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11. Breakpoint analysis

For the sake of completeness we must look briefly at a completely different method of assessing amino acid requirements. There were attempts in the 1970s to use plasma amino acid concentrations as an indicator, the idea being that if concentration is plotted against intake, there will be a sharp inflexion at an intake corresponding to the requirement. Although these expectations were not fulfilled, it seems worthwhile to look at the results of the early tracer balance experiments, since they involved measurements over a wide range of intakes. The results (Figure 6) show a more or less continuous fall of amino acid concentration with decreasing intakes, but nothing that could be interpreted as a breakpoint.

Brooks et al (1972) extended the method to oxidation. They plotted lysine oxidation against intake in the rat and found a clear-cut breakpoint at an intake of about 100 mg/d (although it is not clear how they calculated the lysine oxidized from measurement of the output of labelled CO₂, since there was no analysis of precursor activity). They considered that the method was more sensitive and specific than similar breakpoint measurements on plasma.

The fed-state oxidation rates plotted in Figure 7 show so much variation that it is impossible to predict the requirement from such data. Bayley and coworkers extended the idea. Instead of measuring the oxidation of the test amino acid at different levels of intake, they used oxidation of a labelled indicator amino acid at different levels of the test amino acid. This method gives curves which are the inverse of the original method; at the point where the requirement is fulfilled, oxidation of the indicator amino acid is at its lowest. Some of the breakpoints obtained on pigs are very clear; for example with ¹⁴C-phenylalanine as indicator, there were sharp breakpoints for tryptophan and histidine, but not for lysine (Kim et al, 1983; Ball & Bailey, 1984, 1986).

This approach has been used by Zello et al (1993) (section 8) to measure the lysine requirement with phenylalanine as indicator amino acid, but I know of no other studies in man.

12. Effect of protein/amino acid intake on protein synthesis and breakdown

This subject is relevant to IAA requirements because maintenance of a 'satisfactory' level of protein synthesis, however one defines 'satisfactory', might be a criterion that requirements are being met. This has been suggested by both Young (1987) and by Millward (1994).

As McNurlan & Garlick (1989) have pointed out, there are two questions to be examined. The first is the immediate effect of food, which is given by the difference between fasted and fed rates of turnover. The second is the effect on protein synthesis and breakdown of the prevailing protein or amino acid intake, taken to be the intake over the previous 6-10 days and called the 'habitual' intake.

The first point has been examined in detail by McNurlan & Garlick (1989). The prevailing opinion is that deposition of protein in the fed state results mainly from a decrease in degradation, with only a small increase in synthesis (Nissen & Haymond, 1986). The recent findings of Pacy et al (1994) (Figure 8) lead to a similar conclusion. The original studies of Clugston & Garlick (1982) had indicated an increase in synthesis as a result of feeding, but it now turns out that this was probably the result of recycling during an infusion which lasted 24 h, 12 h fasted and 12 h fed.

For examining the effect of 'habitual' intake on synthesis and degradation it seems best to look at results in the fed state. In the original study of Motil et al (1981b) fed state synthesis rates derived from incorporation of leucine were similar (113 and 102 mmole/kg/h) at protein intakes of 1.5 and 0.6 g/kg/d, but when the intake was reduced to 0.1 g/kg/d, synthesis fell sharply to 64 mmole/ kg/in. This suggests a breakpoint at or near an intake of 0.6 g protein/kg/d. With lysine as the tracer the flux fell from 107 to 75 mmole/kg/h as the intake was reduced from 1.5 to 0.4 g protein/kg/d (Conway et al, 1980).

Figure 9 summarizes the findings of the 1986 studies. With lysine there was a dramatic fall in fed-state synthesis at intakes below 35 mg/kg/d. With the other amino acids there was a continuous fall, and it would take the eye of faith to discern a breakpoint. The results of the other leucine balance studies that have been reviewed here are shown in Table 19 and Figure 10. At all levels of intake the variability is large; the mean synthesis rate at the leucine intake of 30-40 mg/kg/d is 85.5 mmol/kg/ h, and at 14-15 mg it is 75.5, a difference that is clearly not significant. It might be worthwhile to go back to the original data for further analysis. It makes no difference whether only the level of leucine in the diet is altered, or that of all the IAAs. The main difference is that the intake of 40 mg/kg/d allows for modest protein deposition in the fed state, whereas this hardly occurs at lower intakes.

At this intake the fasted synthesis rate, like that in the fed state, averages 85 mmol/kg/h (data not shown). The two rates taken together result in a synthesis rate over 24 h of 3.4 g protein/kg. This is towards the lower end of the range of values usually found in normal men. It is noteworthy, although it is a pure coincidence, that this is exactly the value Young et al (1989) obtained from their calculation that the obligatory loss represents 10% of the protein turnover rate.

