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Occupational sensitization to lactase in the dietary supplement industry a
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Benedikt Stöcker , Sonja Grundmann , Pia Mosters , Paul Nitzsche & Randolf Brehler a
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Department of Dermatology, University Hospital Münster, Münster, Germany
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Werkarztzentrum Rietberg e.V., Rietberg, Germany Accepted author version posted online: 02 Jul 2015.
Click for updates To cite this article: Benedikt Stöcker, Sonja Grundmann, Pia Mosters, Paul Nitzsche & Randolf Brehler (2015): Occupational sensitization to lactase in the dietary supplement industry, Archives of Environmental & Occupational Health, DOI: 10.1080/19338244.2015.1066294 To link to this article: http://dx.doi.org/10.1080/19338244.2015.1066294
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ACCEPTED MANUSCRIPT Occupational sensitization to lactase in the dietary supplement industry Benedikt Stöckera, Sonja Grundmanna, Pia Mostersa, Paul Nitzscheb, Randolf Brehlera
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Department of Dermatology, University Hospital Münster, Münster, Germany
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Werkarztzentrum Rietberg e.V., Rietberg, Germany
Correspondence: Benedikt Stöcker University Hospital Münster Department of Dermatology Von-Esmarch-Str. 58 48149 Münster Germany E-mail: [email protected] Abstract Aerogen lactase exposure carries a risk for the development of allergic asthma and rhinitis; only a few occupationally affected patients have been reported. We report the results of allergy testing with employees of a lactase tablets manufacturing plant.
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ACCEPTED MANUSCRIPT The survey involved 13 workers, including a questionnaire, spirometry, basophil activation test (BAT), and skin prick tests (SPT) with lactase and a panel of common aeroallergens. Furthermore, lactase-specific IgE (s.-IgE) antibodies were analysed. Sensitization to lactase could be proven for 9 workers by SPT and BAT; s.-IgE antibodies could be detected in serum samples of all sensitized. However, IgE levels ≥ 0.35 kU/l were only found
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in 4 sera. Our data confirm that occupational exposure to lactase can induce IgE-mediated respiratory sensitization resulting in allergic diseases. Protective measures should thus be obligatory when working with lactase. Keywords: Aspergillus oryzae, enzyme allergy, lactase, occupational, inhalative allergy
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ACCEPTED MANUSCRIPT Introduction So far, more than 250 substances are described as respiratory allergens in the modern working environment causing allergic rhinitis and conjunctivitis, allergic asthma and contact urticaria due to IgE-mediated sensitization. Those allergens are mostly high-molecular proteins (molecular weight > 5 kDa).1,2 Although exposure minimization or avoidance is a dictum in several
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industries since the 1970s, occupational allergies are a significant problem causing high cost, especially if disability results.1,3 Fungal enzymes, particularly derived from the genus Aspergillus, are important occupational immediate type allergens, commonly used in baking, food, detergent, textile, and pharmaceutical industries.2 The species Aspergillus oryzae is a domesticated fungus, having been used in China and Japan for soy fermentation for thousands of years4 and is the most important resource for the industrial production of α-amylase. A. oryzae-derived α-amylase is the best characterized fungal enzyme used in the working environment, and is the major cause of baker’s asthma with the sensitization prevalence up to 34%.1,2 Another A. oryzae-derived enzyme is lactase, a member of the β-galactosidase family, which is applied in the pharmaceutical and food processing industry.2 Extracellular β-galactosidases from A. oryzae have advantages over the enzyme from other taxonomic groups for industrial applications, in terms of easier isolation from the culture supernatant, higher thermal tolerance and optimum activity at lower pH significantly avoids contamination.5 Due to its disaccharidase activity, lactase hydrolyses lactose into galactose and glucose monomers. Lactose intolerance due to lactase deficiency is widespread especially in African and Asian countries and effects
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ACCEPTED MANUSCRIPT about 75% of the adult world population.6 Clinical symptoms are mainly flatulence, diarrhoea, and abdominal pain due to the consumption of lactose contained in milk products. A supplemental treatment with lactase improves the tolerability of lactose in those patients. Occupational lactase sensitization has been reported in 65 of 207 tested workers of a plant engaged in formulating and packing lactase containing products.7 In a cross-sectional survey of
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94 pharmaceutical workers exposed to lactase, 27 had positive skin tests with lactase.8 In both studies the diagnosis of sensitization based on positive skin prick tests, and an elevated risk for respiratory allergy symptoms was related to sensitization. Furthermore, Laukkanen et al. demonstrated in a single case report a non-atopic pharmaceutical worker suffering from allergic rhinitis, contact urticaria and eczema due to lactase exposure. The diagnosis of an IgE-mediated sensitization was confirmed by skin tests in combination with lactase-specific IgE antibodies.9 Here, we report the results of allergy testing of 13 employees of a plant producing and packing lactase tablets. Methods Study population Thirteen employees of a plant producing coated and uncoated chewable lactase tablets from raw enzyme powder obtained from A. oryzae were sent by the corresponding occupational physician for allergy diagnostics. In the plant lactase was processed several times a year and 10 of these workers suffered from work-related allergic symptoms during these periods. The manufacturing process involves weighing of enzyme powder, mixing it with additives, pressing it to tablets,
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ACCEPTED MANUSCRIPT performing a quality control, and finally packaging. All employees were exposed to airborne lactase during this period, even if they are not handling raw lactase powder like employees engaged directly in the production process or in the quality control unit. Since respiratory or skin protection was not obligatory, the employees had direct contact to lactose by the dermis and respiratory system. Quantitative measurements of the airborne exposure were not performed.
