Latest developments in policy and research on the DIAASmethod to determine protein quality

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2 Latest developments in policy and research on the DIAASmethod to determine protein quality Daniel Tomé AgroParisTech, INRA, France and Wageningen University, The Netherlands Event name

3 Introduction The question of protein supply in diets and the quality of dietary proteins has been debated for decades by public health authorities worldwide Protein quality remains among the main priorities for national and international organizations in charge of agriculture, food and public health. Dietary protein is an indispensable component of the diet by supplying the body with nitrogen and amino acids. Amino acids are used to synthesize and maintain around 10 kg body protein as well as other non protein metabolically active nitrogenous substances.

4 International Authorities initiatives FAO/WHO. Protein Quality Evaluation: Report of the Joint FAO/WHO Expert Consultation, FAO Food and Nutrition Paper 51. Rome: FAO; WHO/FAO/UNU, Protein and amino acid requirements in human nutrition. Report of a Joint WHO/FAO/UNU Expert Consultation, Geneva: World Health Organization (WHO Technical Report Series, No. 935, FAO, Dietary protein quality evaluation in human nutrition: Report of an FAO Expert Consultation, Auckland, New Zealand, FAO Food and Nutrition Paper 92. Rome: FAO; International Atomic Energy Agency (IAEA), Consultants Meeting To review the status of protein and amino acid requirements in infants and young children November 2013, IAEA HQ, Vienna, Austria. FAO, Research approaches and methods for evaluating the protein quality of human foods. Bangalore, India. Report of a FAO Expert Working Group. Rome, FAO; International Atomic Energy Agency (IAEA), Consultants Meeting to Prepare for a CRP on protein bioavailability from plant based foods IAEA HQ, Vienna, Austria December 2014

5 Protein requirement and Protein quality Protein requirement has been established from nitrogen balance at a mean value of 0.66 g protein/kg body weight per day in healthy adult (FAO/WHO/UNU, 2007). Among the 20 amino acids that constitute the proteins, 9 are considered as indispensable as they cannot be synthesized by the body and must be provided by protein in the diet. Indispensable amino acid content - Base for Protein quality Protein quality - Evaluate the capacity of proteins, when consumed at the level of protein requirement (i.e g/kg/day), to also provide the body with the 9 indispensable amino acids (IAA) in a metabolically available form.

6 FAO Expert consultation on protein quality evaluation in Human Nutrition (2007) In the Protein Digestibility-Corrected Amino Acid Score (PD-CAAS) approach: Protein quality is based on indispensable amino acid content of each dietary protein source Indispensable amino acid content is corrected by protein digestibility that predicts the fraction made available to the organism after digestion and absorption Available Indispensable amino acid content is related to a reference amino acid profiles considered to meet indispensable amino acids needs in humans

7 Average amino acid requirements in adults and reference amino acid profile Requirement for adults Average IAA requirements (mg/kg/d) Reference pattern of bioavailable IAA (mg/g Protein) Total Protein 660 (1000) Indispensable AA 184 Histidine Isoleucine Leucine Lysine Methionine+cysteine Phenylalanine+tyrosine Threonine Tryptophan 4 6 Valine WHO/FAO/UNU (2007) Taking into account protein and indispensable amino acid requirements to calculate a reference amino acid profile = reference IAA profile of a protein meeting IAA needs when provided at the level of protein requirement (0.66 g/kg/d)

8 Protein Digestibility Corrected Amino Acid (PD-CAAS) Wheat protein (86% digestibility) Wheat protein (86% digestibility) Total content (mg/g Protein) Available (mg/g Protein) Reference profile Adult (mg/g Protein) PD-CAAS Available/Reference Total protein 1000 (x 0.86) Histidine Isoleucine Leucine Lysine Methionine+cysteine Phenylalanine+tyrosin e Corrected by protein digestibility (WHO/FAO/UNU 2007) Threonine Tryptophan Valine Score 0.43 PD-CAAS: the content of each amino acid is corrected by the digestibility of the protein. The score for each amino acid is the ratio for each amino acid between the available content and the reference profile. The lowest ratio gives the score of the protein. A score below 1 indicates that the amino acid is limiting in the protein.

