STUDIES
ON
THE
ALLERGENICITY
OF
COW’S
MILK
I. The
Allergenic
Properties
of Aipha-casein
Beta-lactoglobulin
and Alpha-lactalbumin
By Bret Ratner, M.D.,5 Murray Dworetzky, M.D., Satoko Oguri, B.A., and
Lydie Aschheim, MS.
Departments of Pediatrics and Microbiology, New York Medical College and Flower and Fifth Avenue
hospitaLs (BR., SO., L.A.) and the Departments of Medicine and Public Health and Preventive Medicine,
Cornell University Medical College (M.D.)
* Posthumously submitted.
(Accepted April 8, 1958; submitted February 19.) Under grant from Ross Laboratories, Columbus, Ohio.
ADDRESS: (M.D.) 50 East 78th Street, New York 21, New York.
PzmAmIcs, September 1958
449
T
WENTY years ago, studies on theana-phylactogenic properties of milki
were carried out with relatively crude
pro-tein fractions. The results obtained were of
only limited value, since the lactalbumin
fraction contained considerable globulin
and there were both lactalbumin and
lacto-globulin in the casein fraction.
We have now obtained highly purified
protein fractions of milk, as tested by
elec-trophoresis. The object of this study is to
determine the allergenicity and allergenic purity of these proteins in milk.
MATERIALS
The protein fractions of bovine (cow’s) milk
under study are alpha-casein, beta-lactoglobulin
and alpha-lactalbumin, which constitute
ap-proximately 60%, 11% and 2.4%, respectively,
of the total proteins of milk.2
Preparation of Pure Proteins of Milk
The fractions of protein shall be designated
as the McMeekin-Gordon milk proteins.
Unpasteurized, mixed, commercial, skimmed
milk was used for the preparation of the milk proteins.
Casein was prepared by the method
de-scribed by Hipp, Groves and McMeekin.3 This
casein fraction contains alpha-casein, beta-casein and gainma-casein.
o They were prepared by Dr. T. L. McMeekin
and Dr. W. G. Gordon, Eastern Utilization Research
and Development Division, Agricultural Research Service, United States Department of Agriculture, Philadelphia, Pennsylvania.
Alpha-casein was isolated and crystallized b
the method of Hipp, Groves and McMeekin.4
Beta-lactoglobulin was isolated and
crystal-lized by the method of Palmer, modified by
using ammonium sulfate instead of sodium
sulfate for precipitation of the protein. Alpha-albumin was isolated and crystallized by the method of Gordon and Zeigler.6
These protein fractions were all shown to be highly purified by electrophoretic analysis.
The final solutions to be tested were
pre-pared#{176}by suspending the protein fractions as
a 5.0% extract in buffered saline and
adjust-ing to pH 7.8 by the addition of 0.1 normal
solution of sodium hydroxide. After 24 hours,
the resulting clear solutions were passed
through a Seitz filter. The total nitrogen
con-tents of the final solutions were alpha-casein,
6.35 mg/ml; beta-lactoglobulmn, 6.13 mg/mI;
alpha-lactalbumin, 6.4 mg/ml.
METHODS
Guinea pigs weighing 200 to 250 gm were
sensitized and challenged with the purified
pro-tein fractions as tabulated (Table I). The diet
had previously contained no milk proteins.
All sensitizing injections were given intra-peritoneally and/or subcutaneously. All chal-lenging injections into intact animals were
given intravenously into the jugular vein.
Anaphylaxis experiments with isolated
ileum7’8 were carried out by removing the
ileums from sensitized guinea pigs and
sus-pending them in a bath containing 5.0 ml of freshly made, oxygenated Tyrode solution
* Center Laboratories, Inc., Port Washington,
450 ALLERGENICITY OF COW’S MILK
TABLE 1*
A. ALLERGENIC SUPERIORITY OF BETA-LACTOGLOBULIN AS COMPARED WITh ALPHA-LACTALBUMIN
AND ALPHA-CASEIN Number of Animals Single Sensitiz-ing Injection,
1.0 mg Sc
Challenge S
Weeks Later,
0.1 ml PSM, Iv
-_______
Result
10 Alpha-lactalbumin All negative
10 Alpha-casein All negative
10 Beta-lactoglobulin 7 + + + +
1 ++
1 +
1 0
B. ENHANCEMENT OF ALLERGENICITY OF ALPHA-LAC’r-ALBUMIN AND ALPHA-CASEIN BY
SENSITIZA-TION WITh MULTIPLE DOSES
Number
of
Animals
Multiple Sensitizing
in-jections; 1 mg Sc+1
2 Days Later 1 mg Sc
Challenge S
Weeks Later,
0.1 ml PSM, lv
Result
10 Alpha-lactalbumin 4 + + + + 1 +++
++
1 +
0
10 Alpha-casein 8 + + + +
++
4 +
1 0
10 Beta-lactoglobulin 8 + + + +
1 +
1 0
* Abbreviations in Table:
Sc= subcutaneously; Iv = intravenously; Ip
= intraperitoneally.
