Vadas P, Wai Y, Burks W, Perelman B. Detection of Peanut Allergens in Breast Milk of Lactating Women. JAMA. 2001;285(13):1746-1748. doi:10.1001/jama.285.13.1746
Author Affiliations: Division of Allergy and Clinical Immunology, St Michael's Hospital, University of Toronto, Toronto, Ontario (Drs Vadas, Wai, and Perelman); and Arkansas Children's Hospital, University of Arkansas for Medical Sciences, Little Rock (Dr Burks).
Context Most individuals who react to peanuts do so on their first known exposure.
A potential but unproven route of occult exposure resulting in sensitization
to peanut is via breast milk during lactation.
Objective To investigate the ability of maternal dietary peanut protein to pass
into breast milk during lactation.
Design and Setting Clinical investigation conducted at 2 North American hospitals from
March 1999 to October 2000.
Patients Twenty-three healthy, lactating women aged 21 to 35 years.
Intervention Each woman consumed 50 g of dry roasted peanuts, after which breast
milk samples were collected at hourly intervals.
Main Outcome Measures Presence in breast milk of total peanut protein, analyzed by a sandwich
enzyme-linked immunosorbent assay, and 2 major peanut allergens, Ara h 1 and Ara h 2, detected by immunoblot
Results Peanut protein was detected in 11 of 23 subjects. It was detected in
10 subjects within 2 hours of ingestion and in 1 subject within 6 hours. The
median peak peanut protein concentration in breast milk was 200 ng/mL (mean,
222 ng/mL; range, 120-430 ng/mL). Both major peanut allergens Ara h 1 and Ara h 2 were detected.
Conclusions Peanut protein is secreted into breast milk of lactating women following
maternal dietary ingestion. Exposure to peanut protein during breastfeeding
is a route of occult exposure that may result in sensitization of at-risk
The prevalence of peanut allergy has increased markedly in recent years.1 Approximately 1% of British and US preschool children
are sensitized to peanuts.2,3
Peanut allergy is a significant public health problem as it starts early in
life, is often associated with severe or life-threatening reactions, and rarely
resolves.4,5 Peanuts account for
the majority of food-induced anaphylactic fatalities.5- 8
Between 72% and 81% of individuals who have reactions to peanuts do
so on their first known exposure.6,9
Since IgE-mediated allergic reactions require prior exposure resulting in
sensitization, earlier peanut exposure must have been occult in most cases.
Potential but unproven routes of occult exposure include exposure in utero
and exposure to peanuts transferred from the maternal diet via breast milk
Delineation of routes of occult exposure leading to sensitization would
be helpful in guiding the formulation of strategies to prevent sensitization
of children at risk. In this study, we investigated the ability of maternal
dietary peanut protein to pass into breast milk during lactation.
This study was approved by the Research Ethics Committee of St Michael's
Hospital, University of Toronto. Healthy, lactating women, ranging in age
from 21 to 35 years (median, 31.1 years), volunteered for the study in response
to posted notices at breastfeeding centers. Inclusion criteria included informed
consent and ability to express breast milk at timed intervals on the day of
protocol. Exclusion criteria were known peanut or tree nut allergy.
Subjects were instructed to avoid ingestion of legumes for 24 hours
prior to breast milk collection, and fasted overnight. Participants collected
breast milk at time 0 on the morning of collection. After the initial breast
milk collection, they consumed 50 g (approximately ½ cup) of dry roasted
peanuts. Strict precautions were taken to avoid any cross-contamination. Samples
of breast milk were collected at 1, 2, 3, 4, 6, 8, and 12 hours after peanut
ingestion. Aliquots of 5 mL of breast milk were collected, centrifuged to
remove cellular elements, and stored at −20°C until assay. Timing
of subsequent meals and infant nursing were standardized to minimize confounding.
Samples were thawed and centrifuged at 8000g,
15 minutes at 4°C to remove fat and cellular debris. Peanut protein was
analyzed quantitatively by sandwich enzyme-linked immunosorbent assay with
the Veratox peanut allergen test kit (Neogen Corp, Lansing, Mich). Breast
milk was added to polyclonal rabbit anti–peanut IgG-coated wells and
incubated for 10 minutes at 22°C. Unbound protein was removed and horseradish
peroxidase-conjugated detection antibody was added. Substrate was added and
the reaction was allowed to develop for 10 minutes at 22°C. Red stop reagent
was added and the color of the resulting solution was measured at 620 nm.
Breast milk samples were mixed with sodium dodecyl sulfate (SDS) buffer
containing 2-mercaptoethanol, treated at 100°C for 2 minutes. Total protein
(20 µg) was applied to each lane of a vertical slab SDS polyacrylamide
gel. Resolving gels were 12% wt/vol polyacrylamide, with 4% wt/vol stacking
gels. Gels were run at 170 V for 55 minutes in Tris-glycine buffer.
