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close this bookEnergy and Protein Requirements, Proceedings of an IDECG workshop, November 1994, London, UK, Supplement of the European Journal of Clinical Nutrition (International Dietary Energy Consultative Group - IDECG, 1994, 198 pages)
close this folderThe requirements of adult man for indispensable amino acids
View the document(introductory text...)
View the document1. Introduction
View the document2. The problem in defining requirements
View the document3. Protein quality
View the document4. The maintenance requirement (MR)
View the document5.Diurnal cycling: the Millward-Rivers model
View the document6. Theoretical basis of the MIT tracer balance studies
View the document7. Technical problems of tracer balance studies
View the document8. Results of the MIT tracer balance studies
View the document9. Relation between leucine oxidation and nitrogen excretion
View the document10. Factors relating to the design of tracer balance experiments
View the document11. Breakpoint analysis
View the document12. Effect of protein/amino acid intake on protein synthesis and breakdown
View the document13. The colon: losses or gains?
View the document14. Conclusion
View the documentReferences
View the documentDiscussion
View the documentReferences

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 8
Response to feeding of protein synthesis, breakdown and amino acid oxidation measured with 1-13C leucine.

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.


Lysine


Leucine


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

Author

Leucine intake (mg/kg/d)

Synthesis (S) (mmol/kg/h)

Breakdown (B) (mmol/kg/h)

S - B

Young (1987)a

80

119

79

40

Marchini (1.993)

83

83

57

26

Mean





Pelletier (1991b)

40

86

81

5

Pelletier (1991b)

40

76

71

5

Marchini (1993)

40

75

63

12

Hiramatsu

40

101

85

16

Mean




9.5

Young (1987)a

30

90

91

- 1

Young (1987)a

14

74

70

4

Pelletier (1991b)

15

76

75

1

Pelletier (1991b)

15

70

73

- 3

Marchini (1993)

14

66

62

4

Hiramatsu

14

92

89

3

Mean




1.3

Young (1987)a

7

79

84

- 5

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.


Figure 10
Rate of leucine uptake into protein in the fed state in relation to leucine intake (later MIT studies: with 1- 13C leucine, comparing FAO, MIT and egg patterns).

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, QA/QU, 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 QA/QU 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 QA/QU 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.


Figure 11
Rates of protein synthesis (uptake) and breakdown (release) in the fed and fasted states in subjects fed three levels of leucine intake: FAO,MIT and egg patterns. Total Amino acid intake equivalent to 0.9 g protein/kg/d. Drawn from data of Marchini et al (1993)

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.