Protein quality & amino acid requirements in relation to needs in India

Obligatory loss model

A theoretical model derived by Young et al13, was called the predicted obligatory AA losses model. In this, the basal rate of N excretion in an individual receiving a zero protein diet was used to estimate the requirement for specific AA by relating it to the composition of AA in body proteins. Since there is always a loss of N from the body when zero protein is administered, as IAA is indispensable, it is called the obligatory loss of N. An individual receiving zero protein intake would therefore, breakdown his/her own body protein, to meet the daily requirement for IAA, resulting in a negative N balance and a subsequent loss of body tissue and weight. While some of the released IAA would be recycled back into protein synthesis, some would be irreversibly oxidized and be excreted as N (it is impossible to bring the irreversible oxidation rate down to zero, due to inefficiencies in metabolic cycling) or the obligatory loss. In theory, the breakdown rate of protein would be determined by that IAA which had the highest rate of obligatory loss and its concentration in the body protein pool13. The other IAAs would be released based on their concentration in the protein that is broken down, and the excess over their daily requirement would be oxidized. In effect, the amount of obligatory loss of N is an indication of how much body protein was broken down, and the pattern of IAA loss is then assumed to be reflected by the IAA composition of body protein. The calculation of the amount of IAA needed to be fed to balance the daily obligatory loss required an assumption of the efficiency of utilization of dietary IAA. If dietary IAAs were used completely to balance the loss in the synthesis of body protein, this would result in an efficiency of 100 per cent; whereas in reality, the efficiency is much lower. This theoretical model pointed to a possible error in the empirically determined N balance estimates of the IAA requirement. It could not be considered to provide primary data needed to make recommendations; however, it led to a sustained effort to find more accurate ways that used isotopic methods, to measure the IAA requirement.

Direct amino acid (AA) balance

A better method of estimating the IAA requirement is by the measurement of AA balance (direct AA balance-DAAB) rather than the N balance14. If IAA oxidation could be accurately measured, then a balance could be constructed as the difference between the 24 h IAA intake and the 24 h irreversible oxidation. This can be done by the use of stable isotope tracers, where a stable isotope of carbon [¹³C] is used to label the studied (test) IAA at the position where it is released on oxidization, such that the tracer would appear in the breath and quantified. Since the labelled CO₂ is mainly released through the breath, and if the quantitation of breath loss was accurate, it would provide a more accurate measurement of IAA oxidation compared to the N loss method, where N could be lost through routes that were difficult to quantify. When different levels of the test IAA are fed to adult human volunteers, the minimum level of intake of the test IAA that result in a zero IAA balance is considered to be the daily requirement of that particular AA (Fig. 1A). An important requirement of this tracer-based technique is the precise measurement of the isotopic enrichment of the IAA pool being subject to oxidation, and this is difficult to determine for most amino acids except leucine15 and probably methionine16.

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Fig. 1

Different tracer methods used in measuring indispensable amino acid requirements. (A) direct amino acid balance (DAAB) or indicator amino acid balance (IAAB) - increasing balance towards zero balance of either ‘test’ or ‘indicator’ amino acid with increasing test amino acid intake levels. Note that at supramaintenance intake levels, balance stays at zero. (B) indicator amino acid oxidation (IAAO)-decreasing oxidation of ‘indicator’ amino acid at supramaintenance test amino acid intake levels. Inflection or breakpoint on the lines indicates the measured requirement level.

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Because the kinetics of leucine are well established17 and can be measured for direct determination of leucine balance over 24 h, this became the first IAA whose requirement was determined accurately over a day. A significant development was the demanding 24 h tracer balance protocol after adapting individuals to levels of test AA intake for one week1819. The value determined for the requirement of leucine (40 mg/kg/day) was similar to that predicted by the theoretical obligatory N loss model13. Other validations included short term leucine balance studies, which showed that the extrapolated daily requirement for leucine was greater than the 1985 FAO/WHO/UNU (World Health Organization/Food and Agriculture Organization/United Nations University) value of 14 mg/kg/day and in the range of about 40 mg/kg/day920. The leucine requirements of healthy, well-nourished Indians were measured in this way, by the 24 h technique using L-AA mixtures supplying graded intake levels of leucine, and where the individuals were adapted to these intakes for one week21, and found a similar value of 40 mg/kg/day.

