range of the requirement, which is 12 mg • kg-1 • d-1.

Objective: Our objective was to explore the 24-h pattern and rate of whole-body lysine [l-13C]oxidation and status of whole-body lysine balance in healthy, young adults given an L-amino acid diet supplying either a low lysine intake (14–15 mg • kg-1 • d-1) or an intermediate lysine intake (29 mg • kg-1 • d-1) for 6 d before a continuous tracer study with L-[1-13C]lysine.

Design: Five subjects received the low lysine intake, 6 subjects received the intermediate intake, and all were studied by using a standard 24-h oral tracer protocol that was described earlier for studies at a generous lysine intake.

Results: The rate of lysine oxidation was not significantly different between the 12-h fasted and 12-h fed states. The daily oxidation rate ( ± SD) was 27.9 ± 8.8 and 27.3 ± 17.6 mg lysine • kg-1 • d-1 for the low- and intermediate-intake groups, respectively (NS). Daily lysine balance was –12.4 ± 92 and 1.8 ± 17.7 mg • kg-1 • d-1, respectively (P < 0.025), for the low and intermediate intakes. The balance was significantly less than zero (P < 0.001) for the low intake.

Conclusion: The FAO/WHO/UNU lysine requirement value is not sufficient to maintain lysine homeostasis in healthy adults. From the results of this and tracer studies done by others, the mean lysine requirement of healthy adults was determined to be 30 mg • kg-1 • d-1.

Key Words: Lysine • oral tracer studies • amino acids • oxidation • healthy adults


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In our more recent investigations of the reassessment of the minimum physiologic requirements for the indispensable amino acids in healthy adults, we used a continuous, 24-h 13C-tracer protocol. We applied this approach to help further define the requirements for leucine (1–3) and the aromatic amino acids (phenylalanine plus tyrosine) (4–6), and we also presented results of a 24-h L-[1-13C]lysine tracer study carried out in healthy adult subjects receiving a generous intake of dietary lysine (7). Thus, to estimate minimum physiologic requirements for lysine it was considered desirable to conduct comparable tracer experiments in subjects who had received, for 6 d, lower amounts of dietary lysine. Hence, this investigation was carried out at 2 dietary lysine intakes. One was {approx}15 mg • kg-1 • d-1, slightly higher than the value of 12 mg • kg-1 • d-1 that was proposed in 1985 by the Food and Agriculture Organization/World Health Organization/United Nations University (FAO/WHO/UNU; 8) as being the upper requirement for lysine in healthy adults. The other test intake was 30 mg • kg-1 • d-1, which we proposed on the basis of limited tracer data (9) and on a predictive approach (10, 11) as being just sufficient to maintain body lysine balance and homeostasis in healthy adults. Here, we summarize the results of our investigation, which support results of our earlier studies on plasma amino acid responses and evaluation of other metabolic data (12) and results from studies involving the indicator amino acid oxidation method carried out in Toronto (13, 14) and Bangalore, India (15).


   SUBJECTS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Subjects
The subjects in these studies were students at the Massachusetts Institute of Technology (MIT) or from the community of the Boston-Cambridge area. The first study included 5 subjects (3 women, 2 men; weight: 64 ± 10 kg; height: 171 ± 8 cm; age: 21 ± 1 y) and the second study included 6 male subjects (weight: 80 ± 13 kg; height: 180 ± 8 cm; age: 23 ± 5 y). All subjects were nonsmokers in good health, as determined by medical history, physical examination, blood cell count, routine blood biochemical profile, and urinalysis.

Because there is no evidence to indicate that the lysine requirements of healthy men and women differ, women of child-bearing age were encouraged to volunteer and the tracer experiments were conducted during the 7–10 d period after the onset of menstrual bleeding. Women who were taking mild doses of contraceptive agents were not necessarily excluded from the study. A negative pregnancy test result was required 2–3 d before the study started.

