protein hydrolysates: do they really work ? vincent c biourge, dvm phd dipl acvn & ecvcn royal...
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Protein hydrolysates:Do they really work ?
Vincent C Biourge, DVM PhD Dipl ACVN & ECVCN
Royal Canin, Centre de Recherche, Aimargues
Adverse Reactions to Food
Adverse Reactions to Food
Food intoleranceFood Allergy
Dermatologic: pruritus, alopecia,otitis, ..Gastrointestinal: vomiting, abd. discomfortRespiratory: asthma, rhinitis, ...General: headache, arthritis, ...
Immune mediated(IgE, cell mediated)
Non immune mediated(Lactose, chocolate, bioamines,
additives, …)Probably the main cause (Olivry T, J Vet derm 2010)
Adverse Reactions to Food
Diagnosis
Clinical signs
Novel ingredients
Improvement of the clinical signs
4 to 12 Weeks
Elimination diet
Challenge
Clin. signs
Double blindedD
r Thie
rry X
imenes
(Fra
nce
)
+ Serum IgE+ Skin test
Definition• Diets composed of ingredients to which
the dog has not been exposed. Dietary history
• Home made Starch sources: Rice, Potato, Tapioca Protein sources: Lamb, Horse, Fish, Turkey, Venison,
.. Essential fatty acids, Minerals and Vitamins
• Commercial Lamb & Rice, Fish & Potatoes, Venison & Potatoes Over the counter
• Not hypoallergenic ! Intact protein sources !
• Sustainability Fish, Rabbit, Duck, Venison, …
Elimination dietsGold standard
Raditic et al, JAPAN 2010
Protein hydrolysates2001
A new strategy to treat adverse reaction to food
EnzymaticHydrolysis
protein
polypeptides
• Easier to digest
Short half-life in the lumen
• Smaller peptides Molecular weight < 16 Kd
Benefits of hydrolysate
Cave, 2006
Do they really work ?• What is the background ?• Should molecular weight be as
low as possible ?• Are they trully hypoallergenic ?• Can they be qualified as allergen
free ?• Are their efficacies substantiated
by clinical studies ?
Protein hydrolysates
Background
0
5
10
15
20
25
30
35
40
Imm
un
orea
ctiv
ity
(mg/
g p
rot)
1 2 3
Glycinine
B-conglycinine
Elisa immunoreactivity of soy protein depending of its source
1. Soy flour2. Soy concentrate3. Soy hydrolyzate
Lallès, 1995
Adverse reaction to soy in milk replacerPoor appetite and growthMalabsorption/maldigestion – DiarrheaSoy antibodiesVillus atrophy
LaMartin.com
Background
Protein hydrolysate based milk replacer.
Babies at risk for allergy Risk of cow milk allergy
Baby allergy to cow’s milkVomiting diarrheaAtopic dermatitis/ UrticariaAsthma/Rhinitis
http://www.eecs.harvard.edu/~eli/pictures/
Dalton• Arbitrary unit of atomic mass• Sir John Dalton, Founder of the atomic theory• 1 Dalton (d) = Weight of 1/12 nuclide of 12C
1.657 10-24 gramme
• Glycin = 75 d - Tryptophan = 204 d• Casein = 19 000 - 25 000 d or 19-25 Kd• Measurement: electrophoresis -
chromatography
Molecular weight
Human serum albumin = 69 Kd
Molecular weightMean Max
Ingredients Vs dietsCave N, 2006
Mean MW
Most common food allergens • Glycoproteins
Protein with glucide moiete ? Lipids (Bacterial glycolipids) ? Carbohydrates
• Water - soluble• Heat and acid resistants• Molecular weight 10 to 40 Kd
Small enough to pass the intestinal wall Large enough to induce immune reaction
