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.
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