From a dietary history and estimation of basal metabolic rates (8), the mean daily energy intake required to maintain body weight was determined to be {approx}188 kJ (45 kcal) • kg-1 • d-1 for these subjects. The subjects were asked to maintain their usual level of physical activity while avoiding excessive or competitive exercise. On the day of the 24-h tracer study (see below), energy intake was reduced by 20% to account for the reduced physical activity during this period. The purpose of the study and the possible risks involved were explained to each subject. The subjects signed a consent form and they were paid for their participation. The experimental protocol was approved by the MIT Committee on the Use of Humans as Experimental Subjects and the Advisory Committee of the MIT Clinical Research Center (CRC). The 24-h stable-isotope-tracer protocol required the admission of the subjects as inpatients to the MIT Medical Department.

Diet and experimental design
Each subject was given a diet based on an L-amino acid mixture for 6 d (Table 1Go). As shown in Table 1Go, the indispensable amino acid profile was close to that for chicken egg protein except that the amount of lysine was adjusted to supply (diet and tracer) 14–15 or 28–29 mg • kg-1 • d-1. The L-amino acids for these studies were obtained from Ajinomoto USA, Inc (Teaneck, NJ). Protein-free wheat-starch cookies and flavored drinks were given as the major source of energy, exactly as described in detail previously (2). Nonprotein energy was supplied as lipid ({approx}40%) and carbohydrate ({approx}60%). Beet sugar and wheat starch were the main sources of dietary carbohydrate to maintain a low 13C content in the diet and a steady background level of breath 13CO2 enrichment over the 24-h period (2). Blood, as well as breath, 13CO2 enrichments obtained during the tracer studies were corrected to account for the small changes in the background 13CO2 output that would be expected to occur without the [13C]lysine tracer. The amino acid mix provided a nitrogen intake of 160 mg • kg-1 • d-1 and on the infusion day the amount of lysine given as tracer was compensated for by an equivalent decrease in the unlabeled lysine content of the dietary amino acid mix so that total daily lysine input remained the same throughout. Other nutrients were given in sufficient amounts to meet or exceed recommended dietary allowances, as described previously (2). Dietary fiber was given as 20 g microcrystalline cellulose daily, and a choline supplement of 500 mg was given daily. During the 6-d diet period, the total daily intake was provided as 3 isoenergetic, isonitrogenous meals at 0800, 1200, and 1800. Each morning, body weight and vital signs were monitored. At least 2 of the 3 daily meals during the 6-d diet period were eaten at the MIT CRC under the supervision of the dietetic staff.


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  		TABLE 1. Composition of the L-amino acid mixtures used to study effects of low and intermediate lysine intakes on lysine kinetics and balance in healthy adults
 
Twenty-four–hour [13C]lysine tracer protocol
In this investigation, we used a primed, hourly oral dosing of tracer and a standard fasting-feeding design, the details of which were described previously (1). For each group of subjects, the last regular meal provided on day 6 was eaten at 1500. The subjects were admitted to the MIT Medical Department on day 6 and the tracer study started at {approx}1800. The subjects slept from {approx}0000 to 0600 of the next day (day 7). Then, 10 isoenergetic, isonitrogenous meals, one meal every hour, were given between 0600 and 1500 (day 7). These meals supplied, in total, the equivalent of the subject's prior 24-h dietary intake. The 24-h tracer study was terminated at 1800. Throughout the 24-h study the subjects remained in bed in a reclined position, except during sleep, when they were supine. With this protocol, the 24-h day was divided into 2 major 12-h metabolic periods; 12 h of fasting and 12 h of feeding. The rationale for and details of this design were presented earlier (1).

The L-[1-13C]lysine tracer (98 atom %), as the monohydrochloride, was obtained from Cambridge Isotope Laboratories (Andover, MA) and MassTrace, Inc (Woburn, MA). In all cases, the tracer was administered at a known rate of {approx}3.1 µmol • kg-1 • h-1; the prime dose was {approx}4.65 µmol/kg. The bicarbonate pool was primed with 0.8 µmol [13C]sodium bicarbonate/kg (99 atom %; Cambridge Isotope Laboratories). The oral tracer, prepared in water, was given each hour and the hourly dose was consumed in a volume of 8 mL. Blood samples ({approx}4 mL each) were drawn through a 20-gauge, 3.2-cm catheter that was inserted into a superficial vein of the dorsal hand or the 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 64°C to achieve arterialization of venous blood for 15 min before withdrawal of each sample. The patency of the sampling catheter was maintained by slow infusion of isotonic saline.

