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8. Results of the MIT tracer balance studies

8.1. Initial studies (1986)

The initial tracer balance studies were concerned with leucine (Meguid et al, 1986a), lysine (Meredith et al, 1986), valine (Meguid et al, 1986b), and threonine (Zhao et al, 1986). The test amino acids were fed at six to nine different levels. The estimates of IAA requirements derived from these experiments were given in Table 1A. The problem with these studies is that no correction was made for precursor enrichment; it is not clear whether in calculating the balance the dose of tracer was included on the input side; and oxidation in the fasted state was not measured directly but estimated from oxidation at the lowest level of amino acid intake. Even a small error in the estimate of fasting oxidation can have quite an important effect on the calculated balance. This can be corrected retrospectively from later MIT studies in which fasting oxidation was measured directly. Those results are shown in Figure 3 and Table 8. In Table 9 the left column gives the balances recorded in the original papers; in the right-hand column I have attempted to make corrections for these various sources of error. For valine and threonine the recalculation makes very little difference to the estimated intake for zero balance. For leucine it increases the estimate; for lysine the balances become more negative.

I do not claim that these recalculated estimates are any more valid than the original ones. I think that the lesson from this exercise is that quite small variations in fasted oxidation may have quite a profound effect on the tracer balance.

Table 8 Fed and fasted oxidation rate: data from the MIT studies

In this table, where several studies have been done at the same intake, the results have been averaged.
^a Oxidation rates corrected on the assumption that APE KIC/APE leucine = 0.8, since APE KIC was not measured in the original studies.

Table 9 Original and recalculated balances from MIT studies (1986)

Recalculated on the assumptions:
1. Fed-state oxidation rates divided by 0.8 as a correction for precursor activity.
2. Fasted-state oxidation rates calculated from values shown in Table 8; taken as 16 mmol/kg/h for intakes above 20, and as 13 mmol/kg/h for intakes of 20 or below. Infused tracer deducted from these estimates of oxidation in calculating balances.
The values used in the original paper were (mmol/kg/h); leucine 7, lysine 5, valine 4, threonine 5 for net oxidation after deducting input of tracer.
* a and b are replicate studies.

8.2. Further MIT studies with leucine

A number of other studies have been undertaken at MIT to validate the original results and to meet certain criticisms. All these were done with leucine and the calculations based on the enrichment in plasma of a-ketoisocaproic acid (KIC), the transamination product of leucine, as a more valid measure of precursor activity. In most cases the protocol was extended to an 8 h infusion, with 3h in the fasted and 5h in the fed state. In the results quoted here, extrapolation to 24 h has been done by the preferred (Marchini et al, 1993) method 1, so there may be some differences from the figures given by the authors.

(i) The experiments of Cortiella et al (1988) were designed primarily to compare results obtained with intravenous and intragastric tracers. This aspect is considered in section 10. Measurements were made at four levels of leucine intake, but fasting oxidation was determined only at the lowest intake (10 mg/kg/d). This study differed from previous ones in that it was the oxidation of the intragastric tracer that was measured. The results, as far as concerns the tracer balances, are shown in Table 10.

(ii) Young et al (1987) measured leucine balance after 1 week on either the FAO or the MIT level for leucine (see Table 1). The special point about this experiment is that the measurements were repeated after 3 weeks on the diets, and again during recovery. This part of the study will be discussed later under the heading 'adaptation'. Here I report only the results after the usual period of 1 week on the diets (Table 11).

(iii) The criticism had been made that in the amino acid diet modelled on egg the relatively high amounts of valine and isoleucine would stimulate the branched chain dehydrogenase complex and artificially elevate the rate of leucine oxidation. Pelletier et al (1991a,b) made measurements in both fed and fasted states at the FAO and MIT levels of leucine requirements (14 and 38 mg/kg/d). Varying the concentrations of valine and isoleucine in the diet had no effect on leucine oxidation at either level of intake. The dehydrogenase complex appears to be more sensitive to leucine or KIC than to the other branched chain amino acids. In a separate experiment oxidation of valine was measured (from the APE of the precursor KIV) and found to be unaffected by leucine intakes between the minimum physiological requirement and what would be supplied by a good quality diet.

Table 10 Results of Cortiella et al (1988) with graded levels of leucine intake given intragastrically

¹³C-leucine was given intragastrically and results calculated from the APE of ¹³C-KIC in plasma.
Fasted-state oxidation was measured only at the 10 mg intake and amounted to 15 mmol/kg/h. This value, less the tracer, has been used for calculating the balances at the other levels.

