Lysozyme, hydrochloride

Sensitisation data (human)

Administrative data

Endpoint:

sensitisation data (humans)

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: Literature case reported.

Data source

Reference

Reference Type:

publication

Title:

Occupational Asthma Induced by Inhaled Egg Lysozyme

Author:

Bernstein J.A., Kraut A.,Bernstein D.I.

Year:

1993

Bibliographic source:

Chest 1993; 103:532-35

Materials and methods

Type of sensitisation studied:

respiratory

Study type:

case report

GLP compliance:

no

Test material

Test material information

Constituent 1

Reference substance name:

Lysozyme

EC Number:

232-620-4

EC Name:

Lysozyme

IUPAC Name:

Lysozyme

Method

Type of population:

occupational

Subjects:

A 26-year-old man, employed in an area of a plant which exclusively manufactured egg white-derived lysozyme, presented with progressive shortness of breath and wheezing 8 months after starting his job. The subject was rotated between the various stages of the work process. When working in the final drying stage of egg lysozyme powder, he wore a protective dust mask. Six months prior to his initial evaluation, he had stopped working directly near these dry ers since they were causing respiratory symptoms.
There was a four pack-year history of cigarette smoking, but he had no documented prior history of allergic rhinitis, asthma or eczema.

Clinical history:

An initial history revealed that the worker experienced chest tightness and shortness of breath during exposure to egg lysozyme powder. These symptoms, which improved a few hours after leaving work, resulted in missed work periodically, but never hospitalization.

Details on study design:

WORK PROCESS
Egg whites were passed through resin in a column for 8 h. This egg white mixture was then precipitated with caustic soda, centrifuged, mixed with water, precipitated again with hydrochloric acid, recentrifuged and dried. The final drying step required large hot air blowers which released lysozyme powder into the air.

CASE HYSTORY
Physical examination one day after exposure to egg lysozyme revealed diffuse wheezing in all lung fields but was otherwise normal.
The subject was skin prick-positive to several seasonal and perennial aeroallergens indicating atopy. Total serum IgE on initial evaluation was elevated at 907 Kvs/l (normal, 2 to 120 Kvs/l), and a peripheral total eosinophil count was 75 x 10’/l (normal, 45 to 330 x 10’/l). No previous laboratory results for IgE or eosinophils were available.
The worker’s symptoms improved after treatment with medications consisting of prednisone, β, agonists, theophyllmne and avoidance measures. Reemployment away from the plant has resulted in complete resolution ofhis asthma symptoms.

MATERIALS AND METHODS
Immunologic Studies
Skin Prick testing: common seasonal and perennial allergens were used to determine if the patient was atopic. The subject was also skin tested for egg white, egg yolk, conalbumin, ovomucoid, ovalbumin and lysozyme. Lysozyme, conalbumin, ovomucoid and ovalbummn were prepared at a concentration of 50 mg/ml and administered as prick tests only. Histamine hydrochloride (10 mg/ml) was used as the positive control with a 5-mm wheal and flare reaction considered a significant reaction and normal saline solution was used as the negative control.
Five negative control subjects, defined as having exposure to egg lysozyme but no symptoms, were also skin tested for lysozyme.

Direct Enzyme-linked Immunosorbent Assay: serum was obtained from the subject, divided into 1-ml aliquots and frozen at -20 °C until further use. All enzyme or proteins used in this assay were obtained from commercial sources except for egg yolk and egg white which were prepared in the laboratory, as previously described. The enzymes were diluted with phosphate-buffered saline solution to an initial concentration of 10 mg/ml. Serum was evaluated by a standard enzymelinked immunosorbent assay (ELISA) technique for IgG and IgE specific antibodies using IgG and IgE alkaline phosphatase immunoconjugates, respectively.

Enzyme-linked immunosorbent Assay Inhibition: the patient’s serum was diluted to a concentration of 1:10 with phosphatebuffered saline and preincubated with the various concentrations of proteins (cellulase, lysozyme, ovalbumin, ovomucoid, conalbumin, whole egge white and egg yolk) for 1 h at room temperature. The pre-inhibited serum was then added to plates coated with lysozyme (10 µg/ml) and the ELISA procedure was performed as described previously. Percentage of inhibition was determined by (B-A)/B x 100 percent, where B represents the ELISA value before incubation with proteins and A represents the ELISA value after incubation with proteins.