Indirect calorimetry
Total carbon dioxide production (•VCO2) and oxygen consumption (•VO2) rates were determined by using an indirect calorimeter (Deltatrac; SensorMedics, Anaheim, CA) with a ventilated-hood system. Measurements were carried out according to a standardized procedure at hourly intervals throughout the 24-h period (16).

Breath 13CO2 background enrichment and [13C]bicarbonate recovery
These studies were performed earlier (1) under essentially the same dietary conditions and in subjects similar to those in the present investigation. As described below, the calculations of oxidation took into account changes in 13CO2 background as well as the bicarbonate recovery factor for the present experimental conditions. As described previously (1), the 13CO2 background changes over 24 h were not expected to exceed 1.5 atom percent excess (APE) x 1000. Bicarbonate recovery factors were taken to be 0.77 for the 12-h fasting phase and 0.85 for the 12-h feeding phase of the experiment (1).

Collection of samples
Breath and blood samples were collected half-hourly after collection of 3 baseline samples at -15, -5, and 0 min, as described previously (1). Blood samples (2 mL) for 13CO2 analysis were collected at 30-min intervals between 0000 and 0600, to substitute for collection of expired air and determination of its 13C enrichment, to give the subjects more time to sleep. After the 2-mL blood sample was drawn through an indwelling line, it was injected immediately via a thin needle (0.5 x 16 mm) into a sodium heparin–coated, 15-mL, capped evacuated tube. Samples were then processed as described and validated previously (1). Blood samples (for analysis of lysine isotopic abundance and lysine concentrations; 4 mL) were drawn into tubes with heparin and centrifuged for 15 min at 1200 x g and 4°C. Plasma was stored at -20°C until analyzed.

Analysis of samples
The total carbon dioxide content of each tube (15 mL nonsilicon-coated glass tubes) was {approx}20 µmol. Samples were stored at room temperature until used for analysis by isotope ratio mass spectrometry (IRMS) (MAT Delta E; Finnigan, Bremen, Germany).

Isolation and analysis of free lysine in plasma (from 250 mL plasma) was as described previously (7). Analysis of plasma free lysine concentrations involved use of a Beckman system Gold HPLC apparatus (Beckman Instruments, Inc, Fullerton, CA) with a Beckman Gold data system. Norleucine was used as the internal standard (2, 3).

Evaluation of primary data
Lysine flux
The lysine flux (QL; µmol • kg-1 • 30 min-1) was calculated according to standard, steady state isotope dilution principles as follows, in the same way as described previously for leucine (4):


where i is the rate of tracer infusion (µmol • kg-1 • 30 min-1) and Ei and Ep are the isotope enrichments as mole fractions (over baseline) of the infusate and plasma amino acid, respectively.

Lysine oxidation
Lysine oxidation was computed for consecutive half-hour intervals to improve the accuracy of the 24-h lysine oxidation value because of the variable rate of lysine oxidation throughout the 24-h period. Furthermore, before the plateau in plasma [13C]lysine enrichment had been reached, during the first 120 min of the 24-h tracer study, lysine oxidation was assumed to be equal to that of the slightly later (120 min) half-hour interval. Therefore, lysine oxidation was measured directly for 44 of the total 48 half-hour intervals. For each half-hourly interval, lysine oxidation (µmol • kg-1 • 30 min-1) was computed as follows:


where


where a indicates the average of the 2 enrichments at the 2 time points determining the half-hour interval (this average was corrected for 13CO2 background, as described previously; 1) and Rb is the mean fractional recovery for the fasted and fed states of 13C in expired air from an infusion of [13C]bicarbonate (1), with an additional 10% indicating a 10% difference between the measured and probable rate of lysine loss based on our previous study (7). The [13C]lysine plasma enrichment is the average value for the 2 time points determining the half-hour interval. Moreover, for the fasted and fed states, •VCO2 over the time interval when it was not directly measured was derived as the arithmetic average of •VCO2 measured just before and just after the interval.

Lysine balance
Measured 24-h lysine balance (input – measured output) was computed as follows:


Measured output (µmol/kg) is the sum of the oxidation of the 48 half-hour intervals. For computation and data summary, we used EXCEL 5.0 (for the Macintosh; Microsoft, Redmond, WA).