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Questionnaire Patient’s history pertaining employment, atopic diseases and work related symptoms were documented by use of a questionnaire. Lung function test Spirometry and IOS Impulse Spirometry was performed using a Master Screen IOS (CareFusion, Hoechberg, Germany). Skin tests Lactase powder, consisting of native enzyme concentrate (66 - 71%), carmellose calcium and glucose, was kindly provided by the plant and was used for all experimental assays. SPTs were performed with 10-fold serial dilutions of lactase in 0.9% NaCl solution, starting with a concentration of 0.1 µg/ml and increasing every 10 minutes until a positive reaction appeared or the maximum concentration of 10 µg/ml was attained. Furthermore, SPTs were conducted with Aspergillus fumigatus (ALK-prick SQ, ALK-Scherax, Wedel, Germany) and common aeroallergens (house dust mite extracts (D. pteronissynus, D. farinae), birch pollen, weed pollen(Allergopharma, Reinbek, Germany) and grass pollen extracts (ALK, Wedel, Germany)).
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ACCEPTED MANUSCRIPT 0.9% NaCl solution served as negative control and 10 mg/ml histamine as positive control. A wheal diameter of ≥ 3 mm was considered positive. Basophil activation test BAT was performed as described by Mertens et al..10,11 In brief, heparinized whole blood was incubated for 25 min at 37°C with 100-fold serial dilutions of lactase in PBS ranging from 0.001
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to 10 µg/ml. To confirm cell responsiveness, 1 µg/ml of a monoclonal anti-IgE antibody (clone BE5; EurobioSciences, Friesoythe, Germany) served as positive control. After cell stimulation, the reaction was stopped by adding 20 mM EDTA in PBS and centrifugation at 400 g. Basophils were stained with 10 µl anti-CD203c-PE (Beckman-Coulter, Krefeld, Germany) for 45 min at room temperature in the dark. Using the whole blood lysing reagent (Beckman-Coulter), erythrocytes were lysed according to the manufacturer’s instructions. After washing and resuspending the cells in PBS with 1% BSA, a total of 60 000 cells were measured using the FACS Calibur flow cytometer equipped with CellQuestPro software (BD Biosciences). The percentage of activated basophils was calculated by subtracting spontaneous CD203c expression (negative control, PBS) from the data obtained with anti-IgE and lactase stimulation; values < 0 were set = 0. The result was considered positive when expression of CD203c was upregulated about more than 10% in relation to the negative control. The specificity of the BAT with lactase was proven using basophils of three non-atopic control donors. SDS-PAGE and Immunoblot analyses
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ACCEPTED MANUSCRIPT For gel separation of enzymes, 10 µg lactase or β-galactosidase (β-gal) (Sigma-Aldrich, Taufkirchen, Germany) from A. oryzea were applied to 12% SDS PAGE12 and stained with Coomassie Brilliant Blue (Sigma-Aldrich). Lactase was also transferred onto Immobilon-P Transfer membranes (Millipore, Billerica, MA, USA). The membrane was saturated with 2% skimmed milk powder in Tris-buffered saline (TBS) with 0.05% Tween-20 (TBST) over night and incubated with patient sera diluted 1:2 in 2% skimmed milk/TBST for 3 hours. Mouse anti-
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human IgE antibody (clone BE5; EurobioSciences, Friesoythe, Germany) diluted 1:1000 in 1% BSA/TBST was used for detection of s.-IgE antibody binding. HRP-labelled goat anti-mouse IgG (Dako, Glostrup, Denmark) diluted 1:30 000 in 5% BSA/TBST was used as secondary antibody. Signals were visualized with Roti-Lumin (Carl Roth, Karlsruhe, Germany). In case of immunoblot inhibition, sera were submitted to overnight incubation with 10 µg β-gal at 4°C before incubated with membrane-bound lactase. IgE and allergen-specific IgE analysis Total and allergen specific IgE were measured with UniCAP 100 (Thermo Fisher Scientific, Phadia GmbH, Freiburg, Germany) according to manufacturer’s instructions using either commercial ImmunoCAPs for alpha-amylase (k87), A. flavus (m228), aspergillus mix (mx4), or self-coupled ImmunoCAPs with lactase. Specific IgE values ≥ 0.35 kU/l were considered positive. As controls, the sera of a non-atopic donor and two Aspergillus-sensitized individuals (mx4 = Cap class 2 and 3) were analysed for lactase-s.-IgE antibodies. Inhibition tests were performed by preincubation the sera over night at 4°C with different concentrations of heat-inactivated β-gal prior to lactase-s.-IgE determination.
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ACCEPTED MANUSCRIPT Preparation of lactase ImmunoCAPs Lactase ImmunoCAPs were prepared by coupling biotinylated lactase to streptavidinImmunoCAPs (o212). Coupling followed basically previous protocols.13,14 In brief, biotinylation was performed using the EZ-Link® Sulfo-NHS-Biotinylation Kit (Pierce, Rockford, IL, USA). Lactase was incubated with biotin in 5-fold molar excess for 1 hour at room temperature. To
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remove excess biotin, desalting spin columns were used, equilibrated with PBS (pH 8). As the ImmunoCAP system is using a β-galactosidase for immuno-fluorescence detection, lactase was heat-inactivated for 10 min at 65°C to avoid false-positive signals. For coupling, 20 µg of biotinylated enzyme were added to a streptavidin-CAP and incubated for 30 min in the UniCAP100 instrument before performing specific IgE assays. Sequence analysis 10 µg of lactase were subjected to SDS-PAGE and bands of 65 and 90 kDa were excised. The proteins were digested in-gel by trypsin and the resulting peptide fragments were submitted to amino acid sequence analysis by MALDI-MS using a Waters Micromass MALDI micro MX (Waters, Milford, MA, USA). Mass spectrometric experiments were performed in the Integrated Functional Genomics (IFG), Core Unit of the Interdisciplinary Centre for Clinical Research (IZKF), Muenster. Data analysis Statistical calculations were performed using SPSS version 16.0 (SPSS Inc., Chicago, IL, USA). Association between lactase SPT reactivity and BAT reactivity as well as presence of lactase-s.-
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ACCEPTED MANUSCRIPT IgE antibodies detected by Western blotting were tested by Fisher’s exact test. All tests were two-sided. Results Characterization of study participants Thirteen employees (4 male and 9 female; age, 26 – 59 years; mean age, 41) of a lactase tablets
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producing plant were involved in this study. Eight of them worked in the production, 3 in the quality control and one each in the management or product development. Occupational lactase exposure was stated from 1 up to 9 years (mean: 4 ± 2 years). Ten employees reported to suffer from mild to severe work-related respiratory symptoms (rhinitis n = 6, conjunctivitis n = 5, sneeze n = 5, cough n = 2, shortness of breath n = 2, pruritus n = 2, oral allergy symptoms n = 1) due to lactase exposure (Table 1). The employee with oral allergy symptoms was engaged in the quality control and tasting of lactase tablets was part of the work process. Pulmonary function tests were normal for all employees with predicted FEV1 values > 80% of the normal value. Based on patient’s history and positive SPT results to one or more common aeroallergens, 8 employees were classified as atopic, whereas sensitization to birch pollen was most frequent. For case numbers 05, 09 and 10, atopy was confirmed by elevated total IgE levels > 100 kU/L (Table 2). A positive SPT to A. fumigatus was found in three cases (Table 1). Remarkably, the employee with case number 09 revealed as well significant s.-IgE levels to Aspergillus mix (0.69 kU/L), A. flavus (1.46 kU/L) and α-amylase (1.32 kU/L) (Table 2).
