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9. Relation between leucine oxidation and nitrogen excretion

It would help to validate estimates of leucine oxidation if parallel changes were found in N excretion as a measure of total amino acid oxidation. Prediction of the oxidation of total N from that of leucine has traditionally relied on the assumption that leucine represents 8% of body protein. As far as I can make out, this value was introduced into the isotope literature by Golden & Waterlow (1977), based on the old analyses of Block & Weiss (1956). It corresponds also to the leucine content of beef muscle (FAO, 1970) and that of the human fetus (Widdowson et al, 1979). Since the body contains about 20% of structural protein that includes very little leucine, the value of 6.5% proposed by Reeds & Harris (1981), based on carcass analysis, is probably not appropriate, although Young has used it in a recent paper (1991)

The data of Reeds & Harris (1981) in pigs show excellent correspondence between measured balances and those predicted from leucine. However, in man Clugston & Garlick (1982) were able to account for only 68% of total N excretion from their measurements of the oxidation of leucine. Young et al (1987) measured N balances in the third week on diets providing, 7, 14 and 30 mg leucine/kg/d and again after repletion. There was qualitative agreement with the leucine balances, but no attempt was made at a quantitative comparison. In the experiments of Marchini et al (1993) N balance as well as leucine balance was measured on the FAO, MIT and egg diets. No significant differences were found between 1 and 3 weeks on the diets (cf. Young et al, 1987), so the results have been averaged in Table 15. The leucine balances put the results in the expected order, which the N balances hardly do, so this may justify the authors' claim for the 'poor reliability' of N balances. However, the s.ds of both sets of balances are so large that the difference between the FAO and MIT diets is significant in only one comparison. Furthermore, there are disturbing quantitative discrepancies between predicted and observed balances.

Price et al (1994) investigated this point in some detail, using the figure of 8% for the leucine content of body protein. Their results are shown in Figure 5. The ratio of predicted to measured N loss was the same over the whole range of protein intakes, with a mean of 0.79. The measured N loss included estimated faecal and miscellaneous losses. It could be argued that in this context it would have been more appropriate to include only the amounts of urea and ammonia N excreted. This would bring the ratio closer to 1. The predicted loss was calculated from leucine oxidation after correcting for any difference between the leucine content of body protein and of food. The important point is that correspondence, even if not exact, is the same over a wide range of intakes. These data, therefore, provide valuable confirmation of the validity of the leucine balances.

El Khoury et al (1994a), during their 24 h infusions, measured urea production with ¹⁵N¹⁵N urea, as well as urea excretion. The results in Table 16 show a remarkable correspondence between N intake, observed N output and predicted N oxidation, calculated on the basis that leucine is 8% of body protein. One might have expected a better correspondence between predicted oxidation and urea production (which does not include urea recycled from the gut) rather than urea excretion, since urea production is presumably a measure of the total amino-N oxidized. However this is a controversial point on which Young and Millward are in agreement and I differ (see section 13). As with Price's data, if the faecal and non-urea N are excluded from the calculation, leucine oxidation comes into good agreement with urea production.

Table 15 Average of leucine and nitrogen balances after 1 and 3 weeks on egg, MIT or FAO diets. Data of Marchini et al, (1993)

Predicted N balance = leucine balance × 100/8 × 1/6.25.

10. Factors relating to the design of tracer balance experiments

10.1. The model

The most important factor affecting the calculation of amino acid oxidation is the enrichment of the precursor, since the measurement of labelled CO₂ output is straightforward. Is there any possibility that the values that have been used for precursor activity are too low? One suggestion of how this could happen is that there is a pool of protein turning over with lifetime kinetics (Waterlow et al, 1978) with an average lifetime of 6-9 h in effect a delay pool (Slevin et al, 1991). This idea arose from the finding of a 'step' in the enrichment curve of urinary ammonia during hourly dosage of ¹⁵N-glycine without a prime. The first step or plateau occurred at 6-9 h, the second from 21 h onwards. This kind of behaviour could not be explained by any model in which exchanges of tracer occur by first order kinetics, nor could it be explained by recycling. Later results have confirmed our preliminary observations (Jackson, unpublished). The APE at the first plateau was 30% less than at the second plateau, and would lead to higher estimates of the flux. We have suggested that this pool, which is turning over rapidly by lifetime kinetics, may be associated with the gastro-intestinal tract, but at present these observations are preliminary and speculative.