Statistical methods and evaluation of the data
Paired t tests were used to compare data in the fasted state with that in the fed state within the same group of subjects. A group t test was used to compare rates of lysine oxidation and lysine balance between the subjects receiving the 2 different lysine intakes. A one-sample, one-tailed t test was used to compare the determined 24-h lysine balance with a zero value because our hypothesis was that a lysine intake of {approx}14–15 mg • kg-1 • d-1 would result in a negative body lysine balance and that the intake of 29 mg • kg-1 • d-1 would result in a zero balance. Values in text are means ± SDs unless otherwise stated. Data analyses were done by using SAS software, version 6 (SAS Institute, Cary, NC).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
[13C]Lysine kinetics
The patterns of change in plasma [13C]lysine and in the output of 13CO2 during the 24-h [13C]lysine tracer period for both diets groups are shown in Figure 1Go and Figure 2Go, respectively. These isotopic data were used to derive the variables of whole-body lysine metabolism discussed below.



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  		FIGURE 1. Temporal pattern for the 13C enrichment of plasma lysine over baseline for subjects receiving the intermediate ({triangleup}) and low (•) lysine intakes. ± 1 SD.

 


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  		FIGURE 2. Mean (±SD) 13CO2 production throughout the 24-h period for subjects receiving the intermediate ({triangleup}) and low (•) lysine intakes.

 
The pattern of change in whole-body lysine flux can be inferred from the data shown in Figure 1Go for 24-h plasma [13C]lysine enrichments. As summarized in Table 2Go, mean lysine fluxes during the fasted phase were 111 and 80 µmol • kg-1 • h-1 for the low and intermediate groups, respectively (NS), and 91 and 74 µmol • kg-1 • h-1 during the 12-h fed period (NS). In addition, the lysine fluxes did not differ significantly between the 12-h fed and 12-h fasted phases within each diet group (Table 2Go).


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  		TABLE 2. Summary of lysine flux and 13CO2 production rates of subjects given the L-amino acid diet supplying low and intermediate lysine intakes1
 
Lysine oxidation, based on plasma [13C]lysine as precursor and 13CO2 production, throughout the 24-h period is depicted in Figure 3Go. The summed rates of oxidation for each 12-h fasted and fed period for subjects given the low and intermediate lysine intakes are presented in Table 3Go. As shown in this table, daily lysine oxidation exceeded total lysine intake (diet + tracer) in the low-lysine group. Consequently, estimated daily lysine balances were negative at this lysine intake (significantly different from zero balance, P < 0.001). The estimated daily rate of lysine oxidation for the intermediate intake approximated that of the daily intake. Hence, the daily lysine balances for this group were not significantly different from zero (P > 0.1), although the individual variation was high.



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  		FIGURE 3. Mean (±SD) lysine oxidation throughout the 24-h period for subjects receiving the intermediate ({triangleup}) and low (•) lysine intakes.

 

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  		TABLE 3. Summary of lysine oxidation and balance, when plasma [13C]-lysine was used as the precursor, for subjects provided low and intermediate lysine intakes1
 
Finally, the pattern of change in plasma free lysine concentrations throughout the 24-h period for both intake groups is depicted in Figure 4Go. Within each group, there was significant variation among individuals. All mean values for plasma lysine concentrations tended to be higher for the intermediate- than for the low-intake group. There was also a trend in both groups for a progressive increase in the mean plasma concentration with progression of fasting and a decrease within 3 h of feeding. The amplitude of this variation with time and physiologic state approximated a 50–100% increase above the baseline values (during fasting) followed by a return to concentrations close to those for baseline (during feeding).



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  		FIGURE 4. The pattern for mean (±SD) plasma free lysine concentration throughout the 24-h period for subjects receiving the intermediate ({triangleup}) and low (•) lysine intakes.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This investigation was the first, to our knowledge, to explore the pattern and quantitative aspects of whole-body lysine kinetics over a continuous 24-h period in healthy subjects who have been given a relatively low ({approx}15 mg • kg-1 • d-1) or intermediate ({approx}29 mg • kg-1 • d-1) lysine intake for 6 d. This study also provided us with an opportunity to compare the present data with those obtained under comparable experimental conditions in healthy adult subjects receiving a generous lysine intake (77 mg • kg-1 • d-1) (7).