PSM=pasteurized skimmed milk.
+ + ++ =Anaphylactic death-dyspnea, convul-sions, collapse, apnea and death. + + + = Severe anaphylaxis-dyspnea,
convul-sions, collapse and recovery.
+ + = Moderate anaphylaxis-dyspnea, con-vulsive movements, moderate collapse, recovery.
+ =Mild anaphylaxis-dyspnea and scratching.
0-No reaction.
maintained at 37.5#{176}C. All contractile reactions
were registered on a smoked drum, rotating at a speed of 2.5 cm/sec. Histamine was used to
test the viability and contractility of each
seg-ment of ileum at the beginning and at the end
of each experiment.
After an anaphylactic contraction was
ob-tamed with a specific antigen, the segment of ileum was allowed to relax completely and then the bath was washed out three times with
fresh Tyrode soluton. The segment was again
tested with the same antigen to demonstrate
specific desensitization, and the bath again
rinsed three times with Tyrode solution. This
procedure was then carried out with each
chal-lenge with subsequent heterologous antigenic
contacts.
RESULTS
Allergenic Specificity of Bovine Crystalline Beta-lactoglobulin, Alpha-lactalbumin and Alpha-casein
Ten guinea pigs were sensitized with two
injections of 1.0 mg each of
beta-lacto-globulin given subcutaneously 2 days apart.
Three weeks later they were each
chal-lenged in turn with intravenous injections
of 0.1 mg first of alpha-lactalbumin, then of
alpha-casein and, finally, of the homologous
beta-lactoglobulin. None reacted to the two
heterologous protein fractions, while all
died in anaphylaxis after challenge with the
homologous beta-lactoglobulin. These
re-suits demonstrate a high degree of
sensitiza-tion to the beta-lactoglobulin with no
evi-dence of cross reaction with
alpha-lactal-bumin or alpha-casein fractions, thus
con-firming the purity of the beta-lactoglobulin.
In order to rule out the possibility of
pri-mary toxicity of beta-lactoglobulin, five
normal animals were given a single
intra-venous injection of 0.1 mg of this fraction.
None showed any reaction.
Sixteen animals were sensitized with two
injections of 1.0 mg each of
alpha-lactal-bumin, given subcutaneously 2 days apart.
Three weeks later they were challenged in
turn with intravenous injections of 0.1 mg
of each of the two heterologous fractions,
first beta-lactoglobulin and then
1#{188}
1#{188}t f
.0f
f
0.25 0.05,3
a
HIST LACT HIST LACT GAS
GLOB.
0 MIN.
1#{188}
1#{188}
ft
W HIST
t
t t t
0.5UICT
GAS.
FIG. 1. Evidence of antigenic purity of alpha-casein fraction. Tracing of contractions of seg-ment of guinea pig ileum sensitized to alpha-casein. 6.0 ml bath. HIST. = histamine followed
by 3 washes. Three washes before each new addition of material. W = 3 washes. From left to
right: two normal histamine contractions; no reaction to alpha-lactalbumin; three histamine contractions; no reaction to beta-lactoglobulin; specific anaphylactic contraction to alpha-casein; four histamine contractions; no contraction with alpha-casein (desensitization); two
normal histamine contractions.
Finally they were challenged with the
homologous alpha-lactalbumin, after which
eight animals had anaphylactic reactions:
three fatal and five mild to moderate in
de-gree; eight had no reactions. These results
demonstrated a moderate degree of
aller-genicity of alpha-lactalbumin, of a lower
order than that of beta-lactogiobulin, and
no evidence of cross reactions with the
het-erologous fractions.
Experiments with the isolated ileum of
the guinea pig fortified the evidence derived
from anaphylaxis in intact animals, and
showed that alpha-casein is also allergenic-ally pure. It was demonstrated that the
iso-lated segment of ileum from an animal
sen-sitized to alpha-casein reacts to neither of
the heteroiogous fractions (Fig. 1).
Relative Allergenicity of Beta-lactoglobu-un, Aipha-lactalbumin and Aipha-casein
In preliminary experiments it was found
that, while a single subcutaneous injection
of 1.0 mg of alpha-lactalbumin or
alpha-casein was an inadequate sensitizing dose,
two or three such injections, given several
days apart, gave a high degree of
sensitiza-tion. In contrast, a single injection of 1.0 mg
of beta-lactogiobulin resulted in a high
de-gree of sensitization, for 7 of 10 animals died of anaphylactic shock after challenge with pasteurized skimmed milk (Table I, A).