9 Protein Digestibility Corrected Amino Acid (PD-CAAS) Milk protein (95% Digestibility) Milk protein (95% digestibility) Corrected by protein digestibility Total content (mg/g Protein) Available (mg/g Protein) (WHO/FAO/UNU 2007) Reference profile (mg/g Protein) PD-CAAS Available/Reference Total protein 1000 (x 0.95) Histidine Isoleucine Leucine Lysine Methionine+cysteine Phenylalanine+tyrosine Threonine Tryptophan Valine Score 1 PD-CAAS: the content of each amino acid is corrected by the digestibility of the protein. The score for each amino acid is the ratio for each amino acid between the available content and the reference profile. The lowest ratio gives the score of the protein. A score below 1 indicates that the amino acid is limiting in the protein.

10 Digestibility issue of proteins Food protein digestion occurs in the stomach and small intestine and produces amino acids absorbed in the small intestine. Digestibility is determined by measuring the digestive losses at the level of the terminal ileum or in the faeces. Digestibility (%) = (ingested digestive losses) / ingested % Faecal and ileal digestibilty The ileal digestibility is measured at the terminal ileum The faecal digestibility is measured in the faeces

11 Digestibility issue of proteins The ileal digestibility is considered more accurate for dietary amino acid digestibility. Protein digestion produces amino acids which are absorbed in the small intestine (duodenum, jenunum, ileum). In the large intestine unabsorbed amino acids are mostly metabolized by colonic bacteria and converted to ammonia that can be absorbed.

12 Digestibility issues of proteins In the PD-CAAS the digestibility of protein is largely determined from faecal digestibility values provided by available faecal protein digestibility data base As unabsorbed amino acids are mostly metabolized by colonic bacteria and converted to ammonia that can be absorbed, faecal digestibility can be over-estimated, particularly for low digestibility proteins : The ileal digestibility is more accurate for dietary amino acid digestibility. In addition, in the PD-CAAS approach the same value of digestibility of the protein is applied to each amino acid. But all amino acids from a dietary protein source are not similarly absorbed: The specific ileal digestibility of each amino acid is more accurate for dietary amino acid digestibility. A modified score, the Digestible Indispensable Amino Acid Score (DIAAS) (FAO, 2011, 2014) considers the specific ileal digestibility of each amino acid but this score is not yet used due to methodologic issues. No data base on ileal AA digestibility

13 Faecal and ileal digestibility The ileal digestibility is more accurate for dietary amino acid digestibility. Milk Soy Ileal digestibility Faecal digestibility Gausserès et al 1997; Gaudichon et al 1999; Bos et al 1999; Mariotti et al 1999; FAO 1990; Hess et al 2000; Kayser et al But difficult to determine ileal digestibility in humans (need access to the ileum content) Ileal digestibility can be measured in animal model (rat, pig) but the translation to human needs validation Alternatively, new non-invasive experimental protocols to determine ileal digestibility in human without sampling ileal digesta are currently being developed (FAO 2014).

14 Specific amino acid digestibility In the PD-CAAS approach the same digestibility of the protein is applied to each amino acid. But all amino acids from a same dietary protein source are not similarly absorbed (Gaudichon et al 1999) In the new discussed index DIAAS (Digestible Indispensable Amino Acid Score, FAO 2014), the specific ileal digestibility of each amino acid is considered. but this score is not yet used due to methodologic issues. No data base on IAA ileal digestibility.

