Latest developments in policy and research on the DIAAS-method to determine protein quality

Daniel Tomé

AgroParisTech, INRA, France and Wageningen University, The Netherlands

Event name

Introduction

The question of protein supply in diets and the quality of dietary proteins hasbeen debated for decades by public health authorities worldwide

Protein quality remains among the main priorities for national and internationalorganizations 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.

International Authorities initiatives• FAO/WHO. Protein Quality Evaluation: Report of the Joint FAO/WHO Expert Consultation, FAO Food and Nutrition Paper 51.

Rome: FAO; 1991.

• WHO/FAO/UNU, 2007. Protein and amino acid requirements in human nutrition. Report of a Joint WHO/FAO/UNU Expert

Consultation, 2002. Geneva: World Health Organization (WHO Technical Report Series, No. 935, 2007.

• FAO, 2013. Dietary protein quality evaluation in human nutrition: Report of an FAO Expert Consultation, Auckland, New Zealand,

FAO Food and Nutrition Paper 92. Rome: FAO; 2013.

• International Atomic Energy Agency (IAEA), 2013. Consultants’ Meeting To review the status of protein and amino acid

requirements in infants and young children’ 12-14 November 2013, IAEA HQ, Vienna, Austria.

• FAO, 2014. Research approaches and methods for evaluating the protein quality of human foods. Bangalore, India. Report of a

FAO Expert Working Group. Rome, FAO; 2014.

• International Atomic Energy Agency (IAEA), 2014. Consultants Meeting to ‘Prepare for a CRP on protein bioavailability from plant

based foods’ IAEA HQ, Vienna, Austria 15-18 December 2014

Protein requirement and Protein quality

Protein requirement has been established from nitrogen balance at a meanvalue 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 asindispensable as they cannot be synthesized by the body and must beprovided by protein in the diet.

Indispensable amino acid content - Base for Protein qualityProtein quality - Evaluate the capacity of proteins, when consumed at thelevel of protein requirement (i.e. 0.66 g/kg/day), to also provide the bodywith the 9 indispensable amino acids (IAA) in a metabolically available form.

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 dietaryprotein 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

Average amino acid requirements in adults and reference amino acid profile

Requirement for adultsAverage IAA

requirements

(mg/kg/d)

Reference pattern of

bioavailable IAA

(mg/g Protein)

Total Protein 660 (1000)

Indispensable AA 184

Histidine 10 15

Isoleucine 20 30

Leucine 39 59

Lysine 30 45

Methionine+cysteine 15 22

Phenylalanine+tyrosine 25 38

Threonine 15 23

Tryptophan 4 6

Valine 26 39

= reference IAA profile of a proteinmeeting IAA needs when providedat the level of protein requirement(0.66 g/kg/d)

WHO/FAO/UNU (2007)

Taking into account protein and

indispensable amino acid

requirements to calculate a

reference amino acid profile

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 profileAdult (mg/g Protein)

PD-CAASAvailable/Reference

Total protein 1000 860 - -

Histidine 21 18 15 1.20

Isoleucine 34 29 30 0.97

Leucine 69 59 59 1.00

Lysine 23 20 45 0.43

Methionine+cysteine 36 31 22 1.40

Phenylalanine+tyrosin

e

7766

38 1.73

Threonine 28 24 23 1.04

Tryptophan 10 9 6 1.43

Valine 48 41 39 1.05

(x 0.86)

(WHO/FAO/UNU 2007)Corrected by protein digestibility

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.