Indicator amino acid (AA) oxidation and balance

The developments in tracer methods ascertained a level of confidence in leucine measurements. However, the requirement of the other IAA was more difficult, as the measurement of the intracellular precursor pool of the labelled test AA was not easy to measure. A technique which had been applied earlier in animals that advanced the measurement of the IAA requirement was the indicator AA oxidation technique (IAAO). In this method, two IAA are considered; the test IAA whose requirement is to be determined, and a tracer labelled ‘indicator’ IAA whose oxidation and balance could be accurately measured when graded test IAAs were fed. This would result in a curve of oxidation or balance, whose inflection indicates the test IAA requirement (Fig. 1A and B). At suboptimal (limiting) intakes of the test IAA, protein synthesis is inefficient, resulting in increased oxidation of the other IAA which is consumed at its normal requirement level. In this approach, the inflection point of the oxidation of the indicator IAA (whose intake is constant) at different test IAA intakes is a nadir, since the indicator IAA oxidation rate would reduce as the intake of the test IAA increased until it reached its minimum; it would stay at that minimum even as the intake of the test IAA increased further. The indicator IAA balance can also be evaluated in a similar way [called the indicator amino acid balance (IAAB) method], where the balance would improve from a negative value towards zero, as the intake of the test IAA increased, and remained at zero for test IAA intakes above the requirement. This would also yield an inflection point at zero balance that would indicate the requirement of the test IAA. It is important to note that oxidation and balance are measured over a full 24 h cycle, such that this method is more correctly called the 24 h IAAO or IAAB method, and is demanding since individuals are adapted to their test IAA intakes for at least a week before IAAO and IAAB are measured22. However, the technical problems of precursor pool identification that are related to the DAAB method are not present, since the indicator IAA chosen is one (leucine) in which the precursor pool enrichment is measurable.

This indicator method has effectively been simplified to a breath test, when it is used as a short-term IAAO method23, in contrast to the longer-term 24 h IAAO and IAAB method described above22. The short-term IAAO method has been used with lysine or phenylalanine as the indicator IAA2425, where the recovery of the oxidized [¹³C] label in the breath, that appears after the oxidation of the 1-¹³C-labelled IAA, is measured postprandially for a few hours. The adaptation period to the test IAA is much shorter, usually over two days. However, the non-invasive breath test with a short adaptation period allows for repeated measurements of IAA oxidation at graded levels of test IAA intake in the same individual, and yields a variance term of the requirement. The possible problems related to the short dietary adaptation period, and the short-term nature of the postprandial evaluation may also influence the requirement estimate.

These studies using the 24 h IAAO and IAAB measurements with an adequate dietary adaptation period have been considered as the best available measurement for the determination of IAA requirements, and based on these measurements as the primary source of data222627282930313233, the 2007 WHO/FAO/UNU expert committee on protein and AA requirements34 recommended a generally increased pattern (2 to 3 times) of IAA requirements for adults (Fig. 2), but in turn, showed that many foods (particularly cereals) that were hitherto thought to be adequate in their IAA content, were limited in lysine. These IAA requirements were determined in healthy, well-nourished Indians222627282930313233, considered representative of well-nourished and healthy populations globally. Similar studies using the short-term IAAO method were also carried out in children to validate the factorial method that was used to determine their IAA requirement36. Since a significant proportion of the population in India is undernourished, these measurements, particularly for lysine, methionine and leucine were performed in chronically undernourished individuals with a low but stable BMI and were found to be reasonably similar to well-nourished individuals373839. In addition, the effect of infections and intestinal function and parasites on the IAA requirement was also studied40, where it was found that the presence of mixed parasites increased the lysine requirement by almost 50 per cent in children and adults4142.

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Fig. 2

Comparison of adult amino acid pattern (mg/g protein) between 2007 and 1985 FAO/WHO/UNU recommendation. Amino acids include His, Histidine; Ileu, Isoleucine; Leu, Leucine; Lys, Lysine; SAA, Sulphur Amino Acids (Methionine and Cysteine); Thr, Threonine; AAA, Aromatic Amino Acid (Phenylalanine and Tyrosine); Trp, Tryptophan; Val, Valine.

Source: Refs 34, 35.

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Protein digestibility corrected amino acid score

The AAS is the ratio between the quantity of each IAA in the food or mixed diet protein (mg/g protein) and a reference requirement pattern of IAA (mg/g protein) for each age group. The lowest (limiting) AAS of the food or mixed diet, when corrected for crude protein faecal N digestibility, is called the protein digestibility-corrected AAS (PDCAAS). This index was introduced by the 1989 Joint FAO/WHO Expert Consultation on Protein Quality Evaluation and has been in use for 20 years now35. The PDCAAS has been subject to constant criticism, of using a single crude protein or N digestibility value instead of a specific digestibility value of each IAA, using faecal instead of true ileal digestibility, the truncation of score and others4344.

Digestible indispensable amino acid score

To overcome the concerns related to PDCAAS, a FAO report recommended replacing the PDCAAS with the new scoring system termed the digestible IAA score (DIAAS)45. In the DIAAS, the limiting AAS is corrected for the specific true ileal digestibility of each IAA. A detailed account of the DIAAS calculation with an evaluation of aspects influencing the calculation is available46. For the computation of DIAAS, there is a need to determine true ileal IAA digestibility of habitually consumed foods and mixed diets, preferably in humans, although a porcine model has been suggested as its digestive physiology is very similar to humans4748.