Thus, the 24-h pattern of lysine oxidation observed in the present experiment differed from that seen earlier in subjects consuming a generous lysine intake (7). In the present study, we observed only a small, nonsignificant mean difference in the rates of whole-body lysine oxidation between the 12-h fed and 12-h fasted states in both diet groups. In contrast, the lysine oxidation rate was shown to increase markedly above the fasting value when subjects received meals providing a generous lysine intake (7). This lysine intake–dependent response of the whole-body oxidation rate matches our earlier observations with both leucine (1, 2) and the aromatic amino acids (4–6).

Another observation worth pointing out is that the daily rate of oxidation was estimated to be essentially identical for the low and intermediate lysine intakes. Studies in sheep (17) and rodents (18, 19) showed that at intakes approximating a requirement intake of lysine and below, lysine oxidation is essentially constant. Above this physiologic requirement intake, oxidation increases with increased amino acid intake. In growing pigs, lysine oxidation did increase when diets supplied 0, 0.2, and 0.8 of the lysine requirement but even in this case the changes in lysine oxidation amounted to only 0.38 g/d with an 8.8-g change in lysine intake (20). Hence, our findings in healthy adults are consistent with the view that the 2 test lysine intakes studied (low and intermediate) fall in the range approximating a minimum physiologic requirement value and below.

The preceding point is further substantiated when the present oxidation data are evaluated with our previous data obtained at a generous lysine intake (77 mg • kg-1 • d-1; 7). At this latter intake, oxidation was determined to be, at minimum, 70 mg • kg-1 • d-1, although it was more likely to approximate the intake. Thus, when intake is reduced to {approx}30 mg • kg-1 • d-1 oxidation declines almost quantitatively, but when intake falls further oxidation remains the same. Thus, when our 2 lysine tracer investigations are viewed together it is apparent that the minimum physiologic requirement either approximates or may be higher than the intermediate lysine intake chosen for the present investigation.

The rate of lysine oxidation during the fasting period was {approx}15 mg lysine • kg-1 • 12 h-1 in both of the intake groups. This rate may be compared with the estimate of 26 mg • kg-1 • 12 h-1 for subjects receiving a generous lysine intake (7). This difference in the postabsorptive rate of lysine oxidation presumably reflects an adaptive reduction in the rate of uptake of lysine into the mitochondria (21), reduced enzyme activity, or both, possibly involving the mitochondrial matrix enzyme (22) L-lysine: 2-oxoglutarate reductase (23–25), which is responsible for the catabolism of lysine. The daily rate measured in the present experiment was 28 mg • kg-1 • d-1, which is similar to the predicted obligatory lysine loss, 30 mg • kg-1 • d-1 (10, 11). Hence, if lysine balance can be achieved at an intake of 29 mg • kg-1 • d-1, as our study suggests, then the efficiency of utilization of dietary lysine approaches 100% (ie, the intake needed to balance obligatory losses) at that intake. This is also consistent with the data from animal experiments that showed that when lysine is limiting in the diet it is particularly well conserved (20, 26, 27).

It might be argued that the mass of tracer lysine given during the fasted period would not have been efficiently retained because of the lack of a simultaneous input of the other indispensable amino acids during this period. Thus, if none of the tracer given was retained and all of it was immediately oxidized during the 12-h fast then, on that basis, our estimate of daily endogenous lysine oxidation for each group was overestimated. Balance, however, with correction for tracer input in this case (fasting), would be even more negative for the low-lysine group. Because the oxidation rate did not exceed, for any subject, the predicted obligatory rate of loss, it seems more likely that the tracer dose given during the fast was retained in the free lysine pools in tissues, perhaps in skeletal muscles, which can serve as a reservoir for retention of free lysine when intake exceeds immediate metabolic demands (28, 29). Therefore, it is reasonable that the lysine tracer given during the 12-h fast be included in the utilizable daily lysine intake for purposes of determination of daily lysine balance.