Table 16 Leucine oxidation and nitrogen excretion. Calculated from data of El Khoury et al (1994a)

We have to consider whether an effect of this kind could be occurring in the tracer balance studies. In these studies it is not possible to identify a step in the enrichment curve of the tracer, because all the infusions were primed and the fluxes calculated from the plateau at 2-3 h. The reason it seems unlikely that there is any serious defect in the model is that the fluxes and protein turnover rates calculated from this early plateau are in good agreement with those in the literature, including results obtained with ¹⁵N (Waterlow, 1995). However, it would surely be a mistake to suppose that the model currently used is the last word and that no more research is needed to establish the validity of the fluxes derived from it (Bier, 1989).

10.2. Route of administration of tracer; the 'first pass' effect

It has been suggested that it is artificial to give the tracer by vein when what is being measured is oxidation of amino acid in the food. Several investigators have given one tracer by vein and another by mouth (Cortiella et al, 1988; Beaufrere et al, 1989; Hoerr et al, 1991, 1993; Biolo et al, 1992; Matthews et al, 1993). These studies are in remarkable agreement in showing that with leucine as tracer 70-80% of the tracer given by mouth enters the plasma and 20-30% is 'sequestered' in the tissues of the splanchnic bed. Of the latter, about 60% is used for protein synthesis and 40% emerges as KIC (Matthews et al, 1993).

According to the data of Yu et al (1990), in the dog in the fed state 14% of the leucine taken up in the splanchnic bed was oxidized. With phenylalanine the proportions taken up and oxidized in the splanchnic bed were much greater (Biolo et al, 1992; Sanchez et al, 1995).

The significance of what has been called the 'first pass effect' for estimates of flux and oxidation is at present far from clear. According to my calculations (which may not be correct), the route of administration of tracer will have no effect on the estimate of flux from measurements on plasma, provided that the enrichment of the precursor is the same in splanchnic tissues, plasma and peripheral tissues. As discussed in section 7.3, one would expect on the basis of most animal experiments that with intravenous infusion the precursor activity would be lower in liver than in plasma, but we do not know how it will behave with an intragastric infusion. If the enrichment is lower with i.g. than with i.v. infusion, then i.v. administration of tracer will lead to an underestimate of flux. This, in fact, has been found, as Table 17A shows. Unfortunately, the corollary, that precursor activity should be lower with i.g. infusion of tracer, is not so far supported by the data. If with leucine infusions KIC is taken as precursor, Hoerr et al (1991) found virtually identical enrichments of plasma KIC by both routes in both fed and fasted states (Table 17B). There is therefore a puzzle here which has not yet been resolved, but which emphasizes the uncertainty still existing about the appropriate precursor for measurements of whole body turnover. One may also recall the old studies of Golden & Waterlow (1977) in which ¹⁴C leucine was infused for 24 h the route of administration being switched from i.v. to i.g., or vice versa, half-way through the infusion, with no effect on the plateau labelling of leucine in plasma. From the practical point of view of the present enquiry, the effect of underestimating the flux in i.v. infusions would be to underestimate oxidation and hence the requirement.

Table 17(A) Rates of flux with either intravenous (i.v.) or intragastric (i.g.) infusions of 1-¹³C or ³H-leucine. All measurements were in the fed state and based on enrichment of plasma KIC

Table 17(B) Enrichments in plasma of leucine and KIC with intragastric or intravenous administration of tracer. Data of Hoerr et al (1991)

E = enrichment.

