Measurement of protein digestibility in humans by a dual-tracer method

Abstract

BackgroundRecent evaluations of the risk of dietary protein deficiency have indicated that protein digestibility may be a key limiting factor in the provision of indispensable amino acids (IAAs), particularly for vulnerable populations living in challenging environments where intestinal dysfunction may exist. Since the digestion of protein occurs only in the small intestine, and the metabolic activity of colonic bacteria confounds measurements at the fecal level, there is a need to develop noninvasive protein digestibility measurements at the ileal level.ObjectiveWe used a dual-tracer method with stable isotopes to characterize the digestibility of uniformly labeled [13C]-spirulina protein as a standard protein, in comparison to a mixture of 2H-labeled crystalline amino acids, and then demonstrated the use of this standard protein to measure the digestibility of selected legumes (chick pea and mung bean) through the use of proteins that were intrinsically labeled with 2H.DesignThe digestibility of uniformly labeled [13C]-spirulina was first measured in 6 healthy volunteers (3 males and 3 females) by feeding it along with a standard mixture of 2H-labeled amino acids, in a dual-tracer, plateau-fed test meal approach. Next, intrinsically labeled legume protein digestibility was studied with a similar dual-tracer approach, with uniformly labeled [13C]-spirulina as the standard, when processed differently before consumption.ResultsThe average digestibility of IAA in spirulina protein was 85.2%. The average IAA digestibility of intrinsically 2H-labeled chick pea and mung bean protein was 56.6% and 57.7%, respectively. Dehulling of mung bean before ingestion increased the average IAA digestibility by 9.9% in comparison to whole mung bean digestibility.ConclusionsAn innovative, minimally invasive “dual-stable-isotope” method was developed to measure protein digestibility, in which the ingestion of an intrinsically 2H-labeled test protein along with a 13C-labeled standard protein of known digestibility allows for an accurate measure of digestion and absorption of the intrinsically labeled protein. This minimally invasive method is critical to redefining protein quality and will aid in revisiting human protein requirements in different settings and in vulnerable populations. This trial was registered at Clinical Trials Registry—India as CTRI/2017/11/010468.

Harnessing Microbes for Sustainable Development: Food Fermentation as a Tool for Improving the Nutritional Quality of Alternative Protein Sources

“…To study the in vivo bioavailability of animal proteins, practices to intrinsically label protein amino acids with stable carbon-13 isotope have been developed, and these approaches are utilized to study the fate of milk, egg, and meat proteins in human metabolism [153][154][155][156][157]. In addition, some studies have employed nitrogen-15 labeling [158], or dual tracer [159] and indicator amino acid oxidation [160] methods to study the metabolic availability of plant proteins in humans. However, although evidence on the protein-digestibility-improving effects of food fermentations is accumulating, there are no meal studies surveying this phenomenon in real physiological conditions of the human gastrointestinal system.…”

Comparison of methodologies used to define the protein quality of human foods and support regulatory claims

“…The dual-isotope tracer method has been recently developed as a method to determine true ileal IAA digestibility in humans (Devi et al 2018). The appearance of labelled AAs in plasma from an intrinsically labelled ingredient protein ( 2 H-chickpeas, 2 H-mung beans) is compared with that of a simultaneously ingested and differentially labelled standard protein ( 13 C-spirulina).…”

Blood 15N:13C Enrichment Ratios Are Proportional to the Ingested Quantity of Protein with the Dual-Tracer Approach for Determining Amino Acid Bioavailability in Humans

“…Even though the dual-tracer method has been applied in several studies ( 6 , 8 , 10 ) and its results were comparable with bibliographic true ileal or apparent fecal digestibility values in pigs and humans ( 9 , 11 ), the approach has not been validated. The approach is based on several assumptions.…”

“…This dual stable isotope tracer method has been applied for measuring the bioavailability of AAs from different intrinsically labeled proteins in humans ( 8 , 9 ). The method relies on the ingestion of a meal containing a test protein of unknown AA bioavailability, intrinsically labeled with 2 H or 15 N, together with a tracer dose of a 13 C-labeled protein or standard free AA mixture of known AA bioavailability.…”

“…In this study we applied the dual-tracer approach for AA bioavailability using 15 N-milk protein concentrate together with 13 C-spirulina as test and standard protein, respectively. These protein sources were chosen as their AA digestibility is known ( 8 , 12 ). The aim was to test the critical assumption of the method that variation in bioavailability should be proportionally reflected in changes in isotope ratios.…”