Nitrogen Balance

Nitrogen balance studies were initiated in the mid-nineteenth century by Carl Voit, and such studies have been central to the definition of protein requirements. The aim of nitrogen balance studies is simple—to define the relationship between intake and all losses (urinary, fecal, and surface—mainly sweat, skin, hair, breath ammonia, nail clippings, etc.) so that the intake that allows equilibrium and provides for all losses can be identified. Thus, when the intake equals the requirement,

As indicated previously, the lowest level of losses observed, the ONL, is approximately 46 mg/kg/day, equivalent to a daily loss of 0.29gprotein/kg/day. When such subjects are re-fed with protein, losses of body protein decrease as the dietary protein provides for some of the metabolic demand. However, nitrogen losses increase with intake so that the required intake for balance is more than the ONL. The main objective of nitrogen balance studies has been to define how much extra protein above the ONL must be fed to achieve equilibrium. The literature on human nitrogen balance studies has been assessed in a meta-analysis, as shown in Figure 3. A linear regression of balance against intake will allow prediction of the ONL as the zero intake intercept. The slope of the balance curve (a/b = e) will indicate the efficiency of utilization, and the maintenance requirement (i.e., the amount that must be fed to balance all losses and produce equilibrium) is ONL/e. Thus, the currently accepted maintenance requirement (0.66 g/kg/day) derives from an analysis of the data shown in Figure 2 (the median requirement calculated from individual regressions on each individual studied at more than three levels of protein intake).

Balance = e x intake-ONL

(e = efficiency of utilisation = abalance(a)/aintake(b))

Balance = e x intake-ONL

(e = efficiency of utilisation = abalance(a)/aintake(b))

Balance = 0.47.intake - 48.1 mg N/kg/d Requirement = intake for zero balance = ONL/e

Figure 3 Meta-analysis of nitrogen balance studies.

Inherent Difficulties with Nitrogen Balance Studies

This apparently simple but laborious approach, which is currently the main method for investigating protein and amino acid requirements, is in fact beset with a large number of quite serious problems, as listed in Table 1.

The lack of precision results in balance being a small value compared with the much larger values of nitrogen intake and nitrogen excretion, resulting in considerable error. The various systematic errors mean that balance is usually overestimated, often with unrealistic positive balances (protein gains) at high intakes. The nonlinearity of the balance curve as losses increase to match intakes when body protein reaches the maximum level means that there is no simple term to define the overall shape of the balance curve allowing prediction of the requirement (as the zero balance intake). In practice, prediction of a zero balance-intake intercept is made

Table 1 Potential problems relating to nitrogen balance methodology

Imprecise

Systematic errors: intake overestimated, loss underestimated due to problem of accounting for all losses, for example, Skin surface and secretions Loss of N2 gas Expired ammonia

Endogenous NO production gives urinary nitrate, faecal ammonia, and nitrite Changing size of the body urea pool Nonlinearity of the balance curve Design

Dietary energy intake and physical activity influences balance Accounting for adaptation from a few balance points by linear regression, and this will result in requirement values that will vary according to where the intake values lie on the balance curve; that is, studies conducted using low intakes will underestimate requirements, whereas studies conducted with supramaintenance intakes will overestimate requirements. The logic of this is that (i) reliable balance studies are those that are conducted with intakes very close to the actual requirement, and (ii) studies with intakes based on preconceived requirement values will tend to confirm such preconceptions.

Energy-Protein Interactions

Body protein equilibrium can be influenced by intakes of energy, and ensuring that energy intakes are sufficient is difficult. Excess energy intake leads to weight and some lean tissue gain, whereas with too little intake, dietary and/or body protein is oxidised as an energy source. This means that the protein requirement is a function of the state of energy balance and the actual influence is quite marked. According to one analysis of nitrogen balance (NB) on intakes of energy (EI; kcal/kg) and N (NI; mg N/ kg), NB = 0.171NI + 1.006EI - 69.13. This means that the intake for N equilibrium (the requirement) will vary from 1.4 to 0.32g/kg/day according to whether energy intakes are equal to the resting metabolic rate (RMR) or equal to twice the RMR. In fact, two-thirds of the overall variability reported in the meta-analysis shown in Figure 2 (SD = 31.9 mgN/kg) could be accounted for by an error of only approximately 0.2 of basal metabolic rate (BMR) in estimating the true energy needs of a subject.

Since nitrogen balance varies as a function of energy intake, it may be argued that protein requirements can only be defined in terms of a specified energy intake level, but what is the appropriate energy intake? Should populations with low protein staples consume more energy to achieve body protein equilibrium? Will this predispose to obesity? To what extent does variation in energy intakes at energy balance (i.e., with increasing levels of physical activity) influence nitrogen balance? These are difficult and currently unanswered questions.

Adaptation

With the metabolic demand for amino acids including both fixed and variable demands, the relationship between intakes and balance will be a function of time and the rate of adaptation. This is undoubtedly why the determination of human protein requirement by nitrogen balance has proved to be so difficult. Thus, when protein intake changes, the metabolic adjustments involved with matching amino acid oxidation and urea excretion rates to the new intakes take considerable time to adapt to the new level of intake. The actual time taken for complete adaptation is poorly understood and controversial. In practice, most balance studies are 'short term,' with dietary periods of 2 weeks at each intake studied and with diet periods randomized to minimize metabolic carryover of prior diets. Two weeks is comparable to the time taken to stabilize excretion in subjects fed a protein-free diet while establishing the magnitude of the obligatory nitrogen losses. It may be that adjustment to a protein-free diet, an extreme metabolic change, occurs more rapidly than the adjustment from one intake to another, with evidence of changes over several months to an intake similar to the ONL, and more than 1 month is required to adjust to a lower but adequate intake (lg/kg/day) after 2 months of a high-protein diet of 3g/kg/day.

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