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

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
Dermatologic: pruritus, alopecia,otitis, ..
Gastrointestinal: vomiting, abd. discomfort
Respiratory: asthma, rhinitis, ...
General: headache, arthritis, ...
Food Allergy
Immune mediated
(IgE, cell mediated)
Food intolerance
Non immune mediated
(Lactose, chocolate, bioamines,
Probably the main cause (Olivry T, J Vet derm 2010)
additives, …)
Dr Thierry Ximenes (France)
Adverse Reactions to Food
Clin. signs
Diagnosis
Challenge
Double blinded
Elimination diet
4 to 12 Weeks
Novel ingredients
+ Serum IgE
+ Skin test
Improvement of
the clinical signs
Clinical signs
Elimination diets
•
•
Definition
Diets composed of ingredients to which the
dog has not been exposed.

•
•
Dietary history
Home made

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•
Gold standard
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Starch sources: Rice, Potato, Tapioca
Protein sources: Lamb, Horse, Fish, Turkey, Venison,
..
Essential fatty acids, Minerals and Vitamins
Commercial
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Lamb & Rice, Fish & Potatoes, Venison & Potatoes
Over the counter
Raditic et al, JAPAN 2010
Not hypoallergenic !
 Intact protein sources !
Sustainability
 Fish, Rabbit, Duck, Venison, …
Protein hydrolysates
2001
A new strategy to treat adverse reaction to food
protein
Enzymatic
Hydrolysis
polypeptides
Benefits of hydrolysate
•
Easier to digest
 Short half-life in the lumen
•
Smaller peptides
 Molecular weight < 16 Kd
Cave, 2006
Protein hydrolysates
•
•
•
•
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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 ?
Background
LaMartin.com
Adverse reaction to soy in milk replacer
Poor appetite and growth
Malabsorption/maldigestion – Diarrhea
Soy antibodies
Villus atrophy
Immunoreactivity (mg/g prot)
Elisa immunoreactivity of soy
protein depending of its source
40
Glycinine
B-conglycinine
35
30
25
20
15
10
5
0
1
1.
2.
3.
2
3
Lallès, 1995
Soy flour
Soy concentrate
Soy hydrolyzate
Background
Protein hydrolysate based milk
replacer.
http://www.eecs.harvard.edu/~eli/pictures
Baby allergy to cow’s milk
Vomiting diarrhea
Atopic dermatitis/ Urticaria
Asthma/Rhinitis
/
Babies at risk for allergy
 Risk of cow milk allergy
Molecular weight
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•
•
•
•
•
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
Human serum albumin = 69 Kd
Molecular weight
Mean MW
Mean
Max
Ingredients Vs diets
Cave N, 2006
Molecular weight
•
•
•
•
Most common food allergens
Glycoproteins
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Protein with glucide moiete
? Lipids (Bacterial glycolipids)
? Carbohydrates
Water - soluble
Heat and acid resistants
Molecular weight 10 to 40 Kd
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Small enough to pass the intestinal wall
Large enough to induce immune reaction
Cave N, 2006
Looringh van Beeck FA, 2009
Antigens in selected food
in man
Protein fraction
MW (Kd)
Caseins
19-20
Ovalbumin
45
Arachis hypogea I
63
Arachis hypogea II
17
Soybean Trypsin inhibitor
20
Soybean B-conglycinine
53
Soybean glycinine
309-363
Fish allergen M
12
Shrimp antigen I
42
Schrimp antigen II
38
Wheat
8-67
Sampson HA, 1993
Molecular weight
Molecular
weight
> 40 kDa
Less common allergy
10- 40 kDa
Most common
3 – 10 kDa
1– 3 kDa
Reduced allergy
Guilford GW ,1996
Serra et al, 2006
Highly reduced allergy
Olivry T, 2010
Cave N, 2006
< 1 kDa
No allergy
Molecular weight
Antigenicity
Hydrolyzed liver
Few
Chicken liver
Sensitized
Hydrolyzed soy
Control
Many
Soy
50 % Hydrolyzed casein
Close in 3D
20 % Hydrolyzed casein
Casein
Hidden
0
1
2
3
Score value
Epitope
Gastroscopic score diameter
Olson ME et al 2000
4
Molecular weight
Antigenicity
AJVR 2006;67:1895-1900
Molecular weight
Digestibility
Soy source and in vitro N
digestibility
100
98
96
94
92
90
88
86
84
82
80
Flour
Concentrate
Hydrolysate
amino acids
N in vitro digestibility (%)
AA ileal digestibility (%)
Soy source and aa ileal
digestibility in calfs
Lallès, 1995
100
98
96
94
92
90
88
86
84
82
80
Soybean
Hydrolysate
N
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
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
Immunogenicity
Absorbance at 405 nm
In vitro testing - ELISA
0,7
0,6
Native soy protein
0,5
Soy hydrolysate
0,4
0,3
0,2
0,1
0
0,01
0,1
1
10
100
1000
10 000 100 000
Protein concentration (ug/mL)
Hannah, 1997
Immunogenicity
Western blot
native
hydrolysed
75
50
50
37
31
30
25
20
8204 8212 8216 8208
Sensitised dogs
8202 8210
8204 8216 8212
8208 8202 8210
Sensitised dogs
AJVR 2006; 67:484-488
Immunogenicity
Skin test
% 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
Puigdemont et al, 2006
Immunogenicity
Clinical reactions
Dogs
Native
soy protein
Hydroly
sed
soy
protein
Soy
specific
IgE
8204
Vomits
Diarrhoea (1)
Pruritus
NR
+++
8212
Diarrhoea (2)
NR
+++
8216
Diarrhoea (2)
NR
++