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ACCEPTED MANUSCRIPT Lactase sensitization In 8 patients suffering from work-related allergy symptoms, the SPT was positive; in 6 cases with 0.01% and in two cases with 1% lactase powder (Table 2). In the BAT, sensitization to lactase was confirmed in 7 of these individuals (Figure 1). One worker suffering from cough and pruritus (no. 12) had a positive BAT in spite of a negative prick test. Hence, it appears a
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significant correlation between a positive SPT and BAT result (P = 0.032; Table 3). Basophils of 3 non-atopic control donors revealed no activation by lactase stimulation (Data not shown). Seven of 9 lactase-sensitized workers were classified as atopic; but in case of the 4 nonsensitized workers, only one was classified as atopic. Lactase-specific IgE antibodies The banding pattern of the used lactase was visualized by Coomassie Brilliant Blue staining following SDS-PAGE (Figure 2a). We found 2 major protein bands with a molecular weight of approximately 65 and 90 kDa, respectively. The heat inactivated lactase used in the ImmunoCAP revealed a similar banding pattern with some minor differences in the range of < 50 kDa. A commercial β-galactosidase enzyme derived from A. oryzae (β-gal), however, showed no recognizable differences in its native or heat-inactivated form compared to native lactase. In order to ensure that the two distinct protein bands could be matched to A. oryzae-derived lactase, these were submitted to amino acid sequence analysis by MALDI-MS. The sequenced fragments of the 65-kDa-band could be assigned to the N-terminal half of the lactase amino acid sequence and the 90-kDa-band fragments to the C-terminal half (see online supplementary figure S1).
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ACCEPTED MANUSCRIPT The IgE-Immunoblot analyses of the 8 SPT-positive study participants are shown in Figure 2b. The strongest signals were detected for case numbers 01, 05 and 09. No significant signals of s.IgE binding were visible for the SPT-negative group. In the serum of the individual with a negative prick test in spite of a positive BAT (no. 12), there is a weak, but ambiguous signal for the 65-kDa-band. That implies a strong correlation between a positive lactase-SPT result and the
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presence of lactase-s.-IgE antibodies in serum (P < 0.001; Table 3). Quantitative lactase s.-IgE determination performed with self-coupled ImmunoCAPs verified significant IgE levels for 4 of the prick test positive employees (no. 01: 0.38 kU/L; no. 05: 0.41 kU/L; no. 07: 0.35 kU/L; no. 09: 3.73 kU/L; Table 2). No lactase-s.-IgE antibodies could be detected in the sera of a non-atopic donor and two Aspergillus-sensitized individuals neither by Western blot nor ImmunoCAP analysis (Data not shown). Inhibition of IgE-binding Preincubation with β-gal resulted in a significant reduction of IgE-binding to lactase analysed by ImmunoCAP and Western blot analysis, thus demonstrating the specificity of the antibodyantigen-complex formation. In case of the Western blot assay, a total inhibition was achieved for all lactase-sensitized workers like shown in Figure 3a for three individuals, who revealed the strongest IgE responses. Inhibition in the ImmunoCAP assay resulted in a dose-dependent reduction with a maximum of 73 – 92% (Figure 3b). Discussion
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ACCEPTED MANUSCRIPT About 200 fungal enzymes have been purified and characterized for their biochemical and catalytic properties until now. Fungal enzymes are widely used in the food, detergent and pharmaceutical industry, therefore a significant exposure to these enzymes is obvious especially in the occupational environment. Important allergens of the fungus A. oryzea are TAKA-amylase A, lipase and lactase.2
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In our study we demonstrated the sensitizing potential of Aspergillus-derived lactase within a group of 13 employees from a supplemental lactase tablets producing plant. On the basis of positive SPT and/or BAT, lactase sensitization was detected in 7 employees by both tests and in 2 by either one test. This reveals a good accordance between the two assays and supports the significance of BAT as a tool in the diagnostic procedures of diagnosing immediate type allergies. The test results were further confirmed by the detection of lactase-s.-IgE in serum samples of affected individuals. The qualitative Immunoblot assay seems to be more sensitive compared with the lactase ImmunoCAP analysis; lactase-s.-IgE antibodies were detected only in the serum of 4 sensitized workers using the ImmunoCAP. These individuals had the stronger sensitization to lactase, indicated by the higher susceptibility in SPT and BAT compared with employees without lactase-s.IgE antibodies in the ImmunoCAP system. The low ImmunoCAP sensitivity for lactase-s.-IgE antibodies may be on the other hand due to the assay-specific antigen preparation. The epitope’s conformation or accessibility of the lactase molecules responsible for IgE-binding may be altered due to heat-inactivation or biotinylation, resulting in decreased CAP values. Heating α-amylase for instance had been shown to reduce the allergenicity of the enzyme.16
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In previous studies, no significant relations were found between sensitization risk and duration of exposure, or exposure level.7,8 However, it is likely that high exposure to lactase increases the risk of sensitization like it was shown for α-amylase. Workers in high-exposure environment who handled α-amylase had a ten times higher risk developing sensitization than workers in the