Physiologic Studies
Peak Flow Monitoring: a mini-Wright peak flowmeter was used to monitor the patient’s respiratory status every 2 h during work over a 2-week interval. Results were recorded by the patient on daily diary cards in relationship to exposure and usage of medication.

Lysozyme Inhalation Challenge: a specific lysozyme bronchoprovocation challenge was performed after approval from the Faculty Committee on the Use ofHuman Subjects and Research at the University ofManitoba, Manitoba, Canada. A modified method described by Pepys and Hutchcroft’ for dry powder was used. The study was performed single blinded on two separate test days with day 1 challenging with lactose placebo and day 2 with lysozyme powder. The subject was instructed to sift 50 mg of either the placebo powderorlysozyme powder back and forth between porous trays. The subject’s FEV, and forced vital capacity were recorded before challenge and every 10 mm using a Vitaigraph. Symptoms were monitored and recorded during the challenge. To monitor for a possible late reaction, the subject was given a mini-Wright peak flowmeter to measure and record peak flow hourly for 9 h while at home.

Results and discussion

Any other information on results incl. tables

IMMUNOLOGIC STUDIES

Skin Prick Testing: Prick skin tests at 50 mg/ml were positive to lysozyme, ovomucoid, ovalbumin and conalbumin antigens, but were negative to whole egg yolk and egg white.

Enzyme-Linked Immunosorbent Assay and its inhibition to Egg Lysozyme: serum of a sensitized egg processing worker with IgE to a spectrum of egg white proteins served as a positive control. Negative control subjects included the pooled sera of exposed asymptomatic egg processing workers (negative control 1; n = 2) and pooled sera of nonexposed asymptomatic laboratory personnel from the University of Cincinnati Medical Science Building, Division of Immunology (negative control 2; n 3). During work exposure, the subject had increased serum IgE binding to egg lysozyme and ovomucoid protein, whereas ovalbumin, conalbumin, egg white and egg yolk yielded low IgE binding. The positive control had increased IgE binding to lysozyme, conalbumin, ovomucoid, ovalbumin, egg white and egg yolk. Both negative control subjects exhibited low IgE binding to the panel ofegg proteins. The IgE binding to all of the egg proteins were undetectable in the subject after one year away from work. The IgE ELISA inhibition, using these egg white proteins as inhibiting reagents, demonstrated greater than 50 percent inhibition of lysozyme specific IgE binding by lysozyme at a concentration of 2.4 mg/mi of inhibitory protein. Egg yolk, egg white, celiulase, ovalbumin and conalbumin showed no evidence of IgE inhibition at this concentration. The IgE ELISA inhibition studies performed using the subject’s serum obtained after prolonged work avoidance were negative to the panel ofegg proteins. Inhibition of lysozyme specific IgE using ovomucoid as the inhibiting reagent was observed at a tenfold greater concentration (20 mg/ml). Heat inactivation of egg lysozyme did not affect its capacity to inhibit.

PHYSIOLOGIC STUDIES

On initial evaluation, pulmonary function testing revealed a baseline FEV₁, of 2.78 l (71.1 percent predicted) which improved after the patient inhaled a β₂ agonist medication to 3.27 l (83.7 percent predicted). Lung volumes were normal. Pulmonary function tests repeated 2 weeks after avoiding work revealed that the baseline FEV₁, had increased to 4.02 l (31 percent increase). A methacholine bronchoprovocation test performed away from work was positive at a provocative challenge dose of 0.8 mg/ml, eliciting a 20 percent drop in FEV₁ confirming the presence of bronchial hyperresponsiveness. Serial peak expiratory flow rate readings, recorded while the worker was at work during an 18-day period, decreased during exposure to egg lysozyme powder. The subject’s peak expiratory flow rate consistently decreased with lysozyme exposure and improved away from work. The FEV₁, was unchanged in response to the lactose powder placebo performed on a separate control test day. The FEV₁, decreased from 4.2 to 2.2 l 15 mm after exposure to egg lysozyme. Serial peak flow determinations monitored hourly for 9 h after the initial early airway response revealed no evidence of a late asthmatic response (LAR).

