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Background: Although corn is often cited as an allergenic food, very few studies have been devoted to the identification of corn allergens and corn allergy has been rarely confirmed by double-blind, placebo-controlled food challenge (DBPCFC). Recently, Pastorello et al. (1) identified some salt-soluble IgE-binding proteins of corn flour as potential allergens. One of these, corresponding to corn Lipid Transfer Protein (LTP), appeared to be the major one. The aim of this study was to verify the clinical significance of the skin prick test (SPT) and CAP-FEIA CAP-System IgE fluozoenzyme immunosorbent assay (Pharmacia Diagnostic, Uppsala, Sweden) positivities to corn and to identify the presence of IgE-binding proteins in the corn flour salt-insoluble protein fractions (comprising up to 96% of the total protein) using sera of patients with DBPCFC-documented food allergy to corn. In addition the effect of cooking and proteolytic digestion on the corn allergens was investigated.
Methods: Sixteen subjects with SPT and CAP-FEIA positivities to corn flour were examined. Only six of them complained of suffering from urticaria and/or other symptoms after ingestion of corn-based foods. The patients were food challenged with cooked corn flour (polenta). IgE-binding proteins were detected by immunoblotting. The digestibility of the IgE-binding proteins was examined during a pepsin attack followed by a pancreatin digestion performed on a cooked corn flour sample.
Results: Oral challenge was positive only for six patients with symptoms after ingestion of corn. A 50 kDa protein, belonging to the corn Reduced Soluble Protein (RSP) fraction was recognized by the serum IgE of all the DBPCFC-positive subjects and resulted to be resistant to both heating and peptic/pancreatic digestion. SPT with the purified RSP fraction gave positive results for all of the DBPCFC-positive patients examined.
Conclusions: SPT and CAP-FEIA positivities to corn flour had no clinical significance for most of the patients and food allergy to corn has to be proved by DBPCFC. A salt-unextractable protein of 50 kDa, belonging to the RSP fraction, represents a potential allergen in food hypersensitivity to corn because of its stability to cooking and digestion.
Corn (Zea mays L.) is the basic ingredient of frequently eaten foods like porridge, polenta, cornflakes, popcorn, taco chips, canned de-embryonated kernels, and so on. Moreover, corn is used in the formulation of various food products such as bread, cakes and beer. As a flour, corn is also inhaled by people handling it while making cakes (bakers, pastry cooks, housewives) or making feed (farmers).
Normal corn flour contains about 7–13% protein that can be fractionated into several solubility classes. The salt-extractable fraction (albumins and globulins), averaging less than 4% of the total endosperm protein (2), mainly comprises proteins with metabolic functions. In contrast, extraction with aqueous alcohol results in the solubilization of the prolamin fraction that contains the storage proteins of the corn seed. These latter, constituting about 60–70% of the corn endosperm proteins (2, 3), are called zeins and comprise various polypeptides differing in molecular weight and isoelectric points (4), and are classifiable into four major groups (α, β, γ, and δ zeins) on the basis of their molecular characteristics (5). Some proteins extracted with reducing alcoholic solutions, however, are also soluble in water in reducing conditions and for this reason are called Reduced Soluble Proteins (RSP), making questionable their identification as true zeins (3, 6). Finally, a heterogeneous alcohol-insoluble protein fraction exists in the corn flour called glutelin fraction (3). This fraction can be extracted only with strong denaturing solvents (4).
Some recent papers have reported cases of allergic respiratory reactions (7) to inhaled corn dust in exposed workers and corn components binding to IgE have been identified (8, 9). Heat shock protein-related epitopes have been reported to be common allergic determinants for corn antigens in a population of pig farm workers exposed to complex bioaereosols (10).
It is very surprising that, although corn is often cited as an allergenic food and RAST positivities to this cereal have been reported, cases of allergic reactions after ingestion of corn have been rarely published and very few studies have been devoted to the identification of corn allergens. On the basis of a retrospective study on patients with histories of food allergy Moneret-Vautrin et al. (11) concluded that food allergy to corn is rare. A case of food-dependent exercise induced anaphylaxis to corn (12) and a few cases of corn allergy in children have been reported (13), although the allergens involved have not been investigated. Lehrer et al. (14) found significant levels of IgE to corn water-soluble and insoluble proteins in the sera of ‘corn-reactive individuals’, but the clinical manifestation of such corn-reactivity was not reported. Recently, Pastorello et al. (1) have identified some salt-soluble IgE-binding components of corn flour: a 9 kDa protein, corresponding to the corn Lipid Transfer Protein (LTP) and a 16 kDa protein, corresponding to a trypsin inhibitor, were bound by the IgE of 86% and 36% of the examined patients, respectively.
In this paper we have examined a population of subjects with SPT and CAP-FEIA positivities to corn flour and we have proved that only a minor fraction of these individuals had a true food allergy to corn. Moreover the potential allergens have been identified by immunoblotting, and the effect of both cooking and proteolytic digestion on them has been studied.