Score 0.43

Protein Digestibility Corrected Amino Acid (PD-CAAS) – Milk protein (95% Digestibility)

Milk protein

(95% digestibility)

Total content(mg/g Protein)

Available(mg/g Protein)

Reference profile(mg/g Protein)

PD-CAASAvailable/Reference

Total protein 1000 950 - -

Histidine 24 22.9 15 1.5

Isoleucine 48 46.0 30 1.5

Leucine 97 92.7 59 1.5

Lysine 76 72.0 45 1.6

Methionine+cysteine 32 38.4 22 1.7

Phenylalanine+tyrosine 105 99.7 38 2.6

Threonine 51 48.2 23 2.1

Tryptophan 14 13.3 6 2.2

Valine 71 67.3 39 1.7

(WHO/FAO/UNU 2007)Corrected by protein digestibility

(x 0.95)

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.

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.

The faecal digestibility is

measured in the faeces

The ileal digestibility is

measured at the terminal ileum

Digestibility (%) = (ingested – digestive losses) / ingested %

Faecal and ileal digestibilty

Digestibility issue of proteins

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.

The ileal digestibility is considered more accurate for dietary amino acid digestibility.

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 ilealdigestibility 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

Faecal and ileal digestibility

Milk Soy

Ileal digestibility 95 91

Faecal digestibility 96-97 95-97

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 1992.

The ileal digestibility is more accurate for dietary amino acid digestibility.

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).

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

• 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.(Gaudichon et al 1999)

Experimental protocols to determine ilealdigestibility of IAA from dietary proteins

FAO 2014, IAEA 2014 - 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

The Dual Tracer ApproachBased 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 13C (of know digestibility) and the tested protein of unknown digestibility labelled with another tracer (15N or 2H).

• the isotopic signature is determined in the meal and in the plasma as the 2H or 15N/13C 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)

Bioavailability of protein amino acids using stable isotope 15N/13Clabelled protein and amino acids isotopic signature

The 15N/13C ratio of amino acid enrichement is the isotopic signature in the plasma

The 15N/13C ratio of amino acid enrichment is the isotopic signature of the meal

B- Collecting bloodsamples and measurement of 13C and 15N enrichment in blood free amino acids

Blood free Aminoacid pool

A- Ingestion of a mealcontaining a mixture of 13C-labelled Spirulline protein and 15N-labelled Milk protein

15N/13C ratio Digestibilityplasma = meal Milk = Spirullineplasma > meal Milk > Spirullineplasma < meal Milk > Spirulline

Study designStudy population: Healthy male and female, age 20-35y, n=415N-labelled milk protein with trace amount of 13C-labelled spirulina

Randomized cross-over trial: test two levels of 15N protein; high protein (50 gram) and low protein (25 gram), while 13C-labelled spirulina remains similar (400mg)

Design test day: Test meal divided in 9 equal portions to reach a steady state in uptake

Isotope measurement in free AA fraction with EA-IRMS

Pudding

Bloo

d

Time -20 0 30 60 90 120 150 180 210 240 270 300 330 360 minutes

Plasma 15N and 13C free AA fractionIn both meals similar dose of 400mg 13C-Spirulline was given, resulting in a similarprofile of plasma 13C-enrichment

50g dose of 15N-milk protein in the mealcompared to 25g milk protein, also resulted in 2 fold higher 15N-enrichment of free AA.

Digestibility of Milk and Spirullineprotein by the dual tracer approach

Milkprotein

Meal Plasma RatioPlasma/Meal15N-APE 13C-APE

15N/13C 15N-APE 13C-APE15N/13C

50g 1.86 0.44 4.24 0.249 0.038 6.92 1.6325g 1.77 0.85 2.09 0.110 0.034 3.45 1.65

From the ratio of relative 15N/13C enrichment of amino acids in plasma and meal in humansubject receiving 15N-labelled milk protein (25g or 50g) with trace amount of 13C-labelled spirulina (400mg), the comparison of Milk and Spirulline protein digestibility is in the relation:

- Milk protein digestibility = 1.64 x Spirulline protein digestibility

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.

Aknowledgments

Claire GaudichonNadejda Khodorova

Nikkie VanderwielenMarco Mensink

Dutch Dairy AssociationStephan PetersJan SteijnsGert Jan Hiddink