Table 11 Leucine balances at FAO (14 mg/kg/d) and MIT (38 mg/kg/ d) levels of intake
Group 1: studies in which the total amino acid intake was kept at a level corresponding to about 0.8 g egg protein/kg/d, and only the level of the test amino acid was altered.
Group 11: studies in which all the IAAs were fed in amounts equivalent to the FAO or MIT pattern.

^a Corrected assuming precursor factor of 0.8.
^b 24 h infusions.
^c non-essential nitrogen.
All studies were made after 6 days on the diet. Except for that of El Khoury et al, the protocol involved an 8h infusion: 3h fasted followed by 5 h fed with measurements of oxidation during the last hour of each period.

(iv) The experiments of Hiramatsu et al (1994a) were designed to test the effect on the oxidation of the IAAs of altering the dietary intake of non-essential nitrogen. Leucine, phenylalanine and tyrosine kinetics were studied, but only the results with leucine will be reported in this section.

The first phase of the study involved four diets; in the first two all the IAA were fed at the FAO level and the nonessentials were provided in amounts that would bring the total N to the equivalent of either 0.65 or 1 g protein/kg/d. With the next two diets the same procedure was followed but all the IAAs were fed at the MIT level. In a second study the IAAs were given at the MlT level and the total N was brought up to the equivalent of 1 g protein/kg/d with varying amounts of glutamine, ranging from 0 to 100% of the non-essential nitrogen. The glutamine content of the diet made no difference to the leucine balance. The results are shown in Table 11

(v) Marchini et al (1993) adopted the same procedure as Hiramatsu et al (1994a) in which all the IAAs were fed at either the FAO or the MIT level. A control group received the egg pattern of amino acids. The total N intake was made up to the equivalent of 0.9 g protein/ kg/d. The ratio of IAAs to total AAs was 0.11 for the FAO diet, 0.21 for MIT diet and 0.53 for the egg diet. As with the study of Young et al (1987), the diets were fed for 3 weeks and measurements of leucine balance made at 1 week, 3 weeks and after recovery; 80 infusions in all, a phenomenal experiment. Only the findings at 1 week are reported here (Table 11). Those at 3 weeks are discussed under the heading of 'adaptation'.

(vi) Finally, we have two papers by El Khoury et al (1994a,b) in which the infusion of leucine was given for 24 h 12 h fasting and 10h fed, followed by a further 2h in which no food was given. In the first study leucine was provided at a single generous level (80 mg/kg/d). The object of the experiment was to check a number of technical points: the baseline ¹³CO₂ enrichment was measured for 24 h to check the isotope abundance of carbon in the food, and a correction applied for this baseline; new correction factors were calculated for ¹³CO₂ recovery during fasting and feeding (see Table 8); the 24 h infusion allowed a check on the accuracy of extrapolation over 24 h from the shorter infusion protocol of the previous studies. The observed oxidation of leucine over 24 h showed satisfactory agreement with that predicted from the third hour of fasting and the fifth hour of feeding. Finally, the opportunity was taken to measure urea production with ¹⁵N¹⁵N urea and to compare the oxidation of leucine with that of total N (see section 9).

The second paper by El Khoury et al explored further the differences between the FAO and MIT leucine intakes when the infusions were continued for 24 h For both levels of intake the 24 h oxidation predicted from the third hour of fasting and the fifth hour of feeding was 12% greater than the measured oxidation over 24 h. Somewhat better agreement was obtained when the fasting oxidation was taken as that observed 12 h after the last meal (Table 7).

These studies show very beautifully the temporal changes with fasting and feeding in the enrichment of plasma KIC and of CO₂ in breath (see section 7, Figure 4). The magnitude of deviations from baseline was greater, the higher the leucine intake, in broad agreement with the predictions of Millward (1992) and the findings of Price et al (1994).

8.3. Tracer balances with other amino acids

Lysine. In the experiments of Meredith et al (1986) the requirement for zero balance was between 20 and 39 mg/kg/d. Halliday & McKeran (1975) found that with unprimed infusions it took some 12 h for lysine labelling to reach a plateau, and this is the order of time that one would expect from the large size of the free lysine pool (Waterlow & Fern, 1981). It seems odd that in Meredith's experiment, with a prime equivalent to 2.5h of infusion, plateau should have been reached in 2h. Perhaps there is a large pool of free lysine which mixes only slowly with the pool into which the infusion is given, producing a situation like that found with glutamine (Daumann et al, 1986). However, the flux and synthesis rates calculated from Meredith's data are entirely reasonable.