10.3. Adaptation

In the classical studies of Nicol & Phillips (1976) in Nigeria, village men were in negative N balance on an essentially protein-free diet (14 mg/kg/d), but came into positive balance and gained weight when the diet was supplemented with egg to provide an intake of 61 mg N/ kg/d, which is little more than the obligatory loss. The Nigerians utilized the egg protein more efficiently (NPU 0.90) than subjects in California (NPU 0.75). A similar high efficiency of utilization of milk protein was observed in children recovering from malnutrition (Chan & Waterlow, 1966).

Two points arise from these studies. The first is whether the state of 'adaptation' described in the Nigerians is acceptable. My view, discussed earlier (section 2), is that this kind of long-term adaptation, with a low body weight and probably a reduced muscle mass, is not acceptable. The fact that the Nigerian men gained weight when given a marginal protein supplement suggests that they were well below Millward's set-point for body protein. Others might contend differently; it is very interesting that, in the Minnesota experiment, when the body weight of the subjects had come down to a body mass index of 16.7, they developed severe psychological and physical symptoms, whereas Indians and Nigerians, who have lived all their lives at this BMI, cope quite well. But the problem remains, that in evaluating states of adaptation it is almost impossible to avoid subjective judgements (Waterlow, 1990).

On the other hand, adjustment of protein metabolism to variations in intake is a part of normal life, and so the second question is how long does this physiological adaptation take? Rand et al (1976) and Bodwell et al (1979) have produced curves showing the change in urinary N output on moving from a normal to a zero protein intake. Rand showed that urinary N decreased according to a single exponential, in agreement with the older findings of Martin & Robison (1922). He suggested that the output might be regarded as 'stabilized' when it was within 1 s.d. of the theoretical output at infinite time. The mean time to reach stability was 4.5 d (range 3-10). The reduction in N output is accompanied, at least in the rat, by coordinated decreases in the activity of the urea cycle enzymes, glutamate dehydrogenase and alanine and as part ate aminotransferases , which presumably play a role in feeding N into the urea cycle (Schimke, 1964; Stephen & Waterlow, 1968; Das & Waterlow, 1974). This adaptation, which we may regard as physiological, takes 30 h in the rat and, from the data of Rand and Bodwell, 6-7 d in man. Presumably the effect of the reductions in enzyme activity is not only to reduce the amount of N excreted but also to improve the efficiency with which N is utilized. In the balance studies of Atinmo et al (1988), in which diets of different protein content were fed for 10-day periods, the NPU improved from 52 at an intake of 0.75 g/kg/d to 91 at 0.3 g over a period of 3-4 weeks.

Further evidence of the need for a period of adaptation in tracer balance studies is the sensitivity of fasting oxidation rates to preceding intake (Figure 3). Therefore it would be unwise to accept the contention of Zello et al (1990) that a period of adaptation is not necessary. Incidentally, Zello recalls that in Rose's experiments, exclusion of a single amino acid from the diet led to anorexia, irritability and fatigue. No such symptoms have been reported in the MIT studies.

To test the adequacy of the 6 d period of adaptation, Young et al (1987) explored the effects of keeping the subjects on the test diets (7, 14 and 30 mg/kg/d) for a further 2 weeks, after which they received leucine at a generous level. The results of this study deserve to be given in some detail. Table 18 shows that leucine balance improved after 3 weeks on the diet compared with 1 week as a result of a fall in fed-state oxidation. However, in the two low intake groups this was achieved at a cost of a sharp decline in the rate of protein synthesis. Young, following Beaton (1985), calls this an 'accommodation', i.e. making the best of a bad job after the limits of physiological adaptation have been passed. A normal rate was restored after 3 days on the generous intake. The groups on the lower intakes went into positive N balance on refeeding, which indicates a degree of depletion or deviation from their setpoint (see also Fisher et al, 1965).

