Determination of indispensable amino acid (IAA) requirements necessitates a range of intakes of the test IAA and monitoring of the physiological response. Short-term methods are the most feasible for studying multiple intake levels in the same individual. Carbon oxidation methods measure the excretion of 13CO2 in breath from a labelled amino acid (AA) in response to varying intakes of the test AA following a period of adaptation. However, the length of adaptation to each AA intake level has been a source of debate and disagreement among researchers. The assertion of the minimally invasive indicator amino acid oxidation (IAAO) technique is that IAA requirements can be estimated after only a few hours (8 h) of adaptation to each test AA intake, suggesting that adaptation occurs rapidly in response to dietary adjustments. On the contrary, the assertion of most other techniques is that 6–7 d of adaptation is required when determining IAA needs. It has even been argued that a minimum of two weeks is needed to achieve complete adaptation. This review explores evidence regarding AA oxidation methods and whether long periods of adaptation to test IAA levels are necessary when estimating IAA requirements. It was found that the consumption of experimental diets containing lower test IAA intake for greater than 7 d violates the terms of a successful adaptive response. While there is some evidence that short-term 8 h IAAO is not different among different test amino acid intakes up to 7 d, it is unclear whether it impacts assessment of IAA requirements.

The factorial approach is used to measure the dietary indispensable amino acid (IAA) requirements in children, although recent measurements based on the indicator amino acid oxidation (IAAO) method have begun to generate more direct evidence. Difficulties with the factorial method are that it depends on accurate estimates of the maintenance protein requirement, as well as of protein deposition during growth. Also, a value for the efficiency of utilizing dietary protein for deposition has to be selected, based on published Nitrogen (N) balance studies. In the recent 2007 WHO/FAO/UNU report, the amino acid requirement pattern for infants was taken to be similar to the amino acid composition of breast milk. For pre-school and older children, the factorial method gave values for the scoring pattern of protein that were fairly close to the earlier 1985 WHO/FAO/UNU report for children, since growth progressively became a smaller component of the factorial calculation as age progressed. However, given that there are several assumptions in the derivation of factorial estimates, direct experimental measurements of the amino acid requirement are desirable. The IAAO method, as it is non-invasive, as made it possible to measure the IAA requirements in children. Over the last decade, some of the IAA requirements have been determined by using the IAAO method in healthy school age children; however, the data on IAA requirements in developing country populations are still being conducted. In the elderly, there are not enough data to make a separate recommendation for IAA requirements from that of adults.

This is a review of previously published data from experiments designed to measure the chicks' requirement for an essential amino acid in diets containing surplus protein (generally in the range 220–300 g crude protein (CP)/kg diet). The evidence shows that, within this range, the requirement for a first limiting amino acid increases nearly in direct proportion to the CP content of the diet. To explain this, the following possibilities are considered: (1) that energy supply was limiting the response to a critical amino acid, (2) that the experiments were conducted in environments which limited heat disposal by the chicks, (3) that results from the trials have been misinterpreted because growth rate rather than protein deposition was used as a response measure, (4) that the availability of amino acids in the diets was lower than had been assumed, (5) that nutrients other than the amino acid under study were limiting performance, and (6) that there was an imbalance of amino acids in the diet. It is concluded that only the last explanation fits the facts. The imbalances reported have led to a decline in the efficiency of the utilisation of the first limiting amino acid in some instances. The depressed growth observed with a high-protein diet limiting in one essential amino acid could not be attributed to lowered feed intake in most cases. The imbalance is, in these respects, different from classical imbalances which result from loading the diet with a mixture of essential amino acids. From the evidence reviewed, some doubt is cast on the validity of the dilution method for estimating amino acid requirements; nevertheless it its concluded that the dilution method remains the most trustworthy procedure for making estimates to be used in practical diet formulation. It is recommended that, in future, programmes for diet formulation should be modified to prevent surplus protein being permitted in the solution, unless a proportional adjustment is made to the minima prescribed for amino acids likely to be present in limiting proportions.

The absorption of lysine synthesised by the gastrointestinal microflora was estimated by comparing the15N incorporated into body lysine in four germ-free (15N-GF) and four conventional (15N-CV) rats. They were fed for 10d on a protein-free diet containing fermentable carbohydrates and 15NHM4Cl; another four conventional rats (control), fed on the same diet but with unlabelled NH4Cl, were used to estimate the natural abundance of 15N. The eviscerated carcass of each rat was homogenized and a sample hydrolysed. Lysine was isolated by ion-exchange chromatography and its 15N enrichment was measured by isotope-ratio mass spectrometry. The 15N-CV rats significantly incorporated 15N into their body lysine. The 15N-GF rats had a statistically significant, although small, incorporation of 15body lysine, probably arising from a measurement artifact. It was concluded, therefore, that all [15N]lysine was of microbial origin. The total lysine content in the body and the 15N enrichment of lysine in the microbial fraction of the faeces of the 15N-CV rats were also determined. The amount of microbial lysine absorbed by the 15N-CV rats was estimated by dividing the total amount of [15N]lysine in the body by the enrichment of microbial lysine. It was estimated that the daily absorption of microbial lysine by the conventional rats was 21·3 (SE 2·04) mg/kg body weight0·75

Nitrogen retention and lysine oxidation were measured in growing pigs given diets which supplied 0, 0·2 or 0·8 of the lysine requirement, with other amino acids in relative excess. Eight groups of three female littermate pigs were used: one of each group was given each of the three diets. In half the pigs (four groups) N retention was measured at body weights (W) of approximately 25,35 and 45 kg. The other four littermate groups of three pigs were given the same three diets; when they reached 35 kg W they were given a continuous (6h) primed infusion of L-[6-3H]lysine. Lysine oxidation was estimated from the production of tritiated water. Rates of both N retention and lysine oxidation increased significantly with lysine intake; mean values (g/kg W0·75 per d) for the three diets respectively were for N retention, 0·00, 0·32 and 1·22, and for lysine oxidation 0·051, 0·058 and 0·078. From the N balance results (assuming a constant lysine concentration in body protein) the efficiency of utilization of absorbed lysine was estimated to be 0·85; from the oxidation results (assuming lysine absorbed but not retained is oxidized) the estimate was 0·95.