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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

7. Technical problems of tracer balance studies

The general procedure in these studies has been for subjects to be placed on a diet in which nitrogen is provided as crystalline amino acids to provide about 0.8 g amino acids per kg, with the level of the test amino acid only being varied. The subjects are kept on each test level for 6-7 days. In the most widely used protocol, after an overnight fast, they are given a primed constant infusion for 8 h of amino acid labelled with 13C in the carboxyl carbon. For the first 3 h the subjects remain fasting, and in the third hour, when isotopic equilibrium has been reached, measurements are made of isotopic enrichment in plasma and expired CO2. For the remaining 5 h the subjects are fed at frequent intervals and the measurements repeated in the final hour. Total CO2 output is determined by standard indirect calorimetry to give the amount of amino acid oxidized.

As mentioned above, early studies were made with Lysine, valine and threonine, as well as with leucine. These results are difficult to interpret, since no fasting measurements were made and there was no correction for precursor activity (see below). Moreover with Lysine and threonine the first step in oxidation does not involve the carboxyl carbon. However, the important point at this stage is to be satisfied about the validity of the method, and for this one amino acid is enough. The following remarks are therefore mainly confined to leucine.

7.1. Mass of tracer

Since stable isotopes are not mass-less, it is necessary in balance measurements to correct for the input from the infusion, particularly in the fasting state and at low intakes. It is unclear whether this was always done, or done correctly, in the early studies.

7.2. Retention of CO2

A proportion of the CO2 derived from oxidation of the labelled amino acid is not excreted in the breath but retained in the body in a form that is not known. The recovery varies a little with feeding state, duration of infusion (Table 4) and exercise (Wolfe et al, 1982; Barstow et al, 1990). The recovery factor is determined beforehand by a separate primed infusion of NaH13 CO3. Use of the same factor from one experiment to another could involve a small error.

Table 4 Percentage recovery of labelled CO2 in constant infusions

Author

Fasting

Feeding


Clugston & Garlick (1983)

0.94

0.88

12 h fasted, 12 h fed

Elia et al (1992)

0.86

3-6 h of infusion

Wenham et al (1991)

0.76

0.90

fasted 0 5 h, fed 4-8 h

Hoerr et al (1989)



fasted 0-4 h, fed 4-8 h


0.70

0.82

intravenous infusiona


0.74

0.79

intragastric infusion

Melville et al (1989)


0.56-0.76

during final 4h of feeding

El Khoury et al (1984a)

0.77

0.85

12 h fasted, 12 h fed

a The difference between i.v. and i.g. infusions is not significant.

It has also been suggested that the recovery from infused bicarbonate may not be a valid measure of the recovery of carbon generated by intramitochondrial enzyme activity. The evidence on this point is conflicting (Marsolais, 1987; Hamel et al, 1993), but the effect, if it exists, is not large.

7.3. Precursor enrichment

It has long been recognized that the labelling of the intra-cellular amino acid, which is presumably the precursor of both protein synthesis and oxidation, is lower than that of the amino acid in plasma, because of dilution with unlabelled amino acids derived from protein breakdown (Aub & Waterlow, 1970). If the intracellular labelled amino acid produces a metabolite which appears in the plasma, the labelling of that metabolite can be taken as that of the amino acid from which it was derived. Thus Fern & Garlick (1974) used the labelling of plasma serine as a measure of the labelling of intracellular glycine which was its precursor. This has come to be known as the 'reciprocal pool' method. For leucine the keto acid produced by transamination, i.e. 1-oxoisocaproic acid (KIC), is the precursor of oxidation, and the labelling of KIC in plasma is regarded as giving a better estimate of the precursor activity than that of the amino acid itself (Matthews et al, 1980, 1982; Schwenk et al, 1985). The ratio APE* of KIC/APE of leucine is usually in the region of 0.7-0.8 (Table 5), but is somewhat variable, with no clear relation to protein intake or fasted/fed state. The keto acid of valine, 1-oxoisovaleric acid (KIV), can be used in the same way, but suitable precursors for the oxidation of Lysine and threonine have not yet been used in tracer balance studies, although Arents & Bier (1991) have suggested that a-amino-adipic acid in urine might be a suitable reciprocal for Lysine. For phenylalanine the oxidation rate can be obtained from the rate of hydroxylation to tyrosine.

* Atom percent excess.

With these and other amino acids, where in contrast to the BCAs the liver is the main site of oxidation, we know from animal experiments (Simon et al, 1978; Lobley et al, 1980) that the isotope abundance of amino acids in the liver may be as little as half that in plasma, but liver biopsies are unlikely to be possible in man. A solution to the problem now seems to be available, based on the finding that apolipoprotein B-100 is synthesized so rapidly in the liver that it reaches isotopic equilibrium with its precursor amino acids in a matter of hours (Reeds et al, 1992). Some of the results obtained by this method are shown in Table 6. Thus, on the assumption that the products of the same precursor have the same enrichment (Waterlow et al, 1978), apo-B can now be used to give a. measure of the precursor activity in the liver of a range of amino acids.