	Lysozyme	Ovalbumin	Conalbumin	Egg white	Egg yolk	Ovomucoid
Subject	0.99± 0.08	0.12± 0.035	0.26± 0.015	0.17± 0.009	0.17± 0.019	0.79± 0.02
Positive control*	0.95± 0.01	0.33± 0.046	0.89± 0.015	0.72± 0.013	0.55± 0.046	1.02± 0.04
N§egative control 1	0.23± 0.017	0.43± 0.035	0.28± 0.044	0.14± 0.003	0.13± 0.003	0.10± 0.006
N§egative control 2	0.19± 0.01	0.11± 0.021	0.09± 0.013	0.13± 0.12	0.13± 0.003	0.12± 0.015

Positive control-serum of an egg processing worker with IgE sensitization to a spectrum ofegg proteins. Negative control 1-pooled sera of two exposed asymptomatic egg processing workers (n 2). Negative control 2-pooled sera of unexposed asymptomatic laboratory workers (n 3).

DISCUSSION

Occupational asthma induced by IgE-mediated sensitization to a spectrum of airborne egg proteins previously has been reported to occur in egg processing workers. This worker has the first reported case of occupational asthma induced by sensitization to inhaled egg lysozyme powder produced exclusively for use in the pharmaceutical industry. This diagnosis was confirmed by significant shifts in the subject’s peak expiratory flow rates on days at work compared to nonexposure days at home and by a positive single-blinded placebo-controlled bronchoprovocation challenge test to egg lysozyme. The subject exhibited an immediate early airway response within 15 min after exposure, but no LAR. The lack of an LAR is consistent with the subject’s clinical course since delayed respiratory symptoms were never manifested after avoidance measures. However, the lack of an LAR could have been influenced by rescue with a β₂ agonist medication after the initial immediate airway response. Finally, a delay in performing the bronchoprovocation challenge for several months after complete avoidance ofthe egg lysozyme powder also could have explained the lack of a late airway response. Specific IgE in this subject was demonstrated in vivo by positive cutaneous reactivity to egg lysozyme and in vitro by serum specific IgE binding to egg lysozyme protein. The ELISA inhibition of lysozyme specffic IgE by lysozyme confirmed the worker’s IgE antibody specificity to egg lysozyme. Although the subject was skin test-positive to several egg proteins, ovomucoid was the only other egg protein which resulted in elevated direct serum specific IgE binding. Ovomucoid also inhibited specific IgE binding to egg lysozyme but at a tenfold higher concentration compared with lysozyme. There are several possible explanations accounting for this finding. Most likely, the subject was sensitized to ovomucoid protein during the work process which in the early stages involved extracting lysozyme from whole egg white. However, the ovomucoid used in these experiments, which was impure according to the manufacturer, also could have been contaminated with egg lysozyme, resulting in increased IgE binding. The possibility of shared epitopic determinants between lysozyme and ovomucoid cannot be excluded from the cross-sensitivity results seen with ELISA inhibition.

In conclusion, this is the first case of IgE-mediated occupational asthma induced by inhaled egg lysozyme manufactured for use in the pharmaceutical industry. This case illustrates that airborne egg proteins are potent occupational allergens that cause occupational asthma.

Applicant's summary and conclusion

Conclusions:

Lysozyme can induce asthma, caused by inhalational exposure to egg lysozyme.

Executive summary:

A 26-year-old man employed in a company which manufactured hen egg white derived lysozyme for use in the pharmaceutical industry was evaluated for occupational asthma. The worker began to experience immediate-onset asthmatic symptoms two months after starting to work with egg lysozyme powder. The work process involved the production of approximately 1000 kg of purified dried lysozyme powder per week. Prick skin testing was positive to egg lysozyme (50 mg/ml) and other egg protein components, but negative to whole egg white and egg yolk reagents. Serum specific IgE to egg lysozyme was documented. Decrements in serial peak expiratory flow rates were associated with lysozyme exposure at work. A specific broncho provocation challenge to lysozyme powder was positive demonstrating an isolated immediate asthmatic response (48 % decrease from baseline FEV₁,). This is the first reported case of lysozyme-induced asthma specifically caused by inhalational exposure to egg lysozyme.

Conclusion

Lysozyme can induced asthma, caused by inhalational exposure to egg lysozyme.