In sum, the present findings do not support the nutritional adequacy of the FAO/WHO/UNU upper lysine requirement for healthy adults of 12 mg • kg-1 • d-1 (8) and they also lead us to conclude that the proposed requirements derived from the classic nitrogen balance studies of Rose et al (30, 31) in healthy men are highly inadequate. According to the authors (31), these latter studies gave a mean lysine requirement of 8.8 mg • kg-1 • d-1; the requirement of 11.2 mg • kg-1 • d-1 was that for the subject with the highest need. The results of our present investigation further support the adoption of an estimated mean lysine requirement of {approx}30 mg • kg-1 • d-1 and we will return to this point below.

Two additional issues need to be raised here. First, our earlier short-term diet and nitrogen balance studies also indicated a lower requirement for lysine (12) than that reported here, and these lower estimates are in general agreement with the conclusions drawn from earlier nitrogen balance studies by many others (32). However, results from nitrogen balance experiments are difficult to interpret, especially for the purposes of estimating the dietary minimum physiologic requirement for lysine in healthy adults, as we discussed previously (33). For example, in college women, Fisher et al (34) determined that a lysine intake as low as 50 mg/d, or even 0 mg/d, would be sufficient to maintain nitrogen balance. These results contradict findings indicating that wheat proteins are limited by their lysine content in the nutrition of younger and adult humans (35, 36).

Bolourchi et al (37) found that balance could be achieved in adults given daily, for 50 d, a 12-g N ({approx}1 g protein • kg-1 • d-1) diet in which wheat proteins supplied {approx}90–95% of total nitrogen intake. We estimate from the data presented in their article that the mean lysine intake was 18 mg • kg-1 • d-1. However, to prevent weight loss, these investigators found it necessary to give their subjects a daily energy intake of 54 kcal/kg (226 kJ/kg). This high energy intake confounds the interpretation of their nitrogen balance data, as discussed previously (10), and also that by Rose et al (31).

Nevertheless, we also pointed out that nitrogen balance studies can be carried out usefully for comparative purposes and for assessing the relative nutritional quality of wheat proteins. Thus, our previous nitrogen balance findings (36) are entirely consistent with a prediction that the nutritional value of wheat protein approximates about half that of good-quality animal protein, such as beef, when it is based on a lysine requirement estimate of 30 mg • kg-1 • d-1 or 50 mg lysine/g protein (12). Furthermore, it is worth pointing to the balance study by Edwards et al (38), which involved giving adults, for 15–29 d, a diet that was based largely on wheat protein but supplemented with other plant foods so that the amount of lysine in the diet approximated 41 mg/g protein or an intake of {approx}26 mg • kg-1 • d-1. Their subjects maintained body nitrogen equilibrium, and in this context their findings would support our conclusions while recognizing that the daily lysine intake in the experiment by Edwards et al (38) exceeded the FAO/WHO/UNU requirement (12 mg/kg) by about 2 times as well as the mean requirement estimate (8.8 mg • kg-1 • d-1) suggested by Rose et al (31) by as much as 3 times.

Furthermore, with respect to the nitrogen balance studies and the minimum physiologic requirement for lysine, Millward (39) considered the nitrogen balance data of Jones et al (40) to be particularly useful for determining the lysine requirement of healthy adults. From his reassessment of these data he proposed a lysine requirement of 19 mg • kg-1 • d-1 (39). We also undertook a recent and more extensive statistical analysis of these nitrogen balance data (41). Again, the results of this statistical study supported our view that the minimum lysine requirement approximates 30 mg • kg-1 • d-1.