Proteins were electrophoretically transferred to Hybond ECL nitrocellulose
membrane (Amersham Pharmacia Biotech, Piscataway, NJ) in a semidry Transblot
apparatus (Bio-Rad Laboratories, Hemel Hempstead, England) for 40 minutes
at 18 V in Tris-glycine buffer containing 20% methanol. Protein transfer was
monitored by transfer of prestained marker proteins and by staining the remaining
gel with coomasie brilliant blue and the membrane with ponceau solution.
Blots were probed with rabbit anti–peanut antibody. After blocking
with a solution of 3% bovine serum albumin in Tris-buffered saline the immunoblots
were incubated with a 1:1000 dilution of rabbit polyclonal anti–peanut
antibody for 2 minutes at 22°C. After washing with phosphate-buffered
saline with Tween, protein G was added and the blots were rotated for 2 hours
at room temperature. After washing, the blots were then exposed to Bio Max
film (Eastman Kodak Co, Rochester, NY) and developed.
Authentic peanut protein was added to breast milk and assayed as described
above. The assay was linear to a peanut protein concentration of 1000 ng/mL.
Addition of peanut standard to breast milk yielded an absorbance curve identical
to that for peanut protein in buffer.
The results of analyses of breast milk samples from 23 subjects are
shown in Figure 1. Peanut protein
was detected in 11 of 23 subjects. Peanut protein appeared within 1 hour of
ingestion in 8 of 11 subjects, within 2 hours in 2 of 11 subjects, and was
delayed for 6 hours in 1 subject. Median peak peanut protein concentration
in breast milk was 200 ng/mL (mean, 222.3 ng/mL; range, 120-430 ng/mL). Peanut
protein was cleared rapidly from breast milk except in 1 subject, who showed
protracted presence of peanut protein.
A representative rabbit polyclonal anti–peanut immunoblot is shown
in Figure 2. As can be seen in this
example, there were recognizable amounts of peanut protein that coincided
with both allergens Ara h 1 and Ara h 2 in the breast milk samples. Although the number of subjects
was small, there were no differences between the 11 secretors and 12 nonsecretors
in age (median, 31.1 and 31.0 years, respectively), time postpartum (median,
17 and 21 weeks, respectively), or history of atopy (3 vs 2 subjects).
Many studies have documented subjects' reactions to foods on the first
A study of 8 exclusively breastfed infants described multisystem allergic
reactions to milk, eggs, or peanuts occurring on the first known exposure.11 More recent studies have shown that such reactions
are increasingly commonplace.6 In 2 studies9,12 of peanut and tree nut allergy, reactions
on first known exposure occurred in 72% to 81% of all cases. Since sensitization
requires prior exposure to generate allergen-specific IgE, the sensitizing
exposure must be occult in many cases.
Exposure to food allergens in breast milk originating from the maternal
diet is thought to be responsible for occult sensitization.6,10,13- 15
Some food allergens have been detected in breast milk of lactating women,
including the major cow's milk allergen, β-lactoglobulin.16- 19
The time from ingestion to peak concentration of β-lactoglobulin in breast
milk was 1 to 6 hours. Two major egg allergens, ovalbumin and ovomucoid, are
readily transferred to breast milk,16,20,21
with peak concentrations attained 2 to 6 hours after ingestion. Similarly,
gliadin from dietary wheat is detectable within 2 to 4 hours of ingestion.22,23 Concentrations of bovine β-lactoglobulin
(5-800 ng/mL), egg ovalbumin (200 pg/mL-6 ng/mL) and ovomucoid (0-2.88 ng/mL),
and wheat gliadin (5-95 ng/mL) are dependent on the amount ingested, and peak
concentrations in breast milk are generally in the nanogram per milliliter
Peanut proteins have long been suspected to be secreted but have never
been identified in breast milk. We now provide definitive evidence for secretion
of peanut protein into breast milk of lactating women. Peanut protein concentrations
found in breast milk ranged from 120 to 430 ng/mL, comparable with the levels
of β-lactoglobulin, ovalbumin, ovomucoid, and gliadin detected in breast
The time courses of appearance of proteins from peanuts, eggs, milk,
and wheat are similar. Peanut proteins appeared within 1 to 3 hours following
oral ingestion and were quickly cleared from breast milk. Both low- and high-molecular-weight
proteins with mobilities corresponding to Ara h 1
and Ara h 2 were secreted intact into breast milk
with no evidence for degradation. Ovalbumin, β-lactoglobulin, and gliadin
also appear in undegraded form in breast milk.16,21,23
Only a portion of lactating women (48%) secreted peanut protein in their
milk. Similarly, only 53% to 63% of women secreted β-lactoglobulin and
59% to 74% of women secreted ovalbumin into breast milk after ingestion of
cow's milk and eggs, respectively.16,17,20,21
Atopic status and other demographic characteristics of lactating women have
not accounted for the variable secretion of food proteins into breast milk
in previous studies, nor in this study, although the number of women investigated
Several studies have documented an epidemiologic relationship between
increased consumption of peanut by pregnant and breastfeeding mothers and
the likelihood of allergic sensitization of their children.1,9,10
These studies, in conjunction with our data, suggest that transfer of maternal
dietary peanut protein to breast milk may predispose at-risk children to occult