Good-quality plant protein sources are important for protein adequacy in a balanced diet. Legumes are known to be a source of good quality plant protein, but the true ileal digestibility of indispensable amino acids (IAAs) of commonly consumed legumes is not known in humans. In this study we measured the true ileal IAA digestibility of ²H-intrinsically labeled chickpea, yellow pea, and mung bean (hulled and dehulled) protein, using the dual-isotope tracer technique referenced to a standard protein ([U-¹³C] spirulina). The study also aimed to validate the use of [U-¹³C] spirulina as a reference protein in this method. ²H-intrinsically labeled legumes, obtained by watering plants with deuterium oxide (²H₂O), were administered in a plateau feeding method to healthy Indian adults to measure their true ileal IAA digestibility with the dual-isotope tracer technique, using [U-¹³C] spirulina protein or a ¹³C-algal IAA mixture as the standard. The true ileal IAA digestibilities (mean ± SD) of chickpea, yellow pea, and mung bean were 74.6 ± 0.8%, 71.6 ± 1.3%, and 63.2 ± 1.5%, respectively. The true mean ileal IAA digestibility of mung bean when referenced to [U-¹³C] spirulina protein or a ¹³C-algal IAA mixture did not differ significantly (63.2 ± 1.5% versus 64.0 ± 2.4%, P > 0.05). The true ileal IAA digestibility of mung bean improved to 70.9 ± 2.1% after dehulling. The true mean ileal IAA digestibility of legumes in healthy Indian adults was lower than expected. Traditional processing techniques such as dehulling improve protein digestibility by about 8%. This study was registered in the Clinical Trials Registry of India (CTRI): CTRI/2017/11/010468 (http://ctri.nic.in, accessed on 28/03/2019).

The recent Food and Agricultural Organization/World Health Organization/United Nations University expert consultations on protein requirements and quality have emphasized the need for the new Digestible Indispensable Amino Acid Score (DIAAS), as a measure of protein quality. This requires human measurements of the true ileal digestibility of individual indispensable amino acids (IAAs) until the end of the small intestine. Digestibility is measured using standard oro-ileal balance methods, which can only be achieved by an invasive naso-ileal intubation in healthy participants or fistulation at the terminal ileum. Significant efforts have been made over the last 2 decades to develop noninvasive or minimally invasive methods to measure IAA digestibility in humans. The application of intrinsically labeled (with stable isotopes like ¹³C, ¹⁵N, and ²H) dietary proteins has helped in circumventing the invasive oro-ileal balance techniques and allowed the differentiation between endogenous and exogenous protein. The noninvasive indicator amino acid oxidation (IAAO) technique, which is routinely employed to measure IAA requirements, has been modified to estimate metabolic availability (a sum of digestibility and utilization) of IAA in foods, but provides an estimate for a single IAA at a time and is burdensome for participants. The recently developed minimally invasive dual isotope tracer method measures small intestinal digestibility of multiple amino acids at once and is suitable for use in vulnerable groups and disease conditions. However, it remains to be validated against standard oro-ileal balance techniques. This review critically evaluates and compares the currently available stable isotope-based protein quality evaluation methods with a focus on the digestibility and metabolic availability measurements in humans. In view of building a reliable DIAAS database of various protein sources and subsequently supporting protein content claims in food labeling, a re-evaluation and harmonization of the available methods are necessary.

