1. Protein hydrolysates: Do they really work ?
Vincent C Biourge, DVM PhD Dipl ACVN & ECVCN
Royal Canin, Centre de Recherche, Aimargues

2. Adverse Reactions to Food
Dermatologic: pruritus, alopecia,otitis, ..
Gastrointestinal: vomiting, abd. discomfort
Respiratory: asthma, rhinitis, ...
General: headache, arthritis, ...
Food Allergy
Food intolerance
Immune mediated
(IgE, cell mediated)
Non immune mediated
(Lactose, chocolate, bioamines,
additives, …)
Probably the main cause (Olivry T, J Vet derm 2010)

3. Adverse Reactions to Food
Diagnosis
Challenge
Dr Thierry Ximenes (France)
Double blinded
Clin. signs
Elimination diet
Clinical signs
4 to 12 Weeks
Novel ingredients
+ Serum IgE
+ Skin test
Improvement of the clinical signs

4. Elimination diets Definition Gold standard
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, …
Raditic et al, JAPAN 2010

5. A new strategy to treat adverse reaction to food
Protein hydrolysates
2001
A new strategy to treat adverse reaction to food
Enzymatic
Hydrolysis
protein
polypeptides

6. Benefits of hydrolysate
Easier to digest
Short half-life in the lumen
Smaller peptides
Molecular weight < 16 Kd
Cave, 2006

7. Protein hydrolysates 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 ?

8. Elisa immunoreactivity of soy protein depending of its source
Background
Elisa immunoreactivity of soy protein depending of its source
LaMartin.com
Adverse reaction to soy in milk replacer
Poor appetite and growth
Malabsorption/maldigestion – Diarrhea
Soy antibodies
Villus atrophy
Lallès, 1995
Soy flour
Soy concentrate
Soy hydrolyzate

9. Protein hydrolysate based milk replacer.
Background
Protein hydrolysate based milk replacer.
Baby allergy to cow’s milk
Vomiting diarrhea
Atopic dermatitis/ Urticaria
Asthma/Rhinitis
Babies at risk for allergy
 Risk of cow milk allergy

10. Molecular weight Dalton Arbitrary unit of atomic mass
Sir John Dalton, Founder of the atomic theory
1 Dalton (d) = Weight of 1/12 nuclide of 12C
gramme
Glycin = 75 d - Tryptophan = 204 d
Casein = d or Kd
Measurement: electrophoresis -chromatography
Human serum albumin = 69 Kd

11. Molecular weight
Mean MW
Mean
Max
Ingredients Vs diets
Cave N, 2006

12. Molecular weight 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
Antigens in selected food
in man
Cave N, 2006
Looringh van Beeck FA, 2009
Sampson HA, 1993

13. Highly reduced allergy
Molecular weight
Molecular
weight
Less common allergy
> 40 kDa
Most common
kDa
Reduced allergy
3 – 10 kDa
Guilford GW ,1996
Serra et al, 2006
Highly reduced allergy
1– 3 kDa
ER: But we are targeting no allergies. Studies of children have established that only amino-acid-based and extensively hydrolysed formulas might be considered truly hypoallergenic (Hill, 2007) and Diets lower than 1kDa will ensure the greatest chance of reducing allergic reaction (Cave 2006)
Olivry T, 2010
Cave N, 2006
No allergy
< 1 kDa

14. Molecular weight Antigenicity Epitope Few Many Close in 3D Hidden
Gastroscopic score diameter
Olson ME et al 2000
Epitope

15. Molecular weight
Antigenicity
AJVR 2006;67:

16. Molecular weight Digestibility Soy source and in vitro N digestibility
Soy source and aa ileal digestibility in calfs
Lallès, 1995
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,

17. Molecular weight Cut-off
« 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

18. In vitro testing - ELISA
Immunogenicity
In vitro testing - ELISA
Hannah, 1997

19. Immunogenicity
Western blot
native
hydrolysed
AJVR 2006; 67:

20. Immunogenicity Skin test 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
Puigdemont et al, 2006
Control showed no wheal on both challenges

21. Immunogenicity Clinical reactions Dogs Native soy protein Hydrolysed
specific IgE
8204
Vomits
Diarrhoea (1)
Pruritus NR
+++
8212
Diarrhoea (2)
8216 ++
Control and 3 other sensitised dogs showed no adverse reactions
Puigdemont et al ,2006

22. Immunogenicity Clinical reactions Jackson et al, 2003 Cutaneous
Score
(Max 35*3*3)
Score max = 315: 3 types de lésions (erythema, excoriation, infection) gradé de 0 à 3.
Pas de réaction avec le corn starch
No corn and starch diet
Corn starch
Corn
Soy
Soy hydrolysate diet
200 mg/kg bw
14 maltese x Beagle dogs with known clinical hypersensitivity to soy and corn
Jackson et al, 2003

23. Immunogenicity Stomach reactivity Gastroscopic score diameter
Olson ME et al 2000

24. Immunogenicity 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.

25. Clinical trials Shown efficacy Managing adverse reaction to food.
Diagnosis of adverse reaction to food.
Inflammatory bowel disease.
Exocrine pancreatic insufficiency.

