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- Materials and methods
Background: Fish is one of the most frequent causes of immunoglobulin E (IgE)-mediated food allergy. Although the fish dark muscle is often ingested with the white muscle, no information about its allergenicity and allergens is available.
Methods: Heated extracts were prepared from both white and dark muscles of five species of fish and examined for reactivity with IgE in fish-allergic patients by enzyme-linked immunosorbent assay (ELISA) and for allergens by immunoblotting. Cloning of cDNAs encoding parvalbumins was performed by rapid amplification cDNA ends. Parvalbumin contents in both white and dark muscles were determined by ELISA using antiserum against mackerel parvalbumin.
Results: Patient sera were less reactive to the heated extract from the dark muscle than to that from the white muscle. A prominent IgE-reactive protein of 12 kDa, which was detected in both white and dark muscles, was identified as parvalbumin. Molecular cloning experiments revealed that the same parvalbumin molecule is contained in both white and dark muscles of either horse mackerel or Pacific mackerel. Parvalbumin contents were four to eight times lower in the dark muscle than in the white muscle.
Conclusions: The fish dark muscle is less allergenic than the white muscle, because the same allergen molecule (parvalbumin) is contained at much lower levels in the dark muscle than in the white muscle. Thus, the dark muscle is less implicated in fish allergy than the white muscle.
Fish is an important food source rich in nutrients such as polyunsaturated fatty acids and lipid-soluble vitamins. However, it is obviously one of the most frequent causes of food allergy mediated by immunoglobulin E (IgE) antibodies, especially in coastal countries, such as Japan and Scandinavia, where the fish consumption is high. In fish-allergic patients, hypersensitivity reactions such as urticaria, asthma, vomiting are rapidly induced after ingestion of fish (1–3). Even fatal cases because of anaphylactic reactions have been reported (4, 5). The first identified fish allergen is parvalbumin (known as Gad c 1) of the cod Gadus callarias (6, 7). Parvalbumin is a calcium-binding sarcoplasmic protein with a molecular mass of about 12 kDa and is distributed universally in the white muscle of fish. In accordance with this, subsequent molecular studies with carp Cyprinus carpio (8), Atlantic salmon Salmo salar (9), Atlantic cod G. morhua (10) and Pacific mackerel Scomber japonicus (11), together with IgE-immunoblotting studies (2, 12–14), have demonstrated that parvalbumin represents the major and cross-reactive allergen in common with various species of fish.
Teleost fish have two types of muscle, white muscle (also called light muscle) and dark muscle (also called red muscle). The white muscle is used for short bursts of swimming, while the dark muscle, which is located directly under the skin, is for continuous swimming. Accordingly, active fish such as tuna and skipjack have more developed dark muscle than bottom fish such as flounder and cod. Probably because the majority of the fish muscle is the white muscle even in active fish, all the previous studies on fish allergens have been performed using only the white muscle or that contaminated with a small amount of the dark muscle. However, the dark muscle of many species of fish is often ingested with the white muscle and hence is assumed to be implicated to some extent in hypersensitivity reactions caused by fish. Therefore, this study was initiated to clarify the allergenicity and allergens of the fish dark muscle for a better understanding of fish allergy. The results showed that the dark muscle was less allergenic than the white muscle and that the major allergen in the dark muscle was parvalbumin as in the white muscle. To obtain molecular evidence for the lower allergenicity of the dark muscle than the white muscle, primary structures and contents of parvalbumins in both white and dark muscles were also determined.
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- Materials and methods
Parvalbumin represents a major and cross-reactive allergen in the fish white muscle. In the present study, therefore, patient sera containing IgE antibodies specific for fish white muscle parvalbumin were used to examine allergenicity and allergens of the dark muscle of five species of fish in comparison with those of the white muscle.
The IgE-immunoblotting experiments revealed that the major allergen in the dark muscle as well as in the white muscle is parvalbumin, except for the yellowtail dark muscle for which no IgE-reactive bands were observed probably due to the scarcity of parvalbumin. However, analysis by SDS-PAGE strongly suggested that, in common with the five species of fish, the parvalbumin content in the dark muscle is significantly low compared with that in the white muscle. These results qualitatively supports the ELISA data that the dark muscle of the five species of fish is much less allergenic than the white muscle.
The significance of this study is in providing molecular evidence for the lower allergenicity of the dark muscle. Molecular cloning experiments clearly demonstrated that the same parvalbumin molecule is contained in both white and dark muscles of two species of fish, horse mackerel and Pacific mackerel. This is the first to report the primary structure of parvalbumin in the fish dark muscle. Because our finding is very likely to be applicable to all species of fish, the allergenicity of the two types of muscles largely depends on the parvalbumin content. Therefore, parvalbumin was then quantified for both white and dark muscles of the five species of fish by ELISA using the antiserum against Pacific mackerel parvalbumin. The results obtained showed that, regardless of the fish species, the parvalbumin content in the dark muscle is much lower than that in the white muscle. In addition, there is a good relationship in each muscle sample between the parvalbumin content and the IgE-binding potency of the heated extract. In preparing this paper, Lim et al. (19) has reported that parvalbumin is found in the white muscle of tuna Thunnus tonggol but absent in the dark muscle, conforming well to our study.
Two important findings were additionally obtained in this study. One finding is that yellowtail is extremely low in allergenicity. In ELISA, all the patient sera tested showed no substantial reactivity to the heated extracts from both white and dark muscles of yellowtail. In IgE-immunoblotting, the patient sera reacted very weakly to parvalbumin in the white muscle of yellowtail but not to that in the dark muscle. These results can be realized by the fact that yellowtail contains very low amounts and no detectable amounts of parvalbumin in the white and dark muscles, respectively. Although patients who have experienced allergic reactions to one species of fish are currently recommended to avoid all species of fish, some of them may be tolerable to yellowtail. Fish with very low allergenicity, such as yellowtail, will be discovered in future screenings with various species of fish. Another important finding is that a new allergen of 14 kDa is contained in both white and dark muscles of red sea bream, although it may be recognized by a limited number of fish-allergic patients. This allergen is not related to parvalbumin, because it does not react with the monoclonal antiparvalbumin antibody. Also, it is obviously distinguishable in molecular mass from collagen (16, 20, 21) and aldehyde phosphate dehydrogenase (22), which have been identified as minor allergens in fish. Purification of the 14 kDa allergen is under progress.
Based on the primary structures and contents of parvalbumin, this study proved at the molecular level that the fish dark muscle is less allergenic than the white muscle. The dark muscle is the minority of the whole muscle and is less frequently eaten than the white muscle. Taken together, we conclude that the dark muscle is much less implicated in fish allergy than the while muscle, being little worth consideration in future studies on fish allergens. Finally, it should be emphasized that a variety of fish species are eaten but only a part of them has been examined for allergenicity and allergens. Even this study with only five species of fish has established the low allergenicity of yellowtail and the presence of a new 14 kDa allergen in red sea bream. Future study with many species of fish is needed to understand complicated features of fish allergy in more details.