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

Am J Clin Nutr. 2018 Jun 1;107(6):984-991. doi: 10.1093/ajcn/nqy062.


Background: Recent 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.

Objective: We 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.

Design: The 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.

Results: The 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.

Conclusions: An 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acids, Essential / chemistry
  • Amino Acids, Essential / metabolism*
  • Dietary Proteins / analysis
  • Dietary Proteins / metabolism*
  • Digestion / physiology*
  • Female
  • Humans
  • Male
  • Plant Proteins / metabolism*
  • Spirulina / chemistry
  • Vigna / chemistry
  • Young Adult


  • Amino Acids, Essential
  • Dietary Proteins
  • Plant Proteins

Associated data

  • CTRI/CTRI/2017/11/010468