Figure 9 Rates of amino acid uptake into protein at different levels of amino acid intake. Data from 1986 MIT studies; (a) lysine; (b) leucine; (c) threonine and valine.

Figure 11 shows the rates of synthesis and breakdown in the fed and fasted states at the three levels of intake: egg, MIT and FAO pattern. These results are very similar to those of Pacy et al (1994) (Figure 12). His experiments covered a wider range of intakes than those of the MIT group, and show that increasing the protein intake produced virtually no change in the fasting synthesis rate and only a small increase in the rate during feeding (Figure 12). In his experiments the upper two intake levels, 1.6 and 2.1 g protein/kg/d, are higher than any of those in the MIT series. The sharp fall in the degradation rate at these high intakes does not come through in the MIT studies because their highest intake, 80 mg leucine/kg/d, equivalent to 1 g protein, is represented by only two studies, which show very divergent rates of degradation.

Table 19 Fed state rates of protein synthesis and breakdown after 1 week at different levels of leucine intake

^a Calculated assuming correction for precursor = 0.8.
In the studies of Young and Pelletier only the leucine content of the diet was altered
In those of Marchini and Hiramatus all the IAAs were altered according to the FAO or MIT pattern.

The conclusion from the ¹³C leucine studies is that although the synthesis rate does show some variation with level of intake, it is not a sensitive indicator of protein or amino acid status.

This, perhaps, is not surprising, since the flux from which the amino acids for protein synthesis are derived is several times greater than the intake from the food. The synthesis rate is maintained even though there may be a substantial fall in plasma amino acid concentration during feeding, as happens particularly with leucine, presumably because of its small pool size. One may speculate that the synthesis rate is preserved because the amino-acyl-t-RNA synthetases, which catalyse the first step in protein synthesis, have such low KM values that they are saturated at all times.

Finally, it is interesting to consider the results obtained by Pacy et al (1994) by the end-product method with ¹⁵Nglycine (Table 20). We have suggested (Fern et al, 1984, 1985a,b) that the estimate of flux obtained by the labelling of ammonia is biased by the metabolism of the peripheral tissues, specially muscle; and the estimate obtained from urea is biassed by the metabolism of the viscera, particularly the liver. It should be made clear that these are independent estimates of whole body flux and are not additive. We consider that the best estimate is given by the average of the two.

The results with ¹⁵N glycine differ strikingly from those with ¹³C leucine; the synthesis rate increases with increasing protein intake, and the degradation rate shows very little change. The reason for this difference clearly needs to be investigated. On the whole, most workers do not have a very high opinion of the endpoint method and relatively little work has been done with it.

Be that as it may, it is claimed that the method can give information about metabolic activity in two different parts of the body and that it is an advantage of the method that the two estimates of flux do not agree. The ratio of the two estimates, Q_A/Q_U, is of some interest (Q being the notation generally used for the flux). From a recent analysis (to be published) of the values obtained under various conditions, it looks as if Q_A/Q_U is in the region of 0.7-0.8 in normal subjects, whereas in undernourished adults or children it tends to be lower. Thus in the study of Soares et al (1991), referred to at the beginning of this report, undernourished Indian labourers had lost muscle mass but not visceral mass. They maintained a rate of whole body protein synthesis per kg equal to or greater than that of controls, but Q_A/Q_U was 0.58, compared with 0.77 in controls. Table 20 suggests that in Pacy's subjects the lowest intake, equivalent to 27 mg leucine/kg/d, fails to support a normal rate of protein turnover, and the reduction is at the expense of turnover in muscle.

There is no independent evidence I know of that it is in some way better to have a high rather than a low rate of protein synthesis, although both Millward and Young favour the idea. All one can say is that the data in Table 19 show that the FAO pattern of IAAs, or the leucine intake equivalent to it, seems not quite able to support a normal or usual rate of protein turnover. I do not think that this can be ignored. From this point of view the MIT pattern is probably close to the lower limit of adequacy.

13. The colon: losses or gains?

Nitrogen is, of course, lost in the faeces, the obligatory loss being 10 - 20 mg/kg/d. Reeds & Harris (1981) and Fuller & Garlick (1994) have published data on the IAA content of ileostomy fluid, showing that small but not negligible amounts are delivered to the colon. It is immaterial, from the point of view of balance, whether these amino acids are endogenous or derived from the food, but if these amounts pass into the faeces, then they should be added to the losses given by the tracer balances. The fact is, we do not know the fate of these IAAs.

