Discussion
In this study, we delineated the allergen profiles of salmon and grass
carp using sera from two Asian populations. With the purified allergens
from both fish species, our study illustrates parvalbumin and collagen
as important biomarkers for fish allergy diagnosis. Our results also
indicate a wider allergen spectrum including sensitization to heat
labile allergens, as well as stronger sensitization to salmon than grass
carp among the Japanese fish allergic subjects where grass carp is
unavailable. Such differences highlights that the relevant fish species
as diagnostic marker and allergen sensitization are distinct in
different populations due to diverse dietary exposures and habits.
Ruethers et al. reported recently 10 allergens from catfish and 6
allergens from salmon13. Specifically, tropomyosin and
TPI identified as fish allergens in their study are known shrimp
allergens23. Despite positive shrimp-sIgE by ImmunoCAP
in six HK subjects, none showed reaction to fish tropomyosin or TPI in
our western blot. Our subjects also showed no detectable IgE binding to
allergens including creatine kinase, pyruvate kinase, L-lactate DH and
G-6-PI that were previously reported as catfish allergens. This might
partly indicate that these are only catfish-specific allergens and/or
the reactions are population-specific. Apart from the well-defined fish
allergens (i.e. parvalbumin, enolase, aldolase and collagen), we
consistently identified GAPDH as a putative fish allergen of both salmon
and grass carp12. Such findings supported GAPDH to be
an important fish allergen.
Our present report further focuses on studying the more well-defined
fish allergens in terms of their sensitization rates. In agreement with
most studies that parvalbumin accounted for 70-95% of allergic
reactions9,24, parvalbumin is the major allergen in
grass carp and salmon with 74% sensitization rate determined by western
blotting and ELISA. Collagen is the second commonest fish allergen with
37.5% of subjects being sensitized. The reported rates of IgE binding
to collagen vary in different populations. For instance, Kalic et al.
analyzed samples from 101 fish allergic patients in Australia and showed
that 21% of subjects displayed IgE binding to collagen from tuna,
barramundi and/or salmon14. Kuehn et al. reported
19.3% sensitization rate to gelatin of cod, salmon and/or tuna in 62
fish allergic patients16. With only 21 patients,
Shimojo et al. reported a higher rate of 52.4% sensitization to
collagen than to parvalbumin25 while Kobayashi et al.
reported also 50% of sensitization rate to mackerel
collagen26. IgE binding to collagen was also more
frequent in patients with more severe fish allergic symptoms than those
with mild-moderate responses while independent reports have shown
anaphylaxis provoked by gelatin/collagen15,27. On the
other hand, Kalic et al. reported that 8/21 of collagen-IgE positive
patients showed IgE binding to collagen only but not
parvalbumin14. Comparatively, seven of our 37
collagen-IgE positive subjects demonstrated moderate IgE binding to
collagen but absence of parvalbumin-specific IgE. Taken together,
collagen is a prevalent allergen in Asian populations. Fish allergic
patients can have IgE binding to collagen only, supporting that testing
for collagen should be included in the diagnostic workup for fish
allergy.
To our knowledge, only one study with 62 fish allergic subjects reported
the sensitization rates of 62.9% and 50.0% to enolase and aldolase,
respectively16. Another smaller study by Ruethers et
al. showed that half of their 16 Australian patients showed weak to
moderate IgE reactivity to enolase in commercial tuna and salmon SPT
extracts28. In our cohort, IgE binding to aldolase was
detected in 38.5% of subjects while that to enolase was present in
17.8% of subjects. We did not anticipate a major difference in the
reactivity of natural and recombinant aldolase and enolase as the reason
for lower sensitization as the recombinant enzymes were confirmed to be
reactive consistently by ELISA (data not shown) and western blots. Such
discrepancy might, on the other hand, be explained by differences in the
ethnicity and age of the studied populations: this study only recruited
children below 18 years old while many subjects studied by Kuehn et al.
were young adults (mean age 28.7, range 13-62 years). Among seven
subjects without parvalbumin-specific IgE in our cohort, only one
Japanese subject showed IgE binding to salmon and grass carp aldolase.