Protein fraction MW (Kd)Caseins 19-20 Ovalbumin 45Arachis hypogea I 63Arachis hypogea II 17Soybean Trypsin inhibitor 20Soybean B-conglycinine 53Soybean glycinine 309-363Fish allergen M 12Shrimp antigen I 42Schrimp antigen II 38Wheat 8-67
Antigens in selected foodin man
Sampson HA, 1993
Molecular weight
Cave N, 2006Looringh van Beeck FA, 2009
10- 40 kDa
3 – 10 kDa
1– 3 kDa
< 1 kDa
> 40 kDa
Highly reduced allergy
No allergy
Reduced allergy
Most common
Less common allergy
Molecular weightMolecular
weight
Guilford GW ,1996Serra et al, 2006
Olivry T, 2010Cave N, 2006
Molecular weightAntigenicity
Epitope
Few
Many
Close in 3D
Hidden 0 1 2 3 4
Casein
20 % Hydrolyzed casein
50 % Hydrolyzed casein
Soy
Hydrolyzed soy
Chicken liver
Hydrolyzed liver
Score value
Sensitized
Control
Gastroscopic score diameter Olson ME et al 2000
Molecular weightAntigenicity
AJVR 2006;67:1895-1900
Molecular weightDigestibility
80
82
8486
88
90
92
94
9698
100
AA
ilea
l dig
esti
bili
ty (
%)
amino acids
Flour
Concentrate
Hydrolysate
Soy source and aa ileal digestibility in calfs
Lallès, 1995808284
868890
92949698
100
N in
vit
ro d
iges
tib
ility
(%
)
N
Soybean
Hydrolysate
Soy source and in vitro N digestibility
Royal Canin, 2004
Cave NJ, Marks SL. Evaluation of the immunogenicity of dietary proteins in cats and the influence of the canning process. AJVR 2004; 10,1427-1433
« The number of IgE binding sites on the allergen, their location, and the tertiary shape of the protein are probably
more important than the molecular weight » S.F. Hefle, 1996
Source of protein (Casein Vs Soy)
Process - type of hydrolysis -exposure of epitopes
Problem measuring MW on finished product
Cut-off
Molecular weight
In vitro testing - ELISA
Immunogenicity
Hannah, 1997
0
0,1
0,2
0,3
0,4
0,5
0,6
0,7
0,01 0,1 1 10 100 1000 10 000 100 000
Protein concentration (ug/mL)
Ab
sorb
ance
at
405
nm Native soy protein
Soy hydrolysate
75
50
2520
8204 8212 82088216
37
8202 8210
Sensitised dogs
31
Sensitised dogs
50
30
8204 8216 82088212 8202 8210
Western blot
Immunogenicity
native hydrolysed
AJVR 2006; 67:484-488
Skin test
Immunogenicity
Puigdemont et al, 2006
% of reduction of the wheal areas for soy hydrolysed
versus native soy protein
Soy protein concentration
Dog
1 g/ml
10 g/ml
100 g/ml
1T2
53,8
45,7
42,0
1T3
53,5
54,6
52.6
2T2
95,6
18,4
44,6
2T6
81,9
61
86,4
3T2
59,7
58,4
37,4
3T4
47
80,1 57,6
Mean SD
65,3 19,1
53,0 20,4
53,4 17,7
Control showed no wheal on both challenges
Clinical reactions
Immunogenicity
Puigdemont et al ,2006
Control and 3 other sensitised dogs showed no adverse reactions
Dogs Nativesoy protein
Hydrolysedsoy
protein
Soyspecific
IgE
8204Vomits
Diarrhoea (1)Pruritus
NR +++
8212 Diarrhoea (2) NR +++
8216 Diarrhoea (2) NR ++
Clinical reactions
Immunogenicity
Jackson et al, 2003
14 maltese x Beagle dogs with known clinical hypersensitivity to soy and corn
No corn and starch diet
Corn starch Corn Soy Soy hydrolysate diet200 mg/kg bw
CutaneousClinicalScore(Max 35*3*3)
ImmunogenicityStomach reactivity
0 1 2 3 4
Casein
20 % Hydrolyzed casein
50 % Hydrolyzed casein
Soy
Hydrolyzed soy
Chicken liver
Hydrolyzed liver
Score value
Sensitized
Control
Gastroscopic score diameter Olson ME et al 2000
Conclusion• Hydrolyzed proteins are less antigenic
than intact proteins. • Hydrolyzed proteins are not anallergenic
except if hydrolyzed to single or few amino acids.