There are some further points of interest about this study. On the two lower intakes plasma leucine concentrations fell, as did the rates of appearance (R_a) of leucine in plasma as a result of protein breakdown (not shown in Table). One might then postulate the following regulatory mechanism: Persistently low leucine intake (r) reduced R_a (r) fall in plasma leucine (r) reduced oxidation. It is noteworthy also from the figure in Young's paper that there is no evidence of longer exposure to the low diet altering the sensitivity of the relation between oxidation rate and plasma leucine level.

Table 18 Leucine kinetics after 1 and 3 weeks on three levels of leucine intake. After Young et al (1987)

Rates of oxidation synthesis (non-oxidative disposal and breakdown (appearance) have been corrected with a precursor factor of 0.8.

Marchini et al (1993) carried out what was essentially a repetition of Young et al's study, except that the intakes of all the IAAs were modified together, according to the FAO or MIT patterns. In this experiment there were no significant differences between 1 week and 3 weeks on the diet at either level, although balances on the FAO diet were lower than on the MIT diet. This again suggests that an unbalanced diet in which the level of only one amino acid is low is less well tolerated than one in which the intakes of all the amino acids are reduced (see section 8).

On the basis of these results it looks as if, on marginal or inadequate intakes, a longer period of adaptation brings the subjects more nearly into balance by reducing oxidative losses. Nevertheless, this balance may not be an adequate criterion. The reduction in the rate of protein synthesis suggests that the physiological is merging into the pathological. Even the 'undernourished' labourers of Soares et al (1991), who presumably have had a marginal intake all their lives, show no reduction in protein synthesis per unit body weight or lean body mass.

From the practical point of view it seems that one week on the diet is probably about right to allow for physiological adaptation. The experiments of Marchini et al (1993) support this view.

10.4. The nature of the meal

It is possible that a diet in which the protein component consists of crystalline amino acids that are very rapidly absorbed might have a different metabolic effect than one in which the proteins are being continuously hydrolysed as they pass down the digestive tract, and much of the nitrogen absorbed as di- and tri-peptides. The only direct comparison that I know of is the study of Bailey & Clark (1976). They obtained identical small positive balances on a rice-wheat diet and an isonitrogenous diet of the same amino acid composition, made up of crystalline amino acids. Millward (personal communication) reports recent experiments by French workers showing that the efficiency of post-prandial protein deposition was some 30% greater with casein than with a hydrolysate containing oligopeptides.

Another problem is whether small hourly meals have a different effect from food eaten in a normal meal pattern. This is a subject that needs further research.

10.5. Immobilization

The standard protocol of the tracer balance studies requires immobilization of the subject in bed for at least 8h, and it is possible that this may have a negative effect on the balance. In people with fractures or in children with muscular dystrophy, muscles that are immobilized have a reduced rate of protein synthesis with atrophy of type I (glycolytic) muscle fibres (Gibson et al, 1987, 1988; Tucker et al, 1981). Not very much research seems to have been done on the effects of immobilization on whole body metabolism. In a study on mobile and immobile elderly people (Lehmann & James, 1985) the immobile had higher rates of protein turnover and lower rates of oxidation than the mobile. This result, which is the opposite of what one might expect, may be because the immobile are more likely to be sick. Deitrick et al (1948), in the aftermath of World War II, carried out a remarkable experiment in which four subjects were immobilized in bed for 6-7 weeks with a pelvic girdle and their legs encased in plaster casts. The urinary N rose to a maximum on the 5th to the 8th day, with an average increase of 2.5 g N/d by the 5th day. There was also an increased output of calcium, phosphorus and potassium and a small increase in the urinary ratio of nitrogen to sulphur. Schonheyder et al (1954) did a similar experiment in which three subjects were put to bed with both their legs in plaster. They showed an even larger increase in N loss after several days, averaging 9 g/d, but inspection of the charts shows no effect at all on the first day.

In another study with a single dose of ¹⁵N-glycine, bed rest and ambulatory conditions were compared (Ang et al, 1992). Synthesis and breakdown both fell during bed rest, but there were no differences in nitrogen excretion.

I conclude that partial immobilization for less than a day is unlikely to have had a significant effect on the tracer balances.

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