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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