Milk is an important high-quality animal protein source in low- and middle-income countries (LMICs). Although the true ileal digestibility and absorption of milk has been shown to be high in French adults, this may be lower in individuals from LMICs who are at risk of environmental enteropathy. To determine the true ileal indispensable amino acid (IAA) digestibility of intrinsically labeled goat milk protein in South Indian women of reproductive age (WRA), using the dual-isotope tracer technique, and to measure intestinal absorption of amino acid and inert sugar in the same participants using L-allo-isoleucine and a dual-sugar assay. Milk with ²H-labeled protein collected from a lactating goat fed intrinsically ²H-labeled fodder (maize and cowpea) was spray dried. Labeled milk protein was administered in a plateau feeding protocol to WRA with normal BMI, in whom urinary lactulose and mannitol recovery and the lactulose/mannitol ratio (LMR) were measured, to determine its true ileal IAA digestibility by the dual-isotope tracer technique with a reference U-¹³C–amino acid mixture. A phenylalanine absorption index was calculated from the plasma to meal ratio of ¹³C₉ phenylalanine within the digestibility protocol. On a separate day, the allo-isoleucine absorption index was estimated from the ratio of plasma allo-isoleucine enrichments after oral ¹³C₆-¹⁵N-L- and intravenous ²H₁₀-L-allo-isoleucine administration. The means ± SDs of true ileal IAA digestibility of goat milk protein, lactulose and mannitol recovery, LMR, allo-isoleucine and phenylalanine absorption index were 94.0 ± 2.9%, 0.09 ± 0.03%, 7.9 ± 2.3%, 0.012 ± 0.004, 88.4 ± 3.8% and 24.5 ± 1.6%, respectively. The LMR correlated with the allo-isoleucine absorption index (r_s = –0.93, P = 0.008). The true ileal digestibility of goat milk protein in South Indian WRA with normal intestinal absorptive function and integrity was comparable to earlier estimates in healthy French adults.

Tea, a commonly consumed beverage, contains high amounts of polyphenols that can impair protein digestibility, as demonstrated in vitro. There are no human studies examining the inhibitory influence of tea polyphenols (TPP) on high-quality protein digestibility. The aim of this study was to determine the effect of black tea on the true indispensable amino acid (IAA) digestibility of whole boiled egg protein, in healthy adult humans, through use of a dual isotope tracer approach. The effect of black TPP (4.6 mg/mL, ingested as a beverage with the meal) on ²H-labeled whole boiled egg protein, administered with ghee rice and tomato curry, was measured with reference to ¹³C-spirulina protein in healthy Indian adults aged 20–27 y of both sexes with BMI of 22.0 ± 2.8 kg/m². The results were then compared to previously determined whole egg mean IAA digestibility measured by the same method, without black tea, in the same subjects (n = 5). To correct for any independent effect of TPP on spirulina protein (used as a standard protein), the true IAA digestibility of ¹³C-spirulina protein was independently measured with reference to a ²H-amino acid mixture, with and without co-ingestion of black tea, in 3 of the same subjects. The true IAA digestibility of whole boiled egg protein significantly decreased by 17% when co-ingested with black tea. However, there was no significant reduction in the true IAA digestibility of spirulina protein when co-ingested with black tea. TPP protein interactions reduced whole egg digestibility in healthy Indian adults but had minimal effect on spirulina protein digestibility. In populations who are at risk of dietary quality protein inadequacy, the consumption of tea during or after a meal can further increase the risk of inadequacy. This trial was registered at Clinical Trials Registry of India (http://ctri.nic.in) as CTRI/2018/03/012265.

Background/Objectives: The bioavailability of crystalline vitamin B₁₂ (B₁₂) through active absorption is reported to have a maximum capacity of 1.5–2.5 µg per dose. A small passive bioavailability has also been suggested at high doses. The present study aimed to determine the dose-dependency of active B₁₂ absorption and to quantify its passive absorption at higher doses. Methods: The dose-dependency of crystalline B₁₂ bioavailability was determined in nine healthy adults, using oral [¹³C]-cyanocobalamin, in a cross-over design at doses of 2.5, 5, and 10 µg. The dose order was randomised, with a washout of one month. Literature data from was added to the present study data in a meta-analysis of the relation between B₁₂ bioavailability and dose, to evaluate its pattern at different doses. Results: Bioavailability, as a function of dose, was significantly different between 2.5, 5, and 10 µg doses of [¹³C]-cyanocobalamin at 50.9 ± 32.5%, 26.7 ± 22.3%, 15.4 ± 13.6%, respectively, (p < 0.01), while the absolute bioavailability trended upward, at 1.16 ± 0.74 µg, 1.22 ± 1.02 µg, and 1.39 ± 1.23 µg (p = 0.46). The meta-analysis showed two distinct phases of bioavailability. Up to a dose of 2.6 µg, there was a significant steep positive correlation, with a slope (bioavailability) of 43%/µg suggesting an active process with a maximum of 1.2 µg. At higher doses, the slope was 1%/µg, not significantly different from zero, possibly a passive process. Conclusions: The active bioavailability of crystalline B₁₂ is not dose-dependent, saturating at ~1.2 µg.