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low-exposure category.17 It is mentioned that already concentrations in the low ng per m-3 range were associated with an increased frequency of sensitization.2 The dimension of dermal or respiratory exposure was not analysed in our study. Moreover, we can not specify the temporal exposure, since no information about how long and often each worker was handling lactase were present. But it is striking that almost all sensitized employees are engaged at least for 5 years in the plant. This was also true for workers 01, 05, and 09, in which the degree of sensitization was most prominent. These patients were between 26 and 37 years old and there is a broad age distribution in the sensitized group, thus the degree of sensitization to lactase seemed not to be influenced by age. Due to incomplete information about smoking habits and the tasks each worker performed within the plant, we cannot estimate the concrete influence of these factors relating to the degree of sensitization. However, we assume an occupational sensitization to lactase in all the cases because none of the employees reported to use lactase due to lactase intolerance. It is probable that sensitization was mediated via the respiratory system, but also the dermal exposure might be relevant for the induction of lactase-s.-IgE. All but one lactase-sensitized employee reported to suffer from work related inhalative symptoms, especially at the time when lactase was manufactured; this is in accordance with the
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ACCEPTED MANUSCRIPT literature.7-9 Allergic reactions due to the consumption of lactase, here swelling of the throat, reported one of our patients (no. 05). This person had a history of hay fever due to grass, weeds and birch pollen allergy, and suffered from food allergy to nuts, celery and fruits. If sensitization in this patient should be induced due to aerogen or oral exposure is not clear. In the literature, allergic swelling of the throat due to the ingestion of lactase tablets has been reported in a single case .18 The patient was not exposed to airborne lactase and the author proposed an inhalant
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Aspergillus allergy as the cause of sensitization. Sensitization to commercial Aspergillus extracts was found in only 2 of the lactase sensitized employees (no. 09 and 12). Both revealed weak skin prick test reactions to A. fumigatus and one of them had significant s.-IgE levels to Aspergillus-mix, A. flavus and even α-amylase (no. 09). It is worth mentioning relating to Aspergillus allergens that A. flavus is genetically almost identical to A. oryzae,4 this may explain the ImmunoCAP reactivity to Aspergillus-mix and A. flavus. By means of incubation the serum with 20 µg heat-inactivated lactase, IgE-binding to Aspergillusmix and α-amylase could be completely inhibited (< 0.01 k/UL) (Data not shown). This indicates rather a cross-reaction between lactase and α-amylase than a co-sensitization. A contamination of the lactase extract with other components of A. oryzea could be excluded by a native polyacrylamide gel electrophoresis, which revealed a single protein band for the enzyme extract (Data not shown). In a study of Muir and colleagues, only 6 of 42 lactase skin test positive individuals were further positive to Aspergillus.7 Our data suggest that lactase sensitization is not associated with a sensitization to Aspergillus or α-amylase.
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ACCEPTED MANUSCRIPT Most lactase allergic patients were atopic, which is in line with former observations.7,8 A similar correlation was also demonstrated for other occupational allergens like α-amylase,2 lipase and cellulase.19 The combination of atopy and a relevant lactase exposure seems be a main risk factor for the development of sensitization and allergy to this allergen. In conclusion, the results of our study demonstrates, that lactase has a relevant sensitizing
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potential. Occupational exposure to lactase powder can induce IgE-mediated sensitization leading to rhinoconjunctivits, asthma and even to oral allergy symptoms, especially in individuals with an atopic predisposition. Exposure should be minimised to reduce the risk for occupational sensitization and the development of allergic symptoms. In Germany suspicious work-related diseases have to be reported to the occupational insurance association, and may be financially compensated in the case of officially accepted occupational diseases. Our test results were reported to the responsible occupational health physician, and the results were discussed with the company to introduce protection measures against airborne lactase exposure. Regulations for the avoidance of lactase exposure are needed for an improved employment protection. Literature data and our results indicate that fungal lactase should be classified with the risk phrase 42 (may cause sensitization by inhalation) by the European Union Directive. Statement of funding No specific funding was given to finance this study. All investigations were performed as part of the regular procedure to diagnose potential allergy in occupationally exposed workers at the University Hospital in Münster, Germany.
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ACCEPTED MANUSCRIPT Acknowledgements The authors would like to thank MTA M. Behring and H. Stuckmann from the Department of
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Dermatology for performing the skin and lung function tests.
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ACCEPTED MANUSCRIPT References 1
Raulf-Heimsoth M, van Kampen V, Kespohl S, et al. Workplace-related respiratory allergies. Current developments. Bundesgesundheitsbl 2012;55:363-372.
2
Green BJ, Beezhold DH. Industrial fungal enzymes: an occupational allergen perspective. J Allergy Published Online First: 21 June 2011;doi:10.1155/2011/682574.
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3
Baur X. Enzymes as occupational and environmental respiratory sensitisers. Int Arch Occup Environ Health 2005;78:279-286.
4
Hedayati MT, Pasqualotto AC, Warn PA, et al. Aspergillus flavus: human pathogen, allergen and mycotoxin producer. Microbiology 2007;153:1677-1692.
5
Todorova-Balvay D, Stoilova I, Gargova S, et al. An efficient two step purification and molecular characterization of β-galactosidases from Aspergillus oryzae. J Mol Recognit 2006;19:299-304.
6
Pribila BA, Hertzler SR, Martin BR, et al. Improved lactose digestion and intolerance among African-American adolescent girls fed a dairy-rich diet. J Am Diet Assoc 2000;100(5):524-528.
7
Muir DCF, Verrall AB, Julian JA, et al. Occupational sensitization to lactase. Am J Ind Med 1997;31:570-571.
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Bernstein JA, Bernstein DI, Stauder T, et al. A cross-sectional survey of sensitization to Aspergillus oryzae-derived lactase in pharmaceutical workers. J Allergy Clin Immunol 1999;103:1153-1157.
