Contact Dermatitis, 2001, 44, 91–93 Copyright C Munksgaard 2001Printed in Denmark . All rights reserved

ISSN 0105-1873

Contact urticaria from riceY Y1,2, H O2, M A2, K T3

Z I2

1Department of Dermatology, Yokohama City University Medical Center, Yokohama;2Department of Dermatology, Yokohama City University School of Medicine, Yokohama;

3AFT (Allergen Free Technology Laboratory Inc.) Tokyo, Japan

A 30-year-old man with atopic dermatitis had had erythema and itching of the hands after washingrice in water, though he had always eaten cooked rice without problems. Handling test with waterused to wash regular rice was performed on abraded hands, and produced urticarial erythemaafter several minutes. Applications of water used to wash allergen-reduced rice were negative forurticarial reaction. Prick test with water used to wash regular rice was πππ. However prick testreaction with water used to wash allergen-reduced rice was π. Histamine-release test of regularrice-washing water was grade 3 and that of allergen-reduced rice grade 1. In immunoblottinganalysis with regular rice washing water, there were no bands with this patient. These resultssuggest that the allergen responsible for contact urticaria in this patient might be water-soluble,heat-unstable, and not contained in allergen-reduced rice.

Key words: immunological contact urticaria; atopic dermatitis; rice; allergen-reduced rice; foods;prevention. C Munksgaard, 2001.

Accepted for publication 29 August 2000

Contact urticaria has been reported with increas-ing frequency, and a growing number of elicitingagents are now recognized. A few reports have ap-peared of contact urticaria from rice (1–3), thoughthe antigenic components have not yet been well-established. This report describes a case of contacturticaria from rice, where we investigated the aller-gen responsible.

Patient and Methods

Case reportA 28-year-old man visited our hospital for treat-ment of atopic dermatitis. The condition hadstarted when the patient was 10 years old, worsen-ing slightly from the age of 17. Remission hadrapidly been obtained with systemic antihistamineand disodium cromoglycate. At the age of 30years, the patient began to experience erythemaand itching of the hands after washing rice inwater, though he had always eaten cooked ricewithout problems. On admission, the blood countshowed 330 eosinophils/mm3. Total serum IgE was4200 IU/ml and RAST was class 2 for rice.

Handling test

A handling test with water used to wash rice wasperformed on the patient’s hands for several min-utes. As previously described, we developed 2 typesof allergen-reduced rice by reduction of major al-lergenic rice proteins (4). One of these is hypoal-lergenic rice from Shiseido (HRS-1, Fine riceA;Shiseido, Japan) (4), and the other is allergen freetechnology rice (AFT-R, AFT-R1A; Allergen-freeTechnology Laboratory Inc., Japan). Applicationsof water used to wash these allergen-reduced ricewere also performed.

Prick tests

Prick tests with various allergens were carried out.Salt-soluble rice protein, one of the rice allergens,was extracted from rice powder with phosphatebuffered saline (PBS) containing NaCl. The salt-soluble rice protein, commercially available riceallergen, water used to wash regular rice, waterused to wash AFT-R, and the 16kDa rice allergenwere used as allergens. Histamine dihydrochloride

92 YAMAKAWA ET AL.

10 mg/ml was used as positive control. We meas-ured the wheal diameter. A reaction of the sameorder of size as that of the positive histamine refer-ence is recorded as πππ, double size of referencereaction as ππππ, half size of reference reactionas ππ, quarter size of reference reaction as π, andnegative reference reaction as ª (5).

Histamine-release test

Further, we evaluated the value of a glassmicrofiber-based histamine-release test (HRT) byusing an HRT kit (RusikaA; Asahikasei KogyoK.K., Japan) (6). We used commercially availablerice allergen, regular rice-washing water, salt-sol-uble rice protein, AFT-R washing water, and 16kDa allergen as allergens. The allergens were di-luted serially 5-fold, and 6 different concentrationsof allergen were prepared in separate microtiter-plates. 25 ml of heparinized whole blood from thispatient was placed onto each well of the plate, andthen incubated for 90 min at 37æC. The net releaseof histamine is calculated on the basis of a stan-dard curve. The results are graded from 0 to 4,according to significant allergen-induced histaminerelease (cut-off level, 15 ng/ml). Grade 4 meansthat histamine release is more than the cut-off level

Fig. 1. Immunoblotting analysis of the patient’s sera, the other3 atopic dermatitis patients’ sera and normal control’s sera forrice-washing water: lane M; molecular weight marker polypep-tides; lane A; regular rice-washing water; lane B; salt-solublerice protein; lane 1; this patient; lanes 2–4; the other 3 atopicdermatitis patients; lane 5; normal control.

at 5ª5 lowest allergen concentration; grade 3means that histamine release is more than cut-offlevel at 5ª3; grade 2 means that it is at 5ª2; grade1 means that it is at 5ª1; and grade 0 means thathistamine release is under the cut-off level at allconcentrations.

Immunoblotting

To determine the molecular weight of antigenscontained in rice-washing water, we performed so-dium dodecyl sulfate-polyacrylamide gel electro-phoresis (SDS-PAGE). Rice-washing water, ex-tracted with 2 volumes of water from rice, was ap-plied to SDS-PAGE. The electrophoresis wascarried out according to the method previously de-scribed (7, 8). With rice-washing water, severalbands at approximately 14 kDa, 20–30 kDa, 30–40 kDa , and 50 kDa were detected. After SDS-PAGE with 10 ml of rice-washing water (in eachlane A) and of salt-soluble rice protein purified (ineach lane B), the proteins fractionated were trans-ferred to polyvinylidene difluoride membrane(PVDF membrane, PVDFA, Cosmo Bio., Japan).After blocking with skim milk, the PVDF mem-brane was subjected to a blotting unit (ScreenerBlotterA, Cosmo Bio.,Japan), this patient’s serawas applied to lane 1 and the other 3 atopic der-matitis patients’ sera were applied, respectively, tolanes 2–4. The 3 atopic dermatitis patients’ IgERAST was class 4 for rice. The membrane was in-cubated overnight at 4æC, washed with PBS con-taining 0.05% Tween 20, and incubated with anti-human IgE biotin conjugate for 2 h at 3æC. Afterwashing with PBS-Tween 20, the membrane wasincubated with avidin-horseradish peroxidase for 1h at 37æC and washed again with rinse PBS-Twe-en20. Then the membrane was developed with0.05M diaminobenzidine and 0.01 M hydrogenperoxide and washed with water.