Testing of enolase and aldolase thus do not substantially improve the
diagnostic yield for fish allergy in Asians while detecting IgE against
parvalbumin and/or collagen might be sufficient to identify patients
with fish allergy.
This study found minimal binding of sera from HK subjects to allergens
other than parvalbumin by western blotting. A possible explanation was
the use of different serum samples for ELISA and western blot, different
sensitivity of the assays, intrinsically low abundance of these
allergens in fish extracts and the insoluble nature of collagen in
aqueous solution29. Nevertheless, both ELISA and
western blot assays robustly indicated a wider spectrum of allergen
sensitization in Japanese than HK subjects. Japanese commonly consume
raw fishes, but this is a less likely reason for this observation given
the age-range of our recruited patients; the consumption of raw fish
might not be a major attribute to the sensitization to a wider spectrum
of allergen including the heat labile allergens. Our present study
showed, for the first time, differences in fish protein and allergen
profiles in relation to cooking methods at the same core temperature.
Despite higher cooking temperature with frying/baking compared with
steaming/boiling, frying/baking retained more heat liable allergens in
salmon meat. Steaming and boiling to make fish congee and soup are the
preferred methods of fish cooking in Hong Kong while baking and frying
of fish are more popular in Japan. For instance, 73.8% of the
interviewed Japanese workers consumed fish by broiling (baking)
comparing to only 14.1% for raw fish and 10% by
stewing30. Although we are aware that our present
study is limited by the lack of direct investigation into the dietary
habits of our studied population, our results support the importance of
dietary exposures, consumption and cooking preferences in determining
fish allergen sensitization in particular when the two populations did
not differ in terms of symptom severity. This factor should be
considered during the selection of allergens for allergy testing and the
interpretation of CRD results.
In the context of fish exposure, Japanese patients should not have eaten
grass carp due to the unavailability of this freshwater fish. Thus, IgE
binding to grass carp allergens in Japanese patients should be a result
of cross-reactivity that can be partly reflected from the stronger
positive correlation between grass carp and salmon sIgE levels than HK
subjects whom could be independently sensitized to the two fish species.
However, we observed that more Japanese patients reacted to grass carp
parvalbumin with higher OD values in ELISA, although salmon-sIgE level
was higher than grass carp-sIgE and dietary exposure to grass carp is
lacking in this population. The higher salmon-sIgE level might thus be
explained by IgE binding to other salmon allergens, particularly
collagen, when considering the lower parvalbumin content in crude salmon
extract, lower IgE affinity of salmon parvalbumin in ELISA, and the more
diverse salmon allergen binding pattern in western blots. On the other
hand, we also showed that one-fifth of subjects reacted only to grass
carp parvalbumin, which indicated that grass carp parvalbumin has higher
allergenicity than salmon parvalbumin. However, we are yet to know the
clinical reactivity to grass carp among the Japanese subjects and so the
question of improving assay sensitivity and specificity using fish
allergen (parvalbumin) with higher allergenicity remains open. The
varying allergenic potential of fish parvalbumins could be explained by
the specific sensitization of patients from their clinical
history8. With the lack of grass carp in the local
Japanese diet, our results suggested the presence of higher diversity,
distinct and/or species-specific IgE-binding epitopes for grass carp
parvalbumin31. It is of note that IgE sensitization
based on parvalbumin did not correlate well with patients’ clinical
allergy status18, and we postulate that the
identification of parvalbumin epitopes may help to resolve the
inconsistency in cross-reactivity of different fish species.
In summary, this study extends our understanding on the allergen profile
of two important fish species that parvalbumin and collagen are
important components for fish allergy diagnosis. We also illustrated
that the fish species extracts relevant for diagnosis and allergen
sensitization pattern differ among populations. Overall, this study
provides additional information for the implementation of relevant
extracts and components for precise fish allergy diagnosis.