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Laukkanen A, Ruoppi P, Remes S, et al. Lactase-induced occupational protein contact dermatitis and allergic rhinoconjunctivitis. Contact Dermatitis 2007;57:89-93.
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Mertens M, Amler S, Moerschbacher BM, et al. Cross-reactive carbohydrate determinants strongly affect the results of the basophil activation test in hymenopteravenom allergy. Clin Exp Allergy 2010;40:1333-1345.
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Kahlert H, Cromwell O, Fiebig H. Measurement of basophil-activating capacity of grass pollen allergens, allergoids and hypoallergenic recombinant derivates by flow cytometry using anti-CD203c. Clin Exp Allergy 2003;33:1266-1272.
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Erwin EA, Custis NJ, Satinover SM, et al. Quantitative measurement of IgE antibodies to purified allergens using streptavidin linked to a high-capacity solid phase. J Allergy Clin Immunol 2005;115:1029-1035.
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Baur X, Barbinova L. Occupational airborne exposure, specific sensitization and the atopic status: evidence of a complex interrelationship. J Occup Med Toxicol Published Online First: 13 Febuary 2013;doi:10.1186/1745-6673-8-2.
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Baur X, Czuppon AB, Sander I. Heating inactivates the enzymatic activity and partially inactivates the allergenic activity of Asp o 2. Clin Exp Allergy 1996;26(2):232-234.
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Nieuwenhuijsen MJ, Heederik D, Doekes G, et al. Exposure-response relations of αamylase sensitisation in British bakeries and flour mills. Occup Environ Med 1999;v.56(3):197-201.
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Binkley KE. Allergy to supplemental lactase enzyme. J Allergy Clin Immunol 1996;97:1414-1416.
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Table 1. Clinical characteristics of lactase exposed workers Case no.
Age
Smoki FEV1 ng pred. (%)
Job Exposure Workcatego to lactase related ry (years) symptoms
SPT common allergens
Atop y
01
37
Y
109.3
1
5
R, S, Co
G, W
Y
02
42
N
158.7
NI
1
SOB
N
03
43
NI
117.2
3
6
-
N
04
32
N
206.7
1
6
R, C, S
G, W, B
Y
05
26
NI
111.9
2
8
OAS
G, W, B
Y
06
45
N
109.1
1
2
R
N
07
37
N
109.8
2
6
R, C, S
N
08
52
NI
97.6
NI
1
R, P
G, W, B, D
Y
09
32
NI
118
1
5
R, C, S, SOB, Co
G, W, B, D, A
Y
10
59
NI
81.9
NI
6
-
D, A
Y
11
47
Y
123.3
2
NI
-
B
Y
12
42
Y
122.3
1
1
C, P
A
N
13
41
NI
129.2
4
9
C, S
B
Y
Job category: 1, production; 2, quality control; 3, production manager; 4, product development. Work-related symptoms: R, rhinitis; S, sneeze; Co, cough; SOB, shortness of breath; C, conjunctivitis; OAS, oral allergy symptoms; P, pruritus; -, no Symptoms. SPT common allergens: G, grass pollen; W, weed pollen; B, birch pollen; D, house dust mite; A, Aspergillus fumigatus. Atopy: Status was defined as positive, if the worker stated a known allergy and/or SPT was positive for at least one common allergen. FEV1, forced expiratory volume in 1 second; pred., predicted; SPT, skin prick test; Y, yes; N, no; NI, no information.
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Table 2. Results of lactase skin-prick testing and total and specific IgE measurements Specific IgE (kU/L)
SPT lactase (mm wheal diameter)
αamylase
A. flavus
Aspergillu s mix
0.38+
0.02
0.07
0.03
23.9
0.14
< 0.01
0.08
< 0.01
NP
114
0.41+
0.04
0.08
0.06
5*
NP
14.1
0.35+
0.06
0.09
0.03
3*
4*
NP
17.9
0.01
< 0.01
0.06
0.03
09
6*
NP
NP
125
3.73+
1.32+
1.46+
0.69+
11
-
2
4*
8.17
0.12
0.01
0.03
0.04
13
2
2
3*
58.9
0.07
< 0.01
0.02
0.04
02
-
-
-
22.9
0.01
< 0.01
0.07
0.02
03
-
-
-
28
0.01
< 0.01
0.07
0.03
06
-
-
-
2.95
0.01
< 0.01
0.02
0.04
10
-
-
-
306
0.04
< 0.01
0.05
0.05
12
-
-
-
55.4
0.09
< 0.01
0.04
0.28
Case no.
0.01 %
0.1 %
1%
01
4*
NP
04
3*
05
Total IgE
Lactase
NP
43.7
4*
NP
5*
NP
07
3*
08
a
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Lactase SPT positive
Lactase SPT negative
*A wheal diameter of at least 3 mm was considered positive when there was no reaction with + the negative control. Values ≥ 0.35 kU/l were defined as positive. # Lactase used for in-house-made ImmunoCAPs was heat inactivated at 65°C for 10 minutes. NP, not performed; -; no wheal.
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Table 3. Association between lactase SPT reactivity and BAT reactivity and presence of lactase-s.-IgE antibodies, respectively Lactase SPT reactivity +
-
P value
+
7
1
.032*
-
1
4
+
8
0
-
0
5
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Lactase BAT reactivity
Lactase-s.-IgE .001**
* significant (P < 0.05), ** highly significant (P < 0.01)
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Figure 1
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Figure 2
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Figure 3
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Archives of Environmental & Occupational Health Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/vaeh20
Occupational sensitization to lactase in the dietary supplement industry a
a
a
b
Benedikt Stöcker , Sonja Grundmann , Pia Mosters , Paul Nitzsche & Randolf Brehler a
a
Department of Dermatology, University Hospital Münster, Münster, Germany
b
Werkarztzentrum Rietberg e.V., Rietberg, Germany Accepted author version posted online: 02 Jul 2015.