The only other isotopic study of lysine requirement in man* is that of Zello et al (1993), which was based on what may be called a 'reverse breakpoint' analysis (see section 11). In this system phenylalanine was used as an indicator amino acid and its oxidation measured at different levels of lysine intake. The idea is that at inadequate lysine intakes the rate of uptake of phenylalanine into protein will be reduced and that of oxidation will be increased; oxidation will fall to a minimal level when the lysine intakes become adequate. Analysis of the data fitted by least squares produced two straight lines which met at a lysine intake of 37 mg/kg/d.

* Studies of lysine turnover in relation to protein intake have been made by Conway et al (1980) and Motil et al (1981a,b)

A criticism of this approach might be that the test diets were only fed for the day on which measurements were made, since Zello maintains that a period of adaptation is unnecessary. However, in confirmation of his result, calculation of protein synthesis rates from the data of Meredith et al (1986) show an abrupt fall at lysine intakes below 35 mg/kg/d (section 12; Figure 9).

Threonine. Table 9 shows very low rates of oxidation of threonine, so that the requirement for zero balance is only of the order of 10 mg. My calculation assumes a precursor correction of 0.8. Ballevre et al (1990), working on young pigs, found a value of 0.76 for the ratio of specific radioactivity of threonine in liver to that in plasma, so that calculation should not involve a serious error. However, threonine is catabolized by two routes: (1) through ketobutyric acid to propionic acid + CO₂ and (2) to glycine and acetyl CoA. In the pigs of Ballevre et al (1990) on high threonine intakes of about 150 mg/kg/d, 80% of threonine oxidation was through the glycine pathway, and an increase in oxidation with increasing intake occurred almost exclusively through this pathway. If the same conditions hold in man, CO2 production will seriously under-estimate threonine oxidation, so that even the requirement of 15 mg/kg/d suggested by Young et al (1989) may be an underestimate. It is noteworthy also that intestinal secretions are rather rich in threonine. Measurements on ileostomy fluid showed a daily loss by this route of 300 mg, or 5 mg/kg/ d (Fuller et al, 1994).

Table 12 Valine balance at different levels of valine and leucine intake. Data of Pelletier et al (1991a)

Valine. The study of Meguid et al (1986b) showed a valine requirement of about 16 mg/kg/d (Table 9). Pelletier et al (1991a) measured valine balance at two levels of valine intake, 10 and 20 mg/kg/d, and two levels of leucine intake, 40 and 80 mg/kg/d. For precursor activity they used the labelling of 1-keto-isovaleric acid in plasma, so there can be no criticism of the results on that score. The results tend to confirm those of Meguid et al. At the lower intake of valine the higher level of leucine significantly depresses the valine balance (t = 3.07).

Methionine. Two recent papers provide new information on the requirement for methionine. In both of them the precursor factor was taken as 0.8. In the first study (Young et al, 1991) methionine was fed at the single level of 13 mg/kg/d, and balance was just achieved. In the second experiment (Hiramatsu et al, 1994b) methionine was fed at two levels, 13 and 6.5 mg/kg/d, and at the lower level cystine intake was varied from 0 to 21 mg/kg/d. Table 13 shows the balances extrapolated by the authors to 24 h by a method that I consider incorrect, compared with those recalculated correctly. This is a good illustration of the uncertainty introduced by the method of extrapolation.

Phenylalanine. The first step in the catabolism of phenylalanine is its hydroxylation to tyrosine. Clarke & Bier (1982) established the method for measuring phenylalanine hydroxylation, and Thompson et al (1989) suggested that it would be a useful method for studying whole body protein turnover in situations where the collection of expired CO₂ was not possible. An alternative approach is to measure the production of ¹³CO₂ from phenylalanine labelled in the 1-carbon position. It seems that this oxidation only occurs after the conversion of phenylalanine to tyrosine. However, if say 20% of the phenylalanine flux is oxidized and 20% of the tyrosine flux is oxidized, the overall oxidation would be only 4%, and ¹³CO₂ production would grossly underestimate phenylalanine oxidation. Zello et al (1990) therefore proposed that the tyrosine derived from phenylalanine was preferentially oxidized, without mixing with the free pool of tyrosine. This introduces an additional complication, particularly since the results of Cortiella et al (1992) showed that the oxidation rate estimated from ¹³CO₂ was 10-15% lower than the rate of hydroxylation.