A second matter concerns the possibility that there is a significant microbial synthesis of lysine within the gastrointestinal tract and that this lysine (together with other indispensable amino acids) is made available to the host through release via microbial protein breakdown and its subsequent uptake from the intestine. Under the assumption that this occurs, it has been argued that in effect the measured whole-body rate of oxidation of an indispensable amino acid such as leucine or lysine would include a contribution made by the intestinally synthesized indispensable amino acid and therefore the 13C-tracer technique would overestimate the net loss of the amino acid from the body (42). There is some evidence to support the fact that in humans the lysine made by the intestinal microflora can appear in body fluids and proteins (43–46), which might then be excreted via urine (42). More data are available from studies in pigs and rats than in humans. In 20-kg growing pigs given a low-protein, highly fermentable carbohydrate diet, it was estimated that microbially derived lysine might account for 3–11% of the daily lysine requirement (45). In rats it appears that coprophagy is necessary to make microbially derived lysine available for host tissue metabolism (47). We confirmed in adult humans that lysine made de novo by the intestinal microflora appears in the peripheral blood circulation as free lysine (48). Preliminary and highly approximate estimates suggest that the daily amount of lysine absorbed via this source may be 12–20 mg • kg-1 • d-1, although there was a large variation between subjects. If accurate, these rates would be physiologically significant and they might also have important implications for [13C]lysine-based determinations of the lysine requirement of adults. However, we do not know whether this approximate rate of absorption of microbially derived lysine represents a net contribution to the total daily lysine intake because there is a significant entry of lysine into the intestinal lumen via endogenous protein secretions and cell turnover. This lysine may be unrecovered, in part, because of microbial and epithelial-mucosal cellular activity as well as its excretion via feces. In ileostomy patients, the lysine lost in feces was estimated to be {approx}4 mg • kg-1 • d-1 (49). However, these subjects were given a protein-free diet for 4 d before the determination, so the extent to which this estimate can be applied to healthy subjects who consume an adequate or generous amount of dietary protein remains uncertain. Furthermore, it appears that our [13C]lysine tracer model and, therefore, our estimate of oxidation, would not necessarily include this route of loss of lysine from the body.

If microbially derived lysine does make a net contribution to body lysine balance, it is reasonable to assume that microbial contributions would apply to other indispensable amino acids. However, if this were the case, it is difficult to understand how it was possible for us to arrive at a 13C-based carbon balance for leucine (1), which is at equilibrium or even in the positive range, when the dietary intake of leucine was supplied in supramaintenance amounts. If microbial leucine contributed to the 13C-based carbon balance estimate, it would be negative by an amount equal to the putative net contribution made by the microbial indispensable amino acid. Additionally, we were able to predict the total nitrogen excretion in subjects given an adequate nitrogen and amino acid intake from 13C-derived measurements of daily leucine (1) and lysine (7) oxidation. This result would not have occurred if the 13C-estimated rate of whole-body lysine (and leucine) oxidation had included a significant oxidative loss of lysine (or leucine) due to a putative net input to the body of the amino acid from a microbial source.

In summary, the findings emerging from this [13C]lysine tracer investigation did not support the adequacy of the FAO/WHO/UNU upper lysine requirement and add strength to our estimate of that requirement. Although our 24-h [13C]lysine tracer and balance studies included only 3 test lysine intakes, 14–15 and 29 mg • kg-1 • d-1 in the present investigation and 77 mg • kg-1 • d-1 in an earlier study (7), a proposed mean lysine requirement of 30 mg • kg-1 • d-1 is consistent with our reassessment of published nitrogen balance data (41). Furthermore, it is intermediate between the estimates of 23 and 27 mg • kg-1 • d-1 recently made by Millward (50) from the data of Fereday et al (51, 52), and the estimates by the Toronto group of 37 (13) and 45 mg (14) lysine • kg-1 • d-1, based on the indicator amino acid oxidation technique (53).

A working group convened in 1994 by the International Dietary Energy Consultative Group concluded in relation to adults that "the values for the amino acid requirements derived from experiments by Rose and collaborators are no longer acceptable or nutritionally relevant because of a series of well-identified methodological errors" (54). We propose a tentative requirement of 30 mg • kg-1 • d-1 or 50 mg lysine/g protein when the total protein intake is just sufficient to meet an FAO/WHO/UNU-estimated average requirement of 0.6 g protein • kg-1 • d-1. It appears prudent, on the basis of the present and related studies, to accept this proposition until more evidence can be gathered to favor new and possibly more definitive recommendations. It is now critical that we and others carry out further studies on the indispensable amino acid requirements in adult humans, especially in view of the importance such values have for the rational and safe planning of food supplies to meet the nutritional needs of the world's growing and future population (55).


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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Received for publication August 31, 1999. Accepted for publication January 3, 2000.




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