To our knowledge, this is the first combined human milk mass spectrometry and protease analysis using clinically relevant NICU preterm milk samples. While the detection of food peptides in the milk samples is interesting and somewhat expected, the size, breadth, and variety of food and aeroallergens as well as other non-food peptides is fascinating, especially when comparing human milk to cow milk formula. Furthermore, we found differential protease activity between the samples with the highest being in maternal expressed breast milk alone, without fortifier (J9) and the lowest in formula (J11).
The presence of allergen peptides in human milk does not appear to be accidental and may be linked to the development of allergy. In one study, there was an increase in atopy in children who were breastfed by atopic mothers and found to have high HM dust mite (Der p 1) levels; this was not noted in the offspring of mothers without atopic history regardless of Der p 1 level in human milk .18 In food allergy, maternal cow’s milk avoidance was associated with increased cow’s milk allergy in offspring, mediated by a lower cow’s milk specific IgA and possibly the lack of cow’s milk protein exposure.23 In our analysis, bovine peptides were the most numerous of the non-human peptides detected in the human milk only samples (without formula or fortifier) and specific allergenic bovine peptides (β-lactoglobulin, α- and β- caseins, α-lactalbumin) were found in the highest relative quantification in regular fortifier and formula samples and lowest in the human milk samples.
Multiple other common food allergens have been identified in human breastmilk (HBM) studies. Ovalbumin has been detected in HBM in 8.3%-76% of subjects,24,30,31,37 while ovomucoid was identified in 78% of subjects in one study.30 There appeared to be a dose-response phenomenon between maternal egg intake and infant serology, whereby for each additional egg ingested, HBM ovalbumin concentration increased by 25% and infant egg-specific IgG4 increased 22%.37 Egg protein (β-enolase) was found in our analysis although specific ovalbumin and ovomucoid peptides were not identified. Regarding peanut protein, one study of 23 lactating females found that after a 50 g oral peanut load, 48% of female subjects’ HM samples contained peanut. 14Another small study demonstrated peanut allergen (Ara h 6) in HM that was functional and IgE-reactive as evidenced by in vitro assays and the observation that administration to mice lead to partial oral tolerance.38 Peanut protein was not identified in our analysis. With wheat protein, gliadin was detected in 67.5% – 100% breast milk samples in two studies.25,39 Multiple different wheat peptides were identified in our samples, but not gliadin.
In the food diaries associated with the maternal milk samples that were not augmented with fortifier or formula, some foods that were reported as consumed did not show up in the samples. Conversely, in other cases, foods that were not reportedly consumed, did show up in the samples. While recall bias and ensuing inaccuracy may partially account for these discrepancies, there is also the issue of timing of food consumption with respect to appearance in human milk. Moreover, the capability of excreting specific proteins may vary between mothers and further impact the presence of allergenic proteins, which further complicates attempts at correlating dietary ingestion and breast milk peptides.
We know that antigen-presenting cells introduce processed allergens to T-helper lymphocytes and proceed down a TH2 pathway in allergic conditions.40 How the allergen is processed, the role of proteases, and the exact conformation of different allergenic proteins in human milk is not known, although the size of the original protein was better elucidated in our study. We demonstrated that many bovine peptides are found digested (original protein size> 40 amino acids) and free (original protein< 40 amino acids), indicating that there are a variety of different parent proteins. These proteins were mostly found shared between the human milk plus fortifier or formula samples. Conversely, human milk samples without fortifier had relatively few digested bovine peptides, supporting that most cow’s milk-derived peptides originated from smaller proteins. Interestingly, cow’s milk allergy is one of the first to appear in infants’ and the majority of those are sensitized to caseins, which may be able to cross the GI border relatively intact as they coagulate in acidic conditions and may be less susceptible to proteolysis.32,41 A variety of caseins of different sizes were identified in our formula, fortifier and human milk samples, although the allergenicity of these specific caseins are not definitively established in this current analysis. Additionally, the exact origin of these proteins, although presumably diet-derived, is unknown. As opposed to various sizes, the majority of non-bovine peptides in our human milk samples were digested, thereby originating from peptides over 40 amino acids in length.
Assuming that most of these peptides were generated by proteases, we looked at the amino acid sequence in the protein that ended up being the substate for cleavage. Sample J9 (pure maternal expressed breast milk) from the MAP study showed the highest protease activity. Previous proteomic studies have shown differences in the presence of proteases and protease inhibitors in HM between allergenic and non-allergenic mothers.42 There is evidence that an imbalance between protease and protease inhibitors in HBM could allow for easier penetration of allergens.43,44 Specifically, reduced cystatin, a protein inhibitor that has been detected in HM, secreted by epithelial cells has been linked to easier penetration of Der p1 through skin.45 Furthermore, protease inhibitors have been detected in the stool of infants who have received HBM indicating that these protease inhibitors may be active in the gastrointestinal tract.46 It is thought this complex interplay between allergens, proteases, and protease inhibitors is important in the pathogenesis of atopy, and protease inhibitors are being evaluated as a potential therapeutic agent to treat asthma and other atopic conditions.
There are several limitations to this study. We started with a small batch of samples to assess feasibility in this pilot trial. There is inconsistency between dietary documentation between the two sample groups. We will have a more consistent and larger sample size in our future analysis. Other sample-based limitations include the lack of multiple “pure” samples that contain only maternal expressed breastmilk without fortifier or the use of pooled donor human milk. Theoretically, a subtractive analysis could be considered, with inference of protein content of breast milk via exclusion of proteins found in fortifier, however, this is limited due to the overlap of proteins between fortifier and human milk and the variability between the samples, including differences in protein content between the two samples of the same fortifier. Moreover, since a large proportion of preterm infants receive supplementation, donor milk, or formula, our results reflect the real world setting in the NICU. Our subject dietary history did not include the temporal relationship of specific food ingestion and sample collection. Thus, secretion kinetics cannot be concluded, and contamination/inadvertent consumption is an issue with the self-reported dietary histories. Closer analysis of maternal diet and timing of consumption may help to determine the kinetics of human milk peptides and the degree of contamination (dietary or via mass spectrometry) that could account for the detection of proteins that are not found in the diet. Database limitations are also possible. We did not manually blast all proteins against NCBI and Uniprot databases, only those which were positively identified, so it is possible that there were false negatives and proteins were not identified due to inaccurate database sequences
Future directions and Conclusions
We have taken a large step forward in identifying what a preterm infant immune system may encounter in their milk feeds, however, it was beyond the scope of this study to determine the origin of the human milk peptides identified. This is an area we plan to investigate in the future. Peptides may be secreted by lactocytes or enter via the bloodstream. It is also not known where proteolytic cleavage occurs, whether it is locally in the breast or in the GI tract/blood, which could be further investigated by paired blood samples in future studies.
The interaction between allergen, protease, and protease inhibitors also warrants further investigation. Identifying which proteases and protease inhibitors are present in our MAP samples would be of great interest, particularly if their presence or absence augments the development of atopic conditions in infants who have been in the NICU. We do plan to follow subjects out to 5 years and look for the development of allergic outcomes in our MAP cohort. The use of formula, fortifiers, and donor milk are important in optimizing the growth and development of preterm infants. However, their use may have unintended long-term consequences, that need further investigation.
In conclusion, the detection of various allergenic peptides and protease activity in our milk samples raises more questions about how modifying feeds in the NICU may impact the development of atopy in preterm infants. Ultimately, whether human milk can serve to induce allergic sensitization or tolerance in an infant is an area of research that needs much further exploration.