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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

^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

^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

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)

^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.

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