26. Adverse reaction to food
Clinical reactions
Cutaneous
Clinical
Score
No corn and starch diet
Corn starch
Corn
Soy
Soy hydrolysate diet
200 mg/kg bw
14 maltese x Beagle dogs with known clinical hypersensitivity to soy and corn
3/14 dogs increased scores on hydrolyzed diet.
Jackson et al, 2003

27. Adverse reaction to food
Evaluation on dogs with demonstrated adverse reactions to food
12 dogs ( breeds, sex, food allergy )
Controlled allergy (no clinical signs)
Clinical 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

28. Diagnosis of ARF Challenge Clin. signs Soy hydrolysate diet
2 dermatology specialty pratices
J Fontaine (Brussels, B), M Vroom (Oisterwijk, NL)
Inclusion in the study:
Suspicion of skin hypersensitivity
J Nutr2002;134:2062S-2064S
Challenge
8 Weeks
Clin. signs
Soy hydrolysate diet
Clinical signs
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
VC Biourge, J Fontaine, MW Vroom, 2004

29. 20 16 22 Diagnosis of ARF Atopy Adverse Reaction to Food
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) 20 16 22
Atopy
Adverse Reaction
to Food
+ 2 cases excluded

30. Adverse reaction to food
Diagnosis of ARF
Adverse reaction to food
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
***

31. Diagnosis of ARF Before After Golden Retriever Male, 4.5 yrs, 29.6 kg
Golden retriver, mâle, 29,6 kg :
J 0 :
Prurit chronique intense et généralisé (initialement à l’abdomen), avec une évolution en alopécie, lichénification-hyperpigmentation et corticosensible seulement à l’apparition des symptômes de l’allergie.
J 30 :
Reduction du prurit et des lésions cutanées spécialement au niveau de l’abdomen et des yeux.
J 60 :
Prurit résiduel.
Les lésions cutanées sont acceptables.
Confirmation de l’allergie alimentaire : réapparition des symptômes suite à la réintroduction de l’ancienne alimentation.
Golden Retriever Male, 4.5 yrs, 29.6 kg
Generalized intense prurit with lichenification, hyperpigmentation
Before
After

32. Adverse reaction to food & Atopy
Diagnosis of ARF
Adverse reaction to food & Atopy
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.
***

33. Diagnosis of ARF Atopy 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

34. ARF: response to the hydrolysate
Diagnosis of ARF
ARF: response to the hydrolysate
94.4 % of ARF dogs responded to the soy hydrolysate based diet

35. Diagnosis of ARF 15 20 47 10 11 38 Other study
Loeffler et al., Vet Derm 2006;17:
Chicken hydrolysate diet Vs homade diets
181 dogs
17 dogs excluded
35 allergic skin disease
Poultry hydrolyzate (109)
Homade (72)
Excluded b/c did not receive exclusively the food PH 13 – HD 4, did not eat the food: PH 5 – HD 4, needed glucocorticoïds PH 1 – HD 3, Diarrhea PH 1 – HD 1, unrelated reason = 2 15 20 47 10
No significant difference 11 38
ARF
Atopy
ARF
Atopy
Excluded: 27
Excluded: 13

36. 1 dog showed severe signs when fed hydrolyzed chicken
Veterinary Dermatology,2010,21:
12 dogs selected and divided In 2 groups
During PH1, 25 of 26 dogs showed an increased CADESI
score after chicken meat once daily for 2 weeks. Twelve
subjects, falling into classes 3 and 4, were selected for
PH2 of the study. Prior to starting PH2, one dog devel-
oped unrelated problems and was substituted with a
class 2 dog. Gastrointestinal disturbances were not recor-
ded during PH1 or PH2.
The average CADESI score (LSMEANS) calculated
during the entire study was not influenced by the group.
Dogs displayed the most prominent clinical response
while eating whole chicken, whereas no significant
differences were detected between PH2.1 and PH2.2
(PH1: versus PH2.1: versus PH2.2:2.69,
P < 0.001; Figure 2).
1 dog showed severe signs when fed hydrolyzed chicken

37. Diagnosis of ARF Cats After Before Dr Aranda
Golden retriver, mâle, 29,6 kg :
J 0 :
Prurit chronique intense et généralisé (initialement à l’abdomen), avec une évolution en alopécie, lichénification-hyperpigmentation et corticosensible seulement à l’apparition des symptômes de l’allergie.
J 30 :
Reduction du prurit et des lésions cutanées spécialement au niveau de l’abdomen et des yeux.
J 60 :
Prurit résiduel.
Les lésions cutanées sont acceptables.
Confirmation de l’allergie alimentaire : réapparition des symptômes suite à la réintroduction de l’ancienne alimentation.
After
Before
Dr Aranda

38. ARF: response to the hydrolysate
Diagnosis of ARF
ARF: response to the hydrolysate
87.5 % of ARF cats responded to the soy hydrolysate based diet
MW 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

39. Food responsive chronic diarrhea

40. 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 ( yrs)
Weight 23 ± 12 kg ( 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).
Mandigers et al., 2010
Objectives
To compare the response of dogs with chronic diarrhea on soy hydrolysate Vs intestinal diet.

41. Food responsive chronic diarrhea
Clinical signs after 2 months
Weight gain *
11%
12.5%
No more clinical signs 23/26 dogs
3 last dogs improved but vomiting and diarrhea persisted

42. Food responsive chronic diarrhea
Follow-up after median 6 months, range 3-15 mo.
15/16 test dogs – 6/7 control dogs
67%
13%
87 % of dogs on hydrolyzate diet remained free of clinical signs on follow-up ! The signs were minor in the remaining 2 dogs.

43. 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:
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

44. Exocrine pancreatic Insufficiency
J Nutr2002;134:2166S-2068S
German Shepherds
EPI and skin disease
GI signs controlled within 7 d
Weight gain with 2 months
Skin within 3 months

45. Protein hydrolysates 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.

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