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- Material and methods
DBPCFC tests with cooked corn flour confirmed that the symptoms showed by six out of the 16 patients examined in this study were clinical manifestations of an IgE-mediated hypersensitivity reaction to ingested corn. Although also the other 10 patients considered were positive to both SPT and CAP-FEIA to corn, they were not suffering from food allergy to this cereal as revealed both by their clinical history and the negative challenge results. Our findings confirm the observations of Jones et al. (20), who found that only five of 17 subjects with positive SPT to corn reacted to an oral challenge with this cereal. These authors, because of the high incidence in the examined population of allergy to pollen of grasses (which are taxonomically related to corn), concluded that clinically insignificant cross-reactivity exists among cereal grains and grasses. Our results confirm this conclusion, since most of our patients were suffering from respiratory allergy to grass pollens (as well as to corn pollen) without any relationships with the results of the food challenge.
Pastorello et al. (1), examining the soluble protein fraction of the corn flour, identified some IgE-binding components by immunoblotting with sera of patients who reported adverse reactions after eating corn. A 9 kDa protein, identified as the corn LTP, and a 16 kDa protein corresponding to a trypsin inhibitor were bound by the IgE of 86% and 36% of the patients, respectively. A 10 kDa protein corresponding to the barley LTP has been found to be recognized by the IgE of patients allergic to beer (18). The results reported here confirm that IgE-binding to a corn protein with a similar molecular weight are present in the sera of some patients with documented food allergy to corn. However, the salt-soluble fraction comprises only a minimal quantity (less than 4%) of the total corn proteins, whereas most of the proteins are extractable with aqueous alcohol in reducing conditions (2, 3). For this reason, the presence of potential allergens in the corn alcohol-soluble fraction has been investigated. We have found that an insoluble 50 kDa protein was bound by the IgE of all the DBPCFC-positive patients examined by immunoblotting. Since this protein could be also extracted with water, but only in reducing conditions, we conclude that it belongs to the RSP fraction of the corn endosperm (6). The allergenicity of this latter protein fraction was confirmed by SPT. Although further studies are needed to precisely characterize this 50 kDa corn protein, our results demonstrate that a salt-insoluble protein can act as allergen in the case of allergy to ingested corn. IgE-binding to insoluble proteins has been reported also in wheat, using sera of patients suffering from either baker's asthma (21), or from food allergy to wheat (22, 23). The IgE-binding components were identified as corresponding to members of the wheat prolamins (gliadins and glutenin subunits). However, in the corn flour, we have not observed any IgE-binding to the main zein components, which are the predominant prolamins of the corn seed.
Most of the studies on food allergy are focused on allergens that are soluble in water/salt solutions. On the basis of the results reported here, and considering that the solubility characteristics of a given protein can be remarkably changed during the digestion process, we recommend performing studies on food allergen identification by considering also the insoluble proteins of raw materials used for preparation of the allergenic food.
Studies on food allergy are commonly carried out by using raw materials as sources of potential allergens, also when the incriminated foods are eaten after cooking. Heat treatments can have important effects on the allergenic potential of processed foods. They can either reduce or destroy protein allergenicity or give origin to new allergens by physicochemical modifications of proteins that are inoffensive in their native state (24). Therefore, the direct effect of cooking on the allergenicity of those foods that are consumed after being cooked should be carefully considered. Our results demonstrate that, whereas most of the soluble IgE reactive proteins of the raw flour seem to be modified after cooking, the insoluble 50 kDa allergen is not affected by the heat treatment, neither in terms of its solubility and electrophoretic features nor for its binding to serum IgE.
Moreover, cooking can modify protein digestibility and then affect indirectly the form in which the ingested allergens interact with the organism (25). For this reason, in vitro digestion experiments on cooked foods are of relevant interest in the cases of food allergies in which the allergens are thought to act or to be absorbed at the level of the gastrointestinal tract (26). It is commonly assumed that proteins that are susceptible to be completely degraded during digestion are inherently safer, especially in relation to food allergy, than those that are more resistant to proteolysis (27). On the basis of these considerations it is widely accepted that the ability of food allergens to reach the intestinal mucosa is a prerequisite to allergenicity (28, 29), although the effect of cooking on this latter characteristic has been rarely considered. The data reported here demonstrate that the insoluble 50 kDa allergen of the corn flour is quite resistant to both gastric and pancreatic digestion in the form in which it is consumed (in a cooked sample). Taking into account that this protein is cross-linked through covalent (S–S) bonds in the flour (6), its characteristic stability could be due to the presence of such bonds. Therefore the factors determining the insolubility of this protein are also likely to be involved in its resistance to both heating and proteolysis.
In conclusion, the insoluble 50 kDa protein present in the RSP fraction of the corn flour is a candidate to act as an allergen in IgE-mediated food allergy to corn products.