Table 13 Methionine balance. Data of Young et al (1991) and Hiramatsu et al (1994b)

Recalculated by correct method of extrapolation (see section 7.5)

In the opinion of Millward (personal communication) Zello's data are uninterpretable.

There are three studies which allow calculation of the tracer balance from the hydroxylation rate: those of Cortiella et al (1992); of Hiramatsu et al (1994a); and of Price et al (1994). The results that we can glean from them are shown in Table 14.

In none of the balances listed in this table has a correction for the precursor been applied. Price et al (1994) calculated that the hydroxylation rates should be divided by 0.69 in the fasted and 0.56 in the fed state. Application of these factors, however, leads to impossibly negative balances. Table 14 suggests that the requirement for phenylalanine for balance is in the region of 20 mg/kg/d, when tyrosine is provided in the same proportion as it occurs in protein.

In addition, Zello et al (1990) have carried out a study based on the principle of breakpoint, which does not require an estimate of precursor activity (section 11). Phenylalanine flux and oxidation rates were measured at seven levels of phenylalanine intakes, from 5 to 60 mg/kg/d, with generous amounts of tyrosine. The subjects were on the test intake for only the day of the measurement; no period of adaptation was considered necessary. When oxidation was plotted against intake, there was a breakpoint at 9 mg/kg/d. Plasma phenylalanine concentrations showed a breakpoint at a similar level. The 1985 FAO/WHO/UNU report estimated that 1/3 of the requirement for the aromatic amino acids must be provided by phenylalanine. If the same ratio is applied to the estimate of Zello, the aromatic amino acid requirement would come out at about 30 mg/kg/d.

8.4. Summary of the results

When one compares the FAO and MIT levels (Table 11), the studies fall into two groups: in those of Cortiella (1988) (Table 10), and of Young (1987), Pelletier (1991a,b) and El Khoury (1994b) (Table 11), only the leucine intake, or that of the branched-chain amino acids was altered. By contrast, in the experiments of Hiramatsu (1994a), and Marchini et al (1993) the whole IAA pattern was fed at two different levels. The balances in the first group are consistently more negative than in the second group. Moreover, in group I there is no indication of any difference in balance between the FAO and MIT patterns, whereas in group II the MIT pattern does have an advantage, although I have not tested it statistically.

Table 14 Phenylalanine balance, based on measurements of hydroxylation. Data from Price et al (1994), Cortiella et al (1992) and Hiramatsu et al (1994a)

In none of these studies has a correction been made for the precursor. All calculations by method 1 (section 7.5).
^a Non-essential nitrogen.

The difference between the results with the two experimental designs suggests that when the intakes of the IAAs, other than the one under test, are high, the activity of the various oxidative enzymes is maintained at a high level across the board, so that oxidation of the test amino acid is increased. The experiments of Cortiella et al (1992) also showed some indication of sparing of oxidation when a diet low in total N and all IAAs was compared with one in which only phenylalanine and tyrosine were low. On the other hand, the experiments of Pelletier and of Hiramatsu show that the high rates of leucine oxidation cannot be explained either by excessive amounts of BCAs or by the level of NEN.

Young (personal communication) has commented: 'An analysis across our multiple experiments of leucine oxidation during the fed state reveals a linear relationship between intake of leucine and its oxidation, with no significant effect of level of N intake, source of nonspecific N and the amino acid patterns involved in these experiments. Hence I do not think that the available data support the implication proposed here'. I do not accept this conclusion, because, as I have pointed out above, fasted state oxidation, not included in this analysis, has an important effect on the balances. Moreover, as Table 11 shows, if Young's argument is correct it weakens his advocacy of the MIT pattern.

Another problem in interpreting the results is the between-subject variation within each experimental group. For example, in the experiment of Marchini et al (1993), with n = 6 or 7, for the FAO diet after l week the s.d. of leucine balance was 6.5 mg/kg/d, and for the MIT diet 13 mg/kg/d (Table 7 in Marchini et al, 1993). On the MIT diet this amounts to 34% of the intake, which means that it will be difficult to get a safe level (mean requirement + 2 s.d.), according to the principles applied to total protein by FAO/WHO/UNU (1985). This kind of variability seems to be almost unavoidable in human studies.

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