Click for updates To cite this article: Benedikt Stöcker, Sonja Grundmann, Pia Mosters, Paul Nitzsche & Randolf Brehler (2015): Occupational sensitization to lactase in the dietary supplement industry, Archives of Environmental & Occupational Health, DOI: 10.1080/19338244.2015.1066294 To link to this article: http://dx.doi.org/10.1080/19338244.2015.1066294
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ACCEPTED MANUSCRIPT Occupational sensitization to lactase in the dietary supplement industry Benedikt Stöckera, Sonja Grundmanna, Pia Mostersa, Paul Nitzscheb, Randolf Brehlera
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Department of Dermatology, University Hospital Münster, Münster, Germany
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Werkarztzentrum Rietberg e.V., Rietberg, Germany
Correspondence: Benedikt Stöcker University Hospital Münster Department of Dermatology Von-Esmarch-Str. 58 48149 Münster Germany E-mail: [email protected] Abstract Aerogen lactase exposure carries a risk for the development of allergic asthma and rhinitis; only a few occupationally affected patients have been reported. We report the results of allergy testing with employees of a lactase tablets manufacturing plant.
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ACCEPTED MANUSCRIPT The survey involved 13 workers, including a questionnaire, spirometry, basophil activation test (BAT), and skin prick tests (SPT) with lactase and a panel of common aeroallergens. Furthermore, lactase-specific IgE (s.-IgE) antibodies were analysed. Sensitization to lactase could be proven for 9 workers by SPT and BAT; s.-IgE antibodies could be detected in serum samples of all sensitized. However, IgE levels ≥ 0.35 kU/l were only found
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in 4 sera. Our data confirm that occupational exposure to lactase can induce IgE-mediated respiratory sensitization resulting in allergic diseases. Protective measures should thus be obligatory when working with lactase. Keywords: Aspergillus oryzae, enzyme allergy, lactase, occupational, inhalative allergy
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ACCEPTED MANUSCRIPT Introduction So far, more than 250 substances are described as respiratory allergens in the modern working environment causing allergic rhinitis and conjunctivitis, allergic asthma and contact urticaria due to IgE-mediated sensitization. Those allergens are mostly high-molecular proteins (molecular weight > 5 kDa).1,2 Although exposure minimization or avoidance is a dictum in several
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industries since the 1970s, occupational allergies are a significant problem causing high cost, especially if disability results.1,3 Fungal enzymes, particularly derived from the genus Aspergillus, are important occupational immediate type allergens, commonly used in baking, food, detergent, textile, and pharmaceutical industries.2 The species Aspergillus oryzae is a domesticated fungus, having been used in China and Japan for soy fermentation for thousands of years4 and is the most important resource for the industrial production of α-amylase. A. oryzae-derived α-amylase is the best characterized fungal enzyme used in the working environment, and is the major cause of baker’s asthma with the sensitization prevalence up to 34%.1,2 Another A. oryzae-derived enzyme is lactase, a member of the β-galactosidase family, which is applied in the pharmaceutical and food processing industry.2 Extracellular β-galactosidases from A. oryzae have advantages over the enzyme from other taxonomic groups for industrial applications, in terms of easier isolation from the culture supernatant, higher thermal tolerance and optimum activity at lower pH significantly avoids contamination.5 Due to its disaccharidase activity, lactase hydrolyses lactose into galactose and glucose monomers. Lactose intolerance due to lactase deficiency is widespread especially in African and Asian countries and effects
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ACCEPTED MANUSCRIPT about 75% of the adult world population.6 Clinical symptoms are mainly flatulence, diarrhoea, and abdominal pain due to the consumption of lactose contained in milk products. A supplemental treatment with lactase improves the tolerability of lactose in those patients. Occupational lactase sensitization has been reported in 65 of 207 tested workers of a plant engaged in formulating and packing lactase containing products.7 In a cross-sectional survey of
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94 pharmaceutical workers exposed to lactase, 27 had positive skin tests with lactase.8 In both studies the diagnosis of sensitization based on positive skin prick tests, and an elevated risk for respiratory allergy symptoms was related to sensitization. Furthermore, Laukkanen et al. demonstrated in a single case report a non-atopic pharmaceutical worker suffering from allergic rhinitis, contact urticaria and eczema due to lactase exposure. The diagnosis of an IgE-mediated sensitization was confirmed by skin tests in combination with lactase-specific IgE antibodies.9 Here, we report the results of allergy testing of 13 employees of a plant producing and packing lactase tablets. Methods Study population Thirteen employees of a plant producing coated and uncoated chewable lactase tablets from raw enzyme powder obtained from A. oryzae were sent by the corresponding occupational physician for allergy diagnostics. In the plant lactase was processed several times a year and 10 of these workers suffered from work-related allergic symptoms during these periods. The manufacturing process involves weighing of enzyme powder, mixing it with additives, pressing it to tablets,
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ACCEPTED MANUSCRIPT performing a quality control, and finally packaging. All employees were exposed to airborne lactase during this period, even if they are not handling raw lactase powder like employees engaged directly in the production process or in the quality control unit. Since respiratory or skin protection was not obligatory, the employees had direct contact to lactose by the dermis and respiratory system. Quantitative measurements of the airborne exposure were not performed.
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Questionnaire Patient’s history pertaining employment, atopic diseases and work related symptoms were documented by use of a questionnaire. Lung function test Spirometry and IOS Impulse Spirometry was performed using a Master Screen IOS (CareFusion, Hoechberg, Germany). Skin tests Lactase powder, consisting of native enzyme concentrate (66 - 71%), carmellose calcium and glucose, was kindly provided by the plant and was used for all experimental assays. SPTs were performed with 10-fold serial dilutions of lactase in 0.9% NaCl solution, starting with a concentration of 0.1 µg/ml and increasing every 10 minutes until a positive reaction appeared or the maximum concentration of 10 µg/ml was attained. Furthermore, SPTs were conducted with Aspergillus fumigatus (ALK-prick SQ, ALK-Scherax, Wedel, Germany) and common aeroallergens (house dust mite extracts (D. pteronissynus, D. farinae), birch pollen, weed pollen(Allergopharma, Reinbek, Germany) and grass pollen extracts (ALK, Wedel, Germany)).