15 Experimental protocols to determine ileal digestibility of IAA from dietary proteins FAO 2014, IAEA protocols either well-tested and already used or promising with future development for measuring specific ileal digestibility of each amino acid to establish a data base of IAA ileal digestibility from dietary protein True Ileal Amino Acid Digestibility in animal models (rodent, pig) kinetic sampling of ileal content Already available - Could be a screening method but translation of the results to human needs validation and possible ethical questions of animal experimentation True Ileal Amino Acid Digestibility in Human with Intestinal tubing or in ileostomate subjects kinetic sampling of intestinal and blood samples Can be used as reference methods but invasive and ethically difficult as routine method Stable isotope-signature dual tracer approach for meauring IAA bioavailability in human blood sampling moderately or not invasive but development and validation is required if validated could be a routine method in human

16 The Dual Tracer Approach Based on the use of intrinsic labeled proteins with two different tracers (FAO, 2014; IAEA, 2014): The principle is to give a mixture of a protein labeled with 13 C (of know digestibility) and the tested protein of unknown digestibility labelled with another tracer ( 15 N or 2 H). the isotopic signature is determined in the meal and in the plasma as the 2 H or 15 N/ 13 C ratio of amino acid isotope enrichment. Differences in these signature between the meal and the plasma allow to calculate protein and amino acid digestibility (bioavailability)

17 Bioavailability of protein amino acids using stable isotope 15N/13C labelled protein and amino acids isotopic signature A- Ingestion of a meal containing a mixture of 13 C-labelled Spirulline protein and 15 N-labelled Milk protein B- Collecting blood samples and measurement of 13C and 15 N enrichment in blood free amino acids Blood free Amino acid pool The 15 N/ 13 C ratio of amino acid enrichment is the isotopic signature of the meal 15 N/ 13 C ratio Digestibility plasma = meal Milk = Spirulline plasma > meal Milk > Spirulline plasma < meal Milk > Spirulline The 15 N/ 13 C ratio of amino acid enrichement is the isotopic signature in the plasma

18 Study design Study population: Healthy male and female, age 20-35y, n=4 15 N-labelled milk protein with trace amount of 13 C-labelled spirulina Randomized cross-over trial: test two levels of 15 N protein; high protein (50 gram) and low protein (25 gram), while 13 C-labelled spirulina remains similar (400mg) Design test day: Test meal divided in 9 equal portions to reach a steady state in uptake Bloo d Isotope measurement in free AA fraction with EA-IRMS Pudding Time minutes

19 Plasma 15N and 13C free AA fraction 50g dose of 15N-milk protein in the meal compared to 25g milk protein, also resulted in 2 fold higher 15N-enrichment of free AA. In both meals similar dose of 400mg 13C- Spirulline was given, resulting in a similar profile of plasma 13C-enrichment

20 Digestibility of Milk and Spirulline protein by the dual tracer approach Milk Meal Plasma Ratio protein 15 N-APE 13 C-APE 15 N/ 13 C 15 N-APE 13 C-APE 15 N/ 13 C Plasma/Meal 50g g From the ratio of relative 15 N/ 13 C enrichment of amino acids in plasma and meal in human subject receiving 15 N-labelled milk protein (25g or 50g) with trace amount of 13 C-labelled spirulina (400mg), the comparison of Milk and Spirulline protein digestibility is in the relation: - Milk protein digestibility = 1.64 x Spirulline protein digestibility

21 Conclusion Protein quality is associated to the capacity of a protein source to provide indispensable amino acids in order to meet metabolic needs, i.e. protein quality is based on digestible indispensable amino acid content of each dietary protein source. In the scoring approach, the content in digestible amino acid of dietary protein is related to the composition of a reference amino acid profile calculated to meet indispensable amino acid needs. The PD-CAAS is the reference score for assessing protein quality but as limitations related to the use a faecal instead of ileal digestibility. Ileal digestibility measured at the terminal ileum is more accurate than faecal digestibility to determine metabolic availability of amino acid. A new score, the DIAAS, has been proposed but is not used due to methodologic issues. New developments aims to develop methods using minimally invasive protocols in order to replace current data base on faecal protein digestibility by AA ileal digestibility in humans.

22 Aknowledgments Nikkie Vanderwielen Marco Mensink Claire Gaudichon Nadejda Khodorova Dutch Dairy Association Stephan Peters Jan Steijns Gert Jan Hiddink