When animals were sensitized with
mul-tiple injections, a definite sensitivity was
established to the aipha-lactalbumin and
aipha-casein (Table I, B), while multiple
sensitizations with beta-lactoglobulin did
not increase the high degree of sensitivity
established after a single injection of that
substance.
From these data it is seen that of the pro-tein fractions of milk studied, beta-lacto-globulin is the most allergenic, while
alpha-lactalbumin and alpha-casein have
appre-ciably lower allergenicity. However, with
multiple sensitizing injections, the latter two
fractions are capable of sensitizing animals.
SUMMARY
It seems apparent from the present study
that each of the McMeekin-Gordon protein
fractions of milk is immunologically
homo-geneous.
Of the three fractions, beta-lactoglobulin is the most allergenic, while alpha-lactal-bumin and alpha-casein are of a much lower
order of allergenicity.
injec-452 ALLERGENICITY OF COW’S MILK
tion is artificial and may not thus apply
strictly to natural sensitization to milk
through ingestion, the data indicate that
lactoglobulin is the dominant allergen of the three studied.
Heretofore, lactalbumin has been
consid-ered the major protein in the production of
milk allergy.9 The present studies furnish
evidence against the lactalbumin fraction
as the potent allergen in clinical practice
and evidence in favor of the lactoglobulin
fraction as the allergen responsible for the
majority of cases of milk allergy.
REFERENCES
1. Ratner, B., and Gruehl, H. L. :
Anaphylacto-genie properties of milk. Am.
J.
Dis.Child., 49:287, 1935.
2. McMeekin, T. L. : Milk proteins, in The
Proteins, Vol. II, Neurath, H., and Bailey,
K., editors. New York, Acad. Press, 1954,
Part A, p. 393.
3. Hipp, N.
J.,
Groves, M. L., and McMeekin,T. L. : Separation of gamma-casein.
J.
Am. Chem. Soc., 72:4928, 1950.4. Hipp, N.
J.,
Groves, M. L., and MeMeekin,T. L.: Separation of alpha-, beta- and
gumma-casein.
J.
Dairy Sc., 35:272, 1952.5. Palmer, A. H.: Preparation of a crystalline
globulin from the lactalbumin fraction of
cow’s milk.
J.
Biol. Chem., 104:359,1934.
6. Gordon, W. G., and Zeigler,
J.:
Alpha-lactalbumin. Biochem. Preparations, 4:
16, 1955.
7. Schultz, W. H.: Physiological studies in
anaphylaxis. I. Reaction of smooth muscle
of the guinea pig sensitized with horse
serum.
J.
Pharmacol. & Exper. Therap.,1:549, 1910.
8. Dale, H. H.: The anaphylactic reaction of
plain muscle in the guinea pig.
J.
Phar-macol. & Exper. Therap., 4:167, 1913.
9. Fries,
J.
H.: Milk allergy; diagnostic aspectsand the role of milk substitutes. J.A.M.A.,
165:1542, 1957.
SUMMARIO IN INTERLINGUA
Studios Relative
Al
Allergenicitate
De
Lacte
Bovin
I.
Tres fractiones proteinic de lacte
bovin-alpha-caseina, beta-lactoglobulina, e
alpha-lactalbumino (le quales esseva obtenite in forma
crystallin e, secundo br analyse electrophoretic, altemente purificate)-esseva investigate con respecto a br puritate allergenic, i.e. br
aller-genicitate relative.
Porcos de India recipeva injectiones del
mentionate fractiones proteinic, sequite per
stimulation con fractiones heterologe e
homo-loge. In le majoritate del casos, le stimulation
esseva effectuate per injectiones intravenose in
animales intacte, sed un certe numero de
ex-perimentos esseva executate con isolate
seg-mentos de ileum prendite ab porcos de India
sensibilisate.
Sensibilisation con le un o Ic altere del tres
fractiones proteinic non resultava in ulle caso
in un reaction anaphylactic sub le effecto de
un heterologe fraction de proteina. Isto provava
que le tres fractiones esseva
immunologica-mente pur.
Inter le tres fractiones studiate,
beta-lacto-globulina se monstrava le plus allergenic,
durante que alpha-lactalbumina e alpha-caseina
exhibiva un ordine multo inferior de
allergeni-citate.
Usque nunc, lactalbumina esseva considerate
como le factor le plus importante in le
dis-veloppamento de allergia a lacte. Le presente
studios ha producite provas contra le
incrimina-tion de lactalbumina como potente allergeno
in le practica clinic e pro le conception que
lactoglobulina es plus probabilemente
respon-sabile pro le majoritate del casos de allergia a
lacte. Tamen, viste que sensibilisation per in-jection parenteral es artificial e possibilemente
non simula strictemente le sensibilisation
natu-ral a lacte per ingestion, un conclusion definite