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ACCEPTED MANUSCRIPT 0.9% NaCl solution served as negative control and 10 mg/ml histamine as positive control. A wheal diameter of ≥ 3 mm was considered positive. Basophil activation test BAT was performed as described by Mertens et al..10,11 In brief, heparinized whole blood was incubated for 25 min at 37°C with 100-fold serial dilutions of lactase in PBS ranging from 0.001
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to 10 µg/ml. To confirm cell responsiveness, 1 µg/ml of a monoclonal anti-IgE antibody (clone BE5; EurobioSciences, Friesoythe, Germany) served as positive control. After cell stimulation, the reaction was stopped by adding 20 mM EDTA in PBS and centrifugation at 400 g. Basophils were stained with 10 µl anti-CD203c-PE (Beckman-Coulter, Krefeld, Germany) for 45 min at room temperature in the dark. Using the whole blood lysing reagent (Beckman-Coulter), erythrocytes were lysed according to the manufacturer’s instructions. After washing and resuspending the cells in PBS with 1% BSA, a total of 60 000 cells were measured using the FACS Calibur flow cytometer equipped with CellQuestPro software (BD Biosciences). The percentage of activated basophils was calculated by subtracting spontaneous CD203c expression (negative control, PBS) from the data obtained with anti-IgE and lactase stimulation; values < 0 were set = 0. The result was considered positive when expression of CD203c was upregulated about more than 10% in relation to the negative control. The specificity of the BAT with lactase was proven using basophils of three non-atopic control donors. SDS-PAGE and Immunoblot analyses
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ACCEPTED MANUSCRIPT For gel separation of enzymes, 10 µg lactase or β-galactosidase (β-gal) (Sigma-Aldrich, Taufkirchen, Germany) from A. oryzea were applied to 12% SDS PAGE12 and stained with Coomassie Brilliant Blue (Sigma-Aldrich). Lactase was also transferred onto Immobilon-P Transfer membranes (Millipore, Billerica, MA, USA). The membrane was saturated with 2% skimmed milk powder in Tris-buffered saline (TBS) with 0.05% Tween-20 (TBST) over night and incubated with patient sera diluted 1:2 in 2% skimmed milk/TBST for 3 hours. Mouse anti-
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human IgE antibody (clone BE5; EurobioSciences, Friesoythe, Germany) diluted 1:1000 in 1% BSA/TBST was used for detection of s.-IgE antibody binding. HRP-labelled goat anti-mouse IgG (Dako, Glostrup, Denmark) diluted 1:30 000 in 5% BSA/TBST was used as secondary antibody. Signals were visualized with Roti-Lumin (Carl Roth, Karlsruhe, Germany). In case of immunoblot inhibition, sera were submitted to overnight incubation with 10 µg β-gal at 4°C before incubated with membrane-bound lactase. IgE and allergen-specific IgE analysis Total and allergen specific IgE were measured with UniCAP 100 (Thermo Fisher Scientific, Phadia GmbH, Freiburg, Germany) according to manufacturer’s instructions using either commercial ImmunoCAPs for alpha-amylase (k87), A. flavus (m228), aspergillus mix (mx4), or self-coupled ImmunoCAPs with lactase. Specific IgE values ≥ 0.35 kU/l were considered positive. As controls, the sera of a non-atopic donor and two Aspergillus-sensitized individuals (mx4 = Cap class 2 and 3) were analysed for lactase-s.-IgE antibodies. Inhibition tests were performed by preincubation the sera over night at 4°C with different concentrations of heat-inactivated β-gal prior to lactase-s.-IgE determination.
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ACCEPTED MANUSCRIPT Preparation of lactase ImmunoCAPs Lactase ImmunoCAPs were prepared by coupling biotinylated lactase to streptavidinImmunoCAPs (o212). Coupling followed basically previous protocols.13,14 In brief, biotinylation was performed using the EZ-Link® Sulfo-NHS-Biotinylation Kit (Pierce, Rockford, IL, USA). Lactase was incubated with biotin in 5-fold molar excess for 1 hour at room temperature. To
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remove excess biotin, desalting spin columns were used, equilibrated with PBS (pH 8). As the ImmunoCAP system is using a β-galactosidase for immuno-fluorescence detection, lactase was heat-inactivated for 10 min at 65°C to avoid false-positive signals. For coupling, 20 µg of biotinylated enzyme were added to a streptavidin-CAP and incubated for 30 min in the UniCAP100 instrument before performing specific IgE assays. Sequence analysis 10 µg of lactase were subjected to SDS-PAGE and bands of 65 and 90 kDa were excised. The proteins were digested in-gel by trypsin and the resulting peptide fragments were submitted to amino acid sequence analysis by MALDI-MS using a Waters Micromass MALDI micro MX (Waters, Milford, MA, USA). Mass spectrometric experiments were performed in the Integrated Functional Genomics (IFG), Core Unit of the Interdisciplinary Centre for Clinical Research (IZKF), Muenster. Data analysis Statistical calculations were performed using SPSS version 16.0 (SPSS Inc., Chicago, IL, USA). Association between lactase SPT reactivity and BAT reactivity as well as presence of lactase-s.-
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ACCEPTED MANUSCRIPT IgE antibodies detected by Western blotting were tested by Fisher’s exact test. All tests were two-sided. Results Characterization of study participants Thirteen employees (4 male and 9 female; age, 26 – 59 years; mean age, 41) of a lactase tablets
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producing plant were involved in this study. Eight of them worked in the production, 3 in the quality control and one each in the management or product development. Occupational lactase exposure was stated from 1 up to 9 years (mean: 4 ± 2 years). Ten employees reported to suffer from mild to severe work-related respiratory symptoms (rhinitis n = 6, conjunctivitis n = 5, sneeze n = 5, cough n = 2, shortness of breath n = 2, pruritus n = 2, oral allergy symptoms n = 1) due to lactase exposure (Table 1). The employee with oral allergy symptoms was engaged in the quality control and tasting of lactase tablets was part of the work process. Pulmonary function tests were normal for all employees with predicted FEV1 values > 80% of the normal value. Based on patient’s history and positive SPT results to one or more common aeroallergens, 8 employees were classified as atopic, whereas sensitization to birch pollen was most frequent. For case numbers 05, 09 and 10, atopy was confirmed by elevated total IgE levels > 100 kU/L (Table 2). A positive SPT to A. fumigatus was found in three cases (Table 1). Remarkably, the employee with case number 09 revealed as well significant s.-IgE levels to Aspergillus mix (0.69 kU/L), A. flavus (1.46 kU/L) and α-amylase (1.32 kU/L) (Table 2).