Control and 3 other sensitised dogs showed no adverse reactions
Puigdemont et al ,2006
Immunogenicity
Cutaneous
Clinical
Score
Clinical reactions
(Max 35*3*3)
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
Immunogenicity
Stomach reactivity
Hydrolyzed liver
Chicken liver
Sensitized
Hydrolyzed soy
Control
Soy
50 % Hydrolyzed casein
20 % Hydrolyzed casein
Casein
0
1
2
3
4
Score value
Gastroscopic score diameter
Olson ME et al 2000
Immunogenicity
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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.
Clinical trials
Shown efficacy
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Managing adverse
reaction to food.
Diagnosis of adverse
reaction to food.
Inflammatory bowel
disease.
Exocrine pancreatic
insufficiency.
Adverse reaction to food
Cutaneous
Clinical
Score
No corn and
starch diet
Clinical reactions
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
Adverse reaction to food
• Evaluation on dogs with demonstrated adverse
reactions to food
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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
Diagnosis of ARF
•2 dermatology specialty pratices
J Nutr2002;134:2062S-2064S
J Fontaine (Brussels, B), M Vroom (Oisterwijk, NL)
•Inclusion in the study:
Suspicion of skin hypersensitivity
Clin. signs
Challenge
8 Weeks
Soy hydrolysate diet
Clinical 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
VC Biourge, J Fontaine, MW Vroom, 2004
 No corticotherapy
Diagnosis of ARF
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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), …
•
20 16 22
Duration of the clinical signs
•2.6±0.4 yrs (3 weeks – 10 yrs)
Adverse Reaction Atopy
to Food
+ 2 cases excluded
Diagnosis of ARF
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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
Prurit score
Adverse reaction to food
5
4,5
4
3,5
3
2,5
2
1,5
1
0,5
0
***
Before
After
Diagnosis of ARF
•
Golden Retriever Male, 4.5 yrs,
29.6 kg
•
Generalized intense prurit with
lichenification, hyperpigmentation
Before
After
Diagnosis of ARF
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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.
Prurit score
Adverse reaction to food & Atopy
5
4,5
4
3,5
3
2,5
2
1,5
1
0,5
0
***
Before
After
Diagnosis of ARF
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•
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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
Prurit score
Atopy
5
4,5
4
3,5
3
2,5
2
1,5
1
0,5
0
Before
After
Diagnosis of ARF
ARF: response to the hydrolysate
40
35
30
25
20
15
10
5
0
Yes
No
94.4 % of ARF dogs responded to the soy hydrolysate based diet
Diagnosis of ARF
Other study
Loeffler et al., Vet Derm 2006;17:273-279
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•
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Chicken hydrolysate diet Vs homade diets
181 dogs
17 dogs excluded
35 allergic skin disease
Poultry hydrolyzate (109)
15
Homade (72)
No significant
10 11
20
47 difference
ARF Excluded: 27
Atopy
ARF Excluded: 13
38
Atopy
Veterinary Dermatology,2010,21:358-366
12 dogs selected and divided In 2 groups
1 dog showed severe signs when fed
hydrolyzed chicken
Diagnosis of ARF
Cats
Before
Dr Aranda
After
Diagnosis of ARF
ARF: response to the hydrolysate
8
7
6
5
4
3
2
1
0
Yes
No
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 2002. 252
Food responsive chronic diarrhea
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).
Mandigers et al., 2010
Objectives
To compare the response of
dogs with chronic diarrhea on
soy hydrolysate Vs intestinal
diet.
Food responsive chronic diarrhea
18
16
14
12
10
8
6
4
2
0
Weight gain
1,4
Hydrolysate
Low residue
1,2
Weight gain (kg)
Number of dogs
Clinical signs after 2 months
1
0,8
0,6
0,4
11% 12.5%
0,2
0
No signs
Hydrolysate
Low residue
*
Signs
No more clinical signs 23/26 dogs
3 last dogs improved but vomiting and diarrhea persisted
Food responsive chronic diarrhea
Follow-up after median 6 months, range 3-15 mo.
15/16 test dogs – 6/7 control dogs
14
Number of dogs
12
Hydrolysate
Low residue
10
8
6
4
2
67%
13%
0
No signs
Signs
87 % of dogs on hydrolyzate diet remained
free of clinical signs on follow-up ! The signs
were minor in the remaining 2 dogs.
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
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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
Exocrine pancreatic Insufficiency
J Nutr2002;134:2166S-2068S
German Shepherds
EPI and skin disease
Case Age
yrs
1 5,0
2 7,0
3 9,0
TLI Body weight (kg)
ug/L Before
After
2,5
33,7
38,0
0,88 32,0
43,0
ND
40,0
44,0
GI signs controlled within 7 d
Weight gain with 2 months
Skin within 3 months
Protein hydrolysates
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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.
If you want to know more …
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