• High digestibility is an important factor in reducing antigenicity.
Immunogenicity
Clinical trials
Shown efficacy• Managing adverse
reaction to food. • Diagnosis of adverse
reaction to food. • Inflammatory bowel
disease.• Exocrine pancreatic
insufficiency.
Clinical reactions
Adverse reaction to food
Jackson et al, 2003
14 maltese x Beagle dogs with known clinical hypersensitivity to soy and corn3/14 dogs increased scores on hydrolyzed diet.
No corn and starch diet
Corn starch Corn Soy Soy hydrolysate diet200 mg/kg bw
CutaneousClinicalScore
• Evaluation on dogs with demonstrated adverse reactions to food
12 dogs ( breeds, sex, food allergy ) Controlled allergy (no clinical signs) Clinical exam @ 0 and 2 months (CADESI) Owners requested to report:
– Pruritus, abnormal behavior of the dog– Digestive tolerance– Palatibility
• None of the 12 dogs relapsed Perfect tolerance
J. Fontaine, CNVSPA 2001
Adverse reaction to food
Diagnosis of ARF
Clinical signs
8 Weeks
Soy hydrolysate diet
Challenge
Clin. signs
1. Recovery challenge + Adverse Reaction to Food (ARF)
2. Marked improvement challenge + ARF + atopy
3. Little or no improvement other elimination diet Recovery
4. Little or no improvement other elimination diet no improvement ? atopy
No corticotherapy
• 2 dermatology specialty pratices J Fontaine (Brussels, B), M Vroom (Oisterwijk, NL)
• Inclusion in the study: Suspicion of skin hypersensitivity
VC Biourge, J Fontaine, MW Vroom, 2004
J Nutr2002;134:2062S-2064S
Diagnosis of ARF
• 60 dogs included• 31 M-3 MC - 13 F - 13FS• Age 4.5±0.4 yrs (3 mo – 11
yrs)• 26 breeds
• German Sheperd (10), Bouledogue Français (2), Bouvier des Flandres (2), Boxer(5), English Cocker Spaniel (2), Golden Retriever (4), Jack Russel (2), Labrador (4), Shar Pei (3), Shi Tsu (2), WHWT (5), …
• Duration of the clinical signs• 2.6±0.4 yrs (3 weeks – 10 yrs)
Adverse Reaction to Food
Atopy
20 2216
+ 2 cases excluded
Diagnosis of ARF
Adverse reaction to food
0
0,5
1
1,5
2
2,5
3
3,5
4
4,5
5
Before After
Pru
rit
scor
e***
• 20 dogs• 9 M-1 MC - 6 F – 4 FS• Age 3.8±0.6 yrs (6 mo – 9yrs)• No more pruritus• No or very mild clinical signs
left.• Challenge +• 18/20 responded to soy
hydrolysate diet Rabbit and Rice Homemade soy diet
• Golden Retriever Male, 4.5 yrs, 29.6 kg
• Generalized intense prurit with lichenification, hyperpigmentation
Before After
Diagnosis of ARF
Diagnosis of ARF
Adverse reaction to food & Atopy
0
0,5
1
1,5
2
2,5
3
3,5
4
4,5
5
Before After
Pru
rit
scor
e
***
• 16 dogs• 8 M-1 MC - 2 F – 5 FS• Age 5.7±0.7 yrs (3 mo –
11yrs)• Pruritus marketly improved• Mild to moderate clinical signs
left.• Challenge +• All dogs responded to the soy
hydrolysate diet.