True ileal digestibility in humans

The direct determination of true ileal protein (nitrogen) or AA digestibility entails the collection of digesta at the level of the ileum, in the animal models or humans49. In the animals, either growing rats or pigs, the method used is slaughter or ileal cannulation technique, with different types of cannulations being used for the latter. These methods have advantages and disadvantages, with key issues related to inaccuracy in quantitative digesta collection and the disruption of normal gut physiology5051.

In humans, mainly two models were adopted; naso-ileal intubation and the ileostomy model (in ileostomates)5253. These methods have made use of intrinsically and uniformly ¹⁵N or ¹³C-labelled (stable isotopes) test foods that allow for true ileal digestibility calculations, which adjust for endogenous protein contribution from the intestine545556. True N and AA digestibility is calculated from the cumulated amounts recovered at the ileal level and thus not absorbed in the small intestine. However, there are considerable methodological limitations concerning both the techniques. The naso-ileal intubation is fraught with technical challenges; such as guided intubation to ensure correct placement of the double-lumen catheters, and the use of only homogenized foods requiring markers to quantify ileal flow of effluents; it is thus time-consuming and invasive. The presence of a tube in situ may disrupt the normal physiological functioning of the gastrointestinal system by increasing small intestinal motility, which could further compromise nutrient absorption57. The ileostomy model involves individuals with surgically exteriorized terminal ileum (stoma), indicated for various pathological conditions of the colon, such as ulcerative colitis, familial polyposis or colonic cancer, and follows the classical oro-ileal balance method58. This method shares the same concerns as the porcine cannulation models.

In order to address these issues non-invasive or minimally invasive methods are recommended, using stable isotopes49. These methods include the non-invasive IAAO technique and the minimally invasive dual stable isotope tracer approach. The IAAO follows the same principle used for IAA requirements but measures the metabolic availability of the limiting AA in the test protein5960. Although non-invasive in nature, IAAO requires repeated experiments in a single individual, and a strict adherence to an adaptation diet that could affect compliance, which limits its use in vulnerable population.

The dual isotope tracer method is based on a similar principle of other dual-tracer approaches such as those used for starch digestion61. This tracer technique was used in protein digestion, but for a single AA, where the phenylalanine digestibility was measured in humans with cystic fibrosis, using uniformly labelled ¹⁵N-spirulina62. To determine the true ileal IAA digestibility of protein source foods, they first have to be intrinsically labelled with a stable isotope. There are multiple ways in which a plant or animal protein can be selectively labelled555663646566 depending on the label of interest (²H, ¹³C or ¹⁵N). The animals are either enterally fed or intravenously infused with labelled isotopes (single or multiple) that in turn intrinsically labels the protein source (eggs, milk and meat)56646566. Deuterium (²H₂O) watering or atmospheric labelling using ¹³CO₂ gas mixtures are used for plants67. A pulsed-dose protocol with ²H₂O for plants, has been standardized to obtain sufficient AA enrichments for use in human digestibility studies63. These intrinsically stable isotope-labelled test proteins are simultaneously fed with a differentially labelled ‘standard’ protein of a pre-determined digestibility, or with crystalline AAs that do not require digestion and are therefore 100 per cent digestible. As standard and test proteins are delivered simultaneously, it is assumed that their splanchnic extraction will be the same. Then, the postprandial ratio of the appearance of differently labelled AAs in the blood in relation to their ratio in the food ingested will allow for the evaluation of digestibility of the test protein. The measurement of IAA enrichment in the blood reflects absorption only from the small intestinal epithelium since colonic AA absorption is controversial. In addition, as the method only measures the appearance of labelled AAs from the intrinsically labelled test and standard protein, it is not affected by the contribution from endogenous protein sources and is therefore, a measure of true ileal digestibility. The technique is promising for determination of true ileal IAA digestibility, as it is minimally invasive, and can be applied across age groups, in pregnancy and in pathological conditions. Studies have been conducted using this approach in south Indian adults where the true ileal IAA digestibility of spirulina, hen egg and meat protein has been determined (Table)6364. The same technique was applied in children (1.5-2 yr) to obtain mean true ileal IAA digestibility of rice (79%), finger millet (68%), mung bean (65%) and whole boiled hen egg (87%). Of particular importance was the digestibility of the two limiting AAs, methionine and lysine. The mean digestibility was, for methionine 80, 60, 54, 86 per cent and 78, 75, 65, 94 per cent for lysine in rice, finger millet, mung bean and whole boiled hen egg, respectively(unpublished data).

Use of protein quality

Protein quality indexes are used in regulatory frameworks for countries and industries, as criteria to identify and endorse foods or formulations to be of a certain quality68. Expert consultations by the WHO/FAO on protein quality considerations have advocated the use of DIAAS where data are available, if not PDCAAS, to member nations3445. These promotions on the use of protein quality assessments are particularly relevant in feeding vulnerable populations such as children, either for optimal growth or when undernourished. For example, ensuring good protein quality of ready-to-use therapeutic food or similar formulations is critical to meet the metabolic needs of the severe acute malnutrition children.