Table 5 Ratio of enrichment of KIC to enrichment of leucine: isotope administered intravenously


Fasted

Fed

Not stated

Pelletier (1991)

0.79

0.69


Hoerr (1991)

0.72

0.79


Reeds (1992)

0.91

0.92


Thompson (1989)



0.77

Bido (1992)



0.81

Price (1994)



0.69

Cortiella (1988)




20-40 mg leucine



0.71

10 mg leucine



0.63

Hoerr (1993)




High protein



0.82/0.77a

Low protein



0.88/0.67a

a Infusions with 2H3-leucine. All others with 13C-leucine.

However, this does not solve the precursor problem. If the enrichment of the tissue amino acid is lower in liver, for example, than in muscle (as many workers, though not all, have found in animal experiments), then no single value for the enrichment of a metabolite such as KIC in plasma can accurately reflect the enrichment of the precursor in both liver and muscle. It is difficult to predict the extent of the error that may arise from this source. One might hazard a guess that, with KIC, the enrichment in plasma is likely to be lower than that of the precursor in muscle and higher than that in liver. The consequence would be that, by using the plasma value across the board, oxidation in muscle would be over-estimated and in liver it would be under-estimated, but these two errors would tend to cancel out. The balance will also vary according to the tracer that is used and its main site of oxidation. For a more extended discussion of the precursor problems see Waterlow (1995).

7.4. Extrapolation from the period of measurement to 24 hours

In the standard protocol of infusion for 8h, 3 h fasted and 5 h fed, measurements made in the last hour of each period are extrapolated to a whole day. Marchini et al (1993) proposed two methods for doing this, which differ according to the weight given to the oxidation of tracer in the fasted state. In my opinion, which agrees with that of Fuller & Garlick (1994), the correct method is Marchini's method 1. This involves calculating the fasting balance as: (infusion - observed oxidation) × 12, and the fed balance as (infusion + dietary intake -observed oxidation) × 12. The 24 h balance is the sum of these two. Marchini's method 2, with a tracer infusion rate of 4 mmol/kg/h, gives a more positive balance of 64 mmol, or about 8 mg/kg/d, which, at low intakes is far from negligible.

Table 6 The ratio of isotopic enrichment of amino acids in apolipoprotein B100 to that of free amino acids in plasma


Fasted

Fed

Leucine

0.90/0.94

0.69/0.72

Lysine

0.73/0.74

0.64/0.64

Phenylalanine

1.07/1.03

0.79/0.86

Data of Reeds et al (1992). The pairs of numbers represent results from two experiments.

Table 7 Comparison of measured and predicted leucine oxidation rates. Data of El Khoury et al (1994a,b)


Leucine oxidation


Leucine intake (mg/kg/d)

Measured (mmol/kg)

Predicted (mmol/kg)

Difference predicted-measured (%)

89 Fast 12 h

34.7

32.5a


Fed 12 h

54.8

58.7b


Sum 24 h

89.5

91.2

+ 1.9

38 Fast 12 h

20.9

25.5a


Fed 12 h

24.3

25.0b


Sum 24 h

45.2

50.5

+ 11.7

14 Fast 12 h

15.0

18.7a


Fed 12 h

12.8

14.2b


Sum 24 h

27.8

31.2

+12.2

a From last hour of fasting × 12.
b From fifth hour of feeding × 12.

In a praiseworthy attempt to solve this problem, El Khoury et al (1994a,b) did two heroic experiments in which leucine was infused for 24 h in order to compare the measured 24 h oxidation with that predicted from the shorter protocol. On the whole, agreement between measured and predicted rates was satisfactory (Table 7), with the prediction tending to overestimate the oxidation rate. Figure 4 shows that there is a temporal pattern of 13CO2 output in both fed and fasted periods, particularly with low intakes, so that the predicted oxidation is critically dependent on which hour is chosen to be representative of the whole period. It would seem that the choice, imposed by the protocol, of the fifth hour of feeding, was rather fortunate, since at this time the 13CO2 output more or less represents the mean, but it is the mean of quite wide variations, e.g. for the 38 mg intake, from about 12 down to about 5 mmol/kg/30 min.

Figure 4 Pattern of 24-h leucine oxidation rate for subjects receiving 38.3 mg (a) or 14 mg (b) leucine/kg/d. Mean ± s.d. Minutes (0-1440) refer to experimental time; time of day is indicated by hours. Reproduced from El Khoury et al (1994) by courtesy of the authors and the publishers of the American Journal of Clinical Nutrition.


Pattern of 24-h leucine oxidation rate - a


Pattern of 24-h leucine oxidation rate - b