Diagnosis of ARF
Atopy
0
0,5
1
1,5
2
2,5
3
3,5
4
4,5
5
Before After
Pru
rit
scor
e
• 22 dogs• 13 M-1 MC - 5 F – 4 FS• Age 4.5±0.6 yrs (1.2 – 11yrs)• No or little improvement of
pruritus, clinical signs.• No response to other elimination
diets and + to skin test
Diagnosis of ARF
ARF: response to the hydrolysate
0
5
10
15
20
25
30
35
40
Yes No
94.4 % of ARF dogs responded to the soy hydrolysate based diet
Diagnosis of ARFOther study
Loeffler et al., Vet Derm 2006;17:273-279
• Chicken hydrolysate diet Vs homade diets
• 181 dogs• 17 dogs excluded• 35 allergic skin disease
10 38
ARF Atopy
114720
ARF Atopy
15
Poultry hydrolyzate (109) Homade (72)
Excluded: 27 Excluded: 13
No significant difference
Veterinary Dermatology,2010,21:358-366
12 dogs selected and divided In 2 groups
1 dog showed severe signs when fed hydrolyzed chicken
Before After
Diagnosis of ARFCats
Dr Aranda
Diagnosis of ARF
ARF: response to the hydrolysate
0
1
2
3
4
5
6
7
8
Yes No
87.5 % of ARF cats responded to the soy hydrolysate based dietMW Vroom, C. Swinnen, A clinical study of a soy protein isolate hydrolysate diet, in dogs and cats with adverse reactions to food. Proc. of Voorjaarsdagen 2002. 252
Food responsive chronic diarrhea
• Dogs:– 26 dogs over a 2 yr-period.– 24/26 dogs with IBD – 4 IF, 10 SF, 8 IM, 4 CM.– Age: 4.3 ± 3.3 yrs (0.6- 11 yrs)– Weight 23 ± 12 kg (4.7- 40 kg)– Duration of the clinical signs:
1 to 36 months before presentation.• Treatments before inclusion:
– Antibiotics (7), Metaclopramide (6), cimitidine (6) , Prednisolone (4), sulfazalazine (3).
• Diets before inclusion:– Low residue intestinal diets (7),
novel protein diets including homemade (12), other diets (7).
Food responsive chronic diarrhea
Mandigers et al., 2010
ObjectivesTo compare the response of
dogs with chronic diarrhea on soy hydrolysate Vs intestinal
diet.
Clinical signs after 2 months
02468
1012141618
No signs Signs
Num
ber
of d
ogs
HydrolysateLow residue
No more clinical signs 23/26 dogs 3 last dogs improved but vomiting and diarrhea persisted
11% 12.5%
Food responsive chronic diarrhea
0
0,2
0,4
0,6
0,8
1
1,2
1,4
Wei
ght
gain
(k
g)
HydrolysateLow residue
*
Weight gain
Follow-up after median 6 months, range 3-15 mo.
15/16 test dogs – 6/7 control dogs
0
2
4
6
8
10
12
14
No signs Signs
Num
ber
of d
ogs
HydrolysateLow residue
87 % of dogs on hydrolyzate diet remained free of clinical signs on follow-up ! The signs were minor in the remaining 2 dogs.
13%67%
Food responsive chronic diarrhea
Food responsive chronic diarrhea• Similar findings by other authors.
Marks SL, Laflamme D, McCandlish A. Dietary trial using a commercial hypoallergenic diet containing hydrolyzed protein for dogs with IBD. Vet Ther 2002; 3:109-18.
• Similar finding in cats 8 cats Chronic diarrhea (4-36 Mo) 1 Colitis, 2 gastritis 6 IBD Soy hydrolyzate based diet Resolution of clinical signs within 4-8 d Median weigh gain 0.75 kg within 2 Mo 11Mo follow-up 6/8 cats free of clinical signs
German Shepherds
EPI and skin disease
GI signs controlled within 7 dWeight gain with 2 monthsSkin within 3 months
Exocrine pancreatic Insufficiency
Case Age TLIyrs ug/L Before After
1 5,0 2,5 33,7 38,02 7,0 0,88 32,0 43,03 9,0 ND 40,0 44,0
Body weight (kg)
J Nutr2002;134:2166S-2068S
Conclusion• Hydrolyzed proteins are less antigenic than intact
proteins.• Molecular weight (except if extremly low (<1Kd) is a
poor predictor of protein immunogenicity. • Hydrolyzed protein based diet are not anallergenic.• Hydrolyzed proteins are sustainable. • Clinical studies to support benefits in:
Diagnosis and management of ARF Idiopathic chronic diarrhea - IBD Exocrine pancreatic insufficiency ? Perianal fistula.
Protein hydrolysates
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