The possibility has been raised that amino acids synthesized by the microbial flora in the colon may be available to the body. One of the sources from which this synthesis could occur is urea. It is well recognized since the pioneer work of Walser & Bodenlos (1959) that in the normal adult about 20% of the urea produced passes into the colon and is hydrolysed to ammonia by bacterial urease. Jackson and coworkers have shown that the proportion of urea production that is 'salvaged' in this way (T/P_u) tends to increase in conditions where N balance is under stress, as in subjects on low protein diets or children recovering from malnutrition (Jackson et al, 1990; Langran et al, 1992). If all the ammonia produced by hydrolysis of urea was delivered to the liver, our studies (unpublished) with ¹⁵N ammonium chloride suggest that about 70% of it would be immediately converted to urea. In fact, the fraction of urea transferred to the colon that is recycled to urea is only about 25% or less (Jackson et al, 1990; Langran et al, 1992). It has usually been assumed that the ammonia which has not been recycled is taken up into the amino-N pool by transamination. However, there is also the possibility that the ammonia is utilized by bacteria for the synthesis of IAA as well as non-IAA.

Table 20 Fed-state protein turnover rates with ¹⁵N-glycine. Data of Pacy et al (1994)

Q_u = flux calculated from enrichment of urea.
Q_a = flux calculated from enrichment of ammonia.
Q_av = arithmetic mean of Q_u and Q_a
S_av = synthesis rate calculated as Q_av -N loss.
B_av = breakdown rate calcuated as Q_av-N intake.

This possibility must be seen against the background of a very large nitrogen flux through the colon. Jackson's calculations (Jackson et al, 1984) estimate this flux at about 15 g N/d in a normal adult, or about 1/3 of the whole body flux (this, incidentally, is why we speculate that the substantial pool turning over by lifetime kinetics (section 12) might be related to the gastrointestinal tract). This evidence for a large flux through the colon is supported by an experiment of Wrong et al (1985), which showed that the enrichment of faecal ammonia was many-fold less than that of the plasma urea from which the ammonia must have been derived. Thus we have the picture of a large intra-colonic flux, of which only about 10%/d is disposed of into the faeces, the remainder being recycled back into the body.

The validity of this picture depends on the possibility of reabsorption of amino acids from the colon. Moran & Jackson (1990a,b) found that in adults 60-80% of ¹⁵N-urea instilled into the colon was retained in the body. Heine et al (1987) instilled ¹⁵N-yeast protein into the colon of infants with Hirschsprung's disease and found that about 90% of the ¹⁵N was retained, with significant labelling of plasma proteins. This could have been ammonia that had been fixed by transamination; even so, on this preliminary evidence it looks as if amino acids as well as urea can pass out of the colon. Read et al (1974) suggested this possibility as early as 1974 but could not pursue it because of the civil war in Lebanon. On the basis of an experiment with ¹⁵N yeast in infants they wrote: 'Although the colon is not capable of digesting the intestinal flora it can absorb amino acids and there is a high probability that a percentage of the amino acids synthesized by the bacteria will be released into the surrounding medium'.

The final stage in the hypothesis that the body can utilize IAAs synthesized by colonic bacteria must be the direct demonstration of their appearance in body protein. So far the evidence is preliminary. Japanese workers in 1980 claimed to have isolated ¹⁵N-labelled lysine from plasma proteins in subjects in Papua New Guinea to whom they had given ¹⁵N-urea (Tanaka et al, 1980), but this claim was greeted by most workers with some scepticism. In a recent study (Torrallardona et al, 1993) a pig was given ¹⁵N-ammonium chloride and ¹⁴C polyglucose for 10 d. Significant labelling of both N and C was found in the carcass protein. It was estimated that synthesis by the bacterial flora provided lysine to the amount of 43 mg/kg/d if calculated from ¹⁵N and 33 mg/kg/d if calculated from ¹⁴C (see also Fuller & Garlick, 1994).

Young has commented that if bacterial synthesis of IAAs occurred to any significant degree, El Khoury et al (1994a) would not have found an almost exact correspondence between leucine intake from the food and leucine oxidized (Table 16). It is difficult to suppose any mechanism by which the colonic bacteria could 'recognize' that their host's dietary intake of an amino acid was high, and therefore reduce their own production. If, then, we visualize a more or less constant and significant addition of IAA from the colon, in striking a balance this addition will be taken into account on the output side (oxidation) but not on the input side (food). The result will be that when oxidation is substracted from the amino acid intake in the food, there will be a negative tracer balance, and this balance will always be negative, however high the intake. In fact this does not happen: the work of both Young and Millward shows that at higher intakes, e.g. 80 mg/kg/d and above, tracer balances become progressively more positive - a well known fact that has never been explained.

I conclude that, in people living on Western diets, colonic synthesis and release of IAAs is unlikely to be significant. However, the microbial flora of the large gut is variable, depending, among other things, on the nature of the carbohydrate and the fibre content of the diet. An effect which does not occur in habitually well nourished subjects might well become significant in those whose diets are less privileged. Research on this subject is now active and the results will be awaited with interest.

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