Results

The handling test with water used to wash rice pro-duced urticarial erythema after several minutes.But applications of water used to wash HRS-1 orAFT-R were negative for urticarial reaction.

Prick test with commercially available rice aller-gen was ππ. The results of water used to washregular rice and the salt-soluble rice protein, re-spectively, were πππ. However, reaction to theprick test with water used to wash AFT-R was πand that to the 16kDa rice allergen was ª.

Further, the HRT results of commercially avail-able rice allergen and of regular rice-washing waterwere grade 3. That of salt-soluble rice protein wasgrade 4. However, the result with AFT-R washing

93CONTACT URTICARIA FROM RICE

water was grade 1, and that with 16kDa allergengrade 2.

By immunoblotting analysis, in the 3 atopic der-matitis patients with high IgE RAST for rice, sev-eral polypeptides were detected (Fig. 1, lanes 2–4).However, no bands were detected in the samplefrom this patient (Fig. 1, lane 1).

Discussion

Rice, a staple of the Japanese diet, is difficult toeliminate from the daily fare. Because rice is vigor-ously hand-washed several times before boiling,water-soluble allergens are considered to be re-sponsible for producing the contact urticaria inthis patient.

Recently, severe cases of atopic dermatitis havebeen noted to be on the increase. Based on a stat-istical analysis of the correlation between rice-RAST score and clinical severity in atopic derma-titis patients, we have reported the probable in-volvement of rice allergy in many such severe cases(9). Given that, in severe atopic dermatitis pa-tients, eating boiled rice can induce cutaneoussymptoms, the rice antigen may be stable to heator digestive enzymes. In this patient, the oral in-take of boiled rice allergens did not influence theclinical course of atopic dermatitis. We could notdetermine the rice antigenic components for thispatient by immunoblotting analysis, in which theproteins in SDS-PAGE are treated by heat. Thisresult means that, in the case of this patient, anti-genicity may be unstable to heat; after boiling, therice allergen may lose antigenicity.

We developed 2 types of allergen-reduced rice.One, HRS-1, is treated by enzymatic decompo-sition of the constituent proteins presumed to bethe major allergens of rice (4). The other, AFT-R, is treated with alkali. We reported that proteinanalysis with SDS-PAGE of such rice indicated thenear absence of 16kDa, 25kDa, and other highermolecular weight allergen bands corresponding tothe globulin fraction (4). In this study, the patientshowed no hypersensitivity response to allergen-re-duced rice in the handling test, prick test or HRT.

Moreover, prick test with the 16 kDa protein wasnegative and its HRT was weak. The results of thisinvestigation suggest that the allergen responsiblefor contact urticaria in this patient might be water-soluble, salt-soluble, heat-unstable, not be con-tained in allergen-reduced rice and not be the16kDa allergen.

The patient’s atopic dermatitis has improvedover the course of several years, and he has hadno problems with his hands since avoiding directcontact with water used for washing rice.

References1. Lernia V I, Albertini G, Bisighini G. Immunologic contact

urticaria syndrome from raw rice. Contact Dermatitis 1992:27: 196.

2. Lezaun A, Igea J M, Quirce S, Cuevas M, Parra F, AlonsoM D, Martin J A, Cano M S. Asthma and urticaria causedby rice in a housewife. Allergy 1994: 49: 92–95.

3. Sasai S, Takahashi K, Takahashi K, Tagami H. Contacturticaria to rice. British J Dermatol 1995: 132: 827–839.

4. Ikezawa Z, Ikebe T, Ogura H, HRS-1 Reseach Group. Clin-ical effect of hypo-allergenic rice (HRS-1) in atopic derma-titis. Allergy 1991: 40: 633–642.

5. Alas K, Belin L. Suggestions for biological quantitativetesting and standardization of allergen extracts. Acta Aller-gol 1974: 29: 238–240.

6. Yamakoshi M, Fujii Y, Nagai H, Ooyama K. A novel diag-nostic method for allergy ‘‘LUCICA HRT’’. Jpn J ClinPathol 1997: 45: 163–173.

7. Laemmli U K. Cleavage of structural proteins during theassembly of the head of bacteriophage T4. Nature 1970:227: 681–685.

8. Tsubaki K, Takahashi Y, Aihara Y, Matsuyama S, YokotaS, Ikezawa Z. Detection of IgE antibodies to the salt-insol-uble wheat antigen in sera of patients with atopic derma-titis (AD) by ELISA and immunoblotting techniques (inJapanese). Jpn J Allergol 1995: 44: 134–142.

9. Ikezawa Z, Ikebe T, Ogura H, Odajima H, Furosaka F,Komatau H. Mass trial of hypoallergenic rice (HRS-1)produced by enzymatic digestion in atopic dermatitis withsuspected rice allergy. Acta Dermato-venereologica 1992:(Suppl 176): 108–112.

Address:

Yuko YamakawaDepartment of DermatologyYokohama City University Medical Center4-57 Urafune-choMinami-ku, Yokohama 232-0024Japan