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ACCEPTED MANUSCRIPT Lactase sensitization In 8 patients suffering from work-related allergy symptoms, the SPT was positive; in 6 cases with 0.01% and in two cases with 1% lactase powder (Table 2). In the BAT, sensitization to lactase was confirmed in 7 of these individuals (Figure 1). One worker suffering from cough and pruritus (no. 12) had a positive BAT in spite of a negative prick test. Hence, it appears a
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significant correlation between a positive SPT and BAT result (P = 0.032; Table 3). Basophils of 3 non-atopic control donors revealed no activation by lactase stimulation (Data not shown). Seven of 9 lactase-sensitized workers were classified as atopic; but in case of the 4 nonsensitized workers, only one was classified as atopic. Lactase-specific IgE antibodies The banding pattern of the used lactase was visualized by Coomassie Brilliant Blue staining following SDS-PAGE (Figure 2a). We found 2 major protein bands with a molecular weight of approximately 65 and 90 kDa, respectively. The heat inactivated lactase used in the ImmunoCAP revealed a similar banding pattern with some minor differences in the range of < 50 kDa. A commercial β-galactosidase enzyme derived from A. oryzae (β-gal), however, showed no recognizable differences in its native or heat-inactivated form compared to native lactase. In order to ensure that the two distinct protein bands could be matched to A. oryzae-derived lactase, these were submitted to amino acid sequence analysis by MALDI-MS. The sequenced fragments of the 65-kDa-band could be assigned to the N-terminal half of the lactase amino acid sequence and the 90-kDa-band fragments to the C-terminal half (see online supplementary figure S1).
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ACCEPTED MANUSCRIPT The IgE-Immunoblot analyses of the 8 SPT-positive study participants are shown in Figure 2b. The strongest signals were detected for case numbers 01, 05 and 09. No significant signals of s.IgE binding were visible for the SPT-negative group. In the serum of the individual with a negative prick test in spite of a positive BAT (no. 12), there is a weak, but ambiguous signal for the 65-kDa-band. That implies a strong correlation between a positive lactase-SPT result and the
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presence of lactase-s.-IgE antibodies in serum (P < 0.001; Table 3). Quantitative lactase s.-IgE determination performed with self-coupled ImmunoCAPs verified significant IgE levels for 4 of the prick test positive employees (no. 01: 0.38 kU/L; no. 05: 0.41 kU/L; no. 07: 0.35 kU/L; no. 09: 3.73 kU/L; Table 2). No lactase-s.-IgE antibodies could be detected in the sera of a non-atopic donor and two Aspergillus-sensitized individuals neither by Western blot nor ImmunoCAP analysis (Data not shown). Inhibition of IgE-binding Preincubation with β-gal resulted in a significant reduction of IgE-binding to lactase analysed by ImmunoCAP and Western blot analysis, thus demonstrating the specificity of the antibodyantigen-complex formation. In case of the Western blot assay, a total inhibition was achieved for all lactase-sensitized workers like shown in Figure 3a for three individuals, who revealed the strongest IgE responses. Inhibition in the ImmunoCAP assay resulted in a dose-dependent reduction with a maximum of 73 – 92% (Figure 3b). Discussion
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ACCEPTED MANUSCRIPT About 200 fungal enzymes have been purified and characterized for their biochemical and catalytic properties until now. Fungal enzymes are widely used in the food, detergent and pharmaceutical industry, therefore a significant exposure to these enzymes is obvious especially in the occupational environment. Important allergens of the fungus A. oryzea are TAKA-amylase A, lipase and lactase.2
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In our study we demonstrated the sensitizing potential of Aspergillus-derived lactase within a group of 13 employees from a supplemental lactase tablets producing plant. On the basis of positive SPT and/or BAT, lactase sensitization was detected in 7 employees by both tests and in 2 by either one test. This reveals a good accordance between the two assays and supports the significance of BAT as a tool in the diagnostic procedures of diagnosing immediate type allergies. The test results were further confirmed by the detection of lactase-s.-IgE in serum samples of affected individuals. The qualitative Immunoblot assay seems to be more sensitive compared with the lactase ImmunoCAP analysis; lactase-s.-IgE antibodies were detected only in the serum of 4 sensitized workers using the ImmunoCAP. These individuals had the stronger sensitization to lactase, indicated by the higher susceptibility in SPT and BAT compared with employees without lactase-s.IgE antibodies in the ImmunoCAP system. The low ImmunoCAP sensitivity for lactase-s.-IgE antibodies may be on the other hand due to the assay-specific antigen preparation. The epitope’s conformation or accessibility of the lactase molecules responsible for IgE-binding may be altered due to heat-inactivation or biotinylation, resulting in decreased CAP values. Heating α-amylase for instance had been shown to reduce the allergenicity of the enzyme.16
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In previous studies, no significant relations were found between sensitization risk and duration of exposure, or exposure level.7,8 However, it is likely that high exposure to lactase increases the risk of sensitization like it was shown for α-amylase. Workers in high-exposure environment who handled α-amylase had a ten times higher risk developing sensitization than workers in the
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low-exposure category.17 It is mentioned that already concentrations in the low ng per m-3 range were associated with an increased frequency of sensitization.2 The dimension of dermal or respiratory exposure was not analysed in our study. Moreover, we can not specify the temporal exposure, since no information about how long and often each worker was handling lactase were present. But it is striking that almost all sensitized employees are engaged at least for 5 years in the plant. This was also true for workers 01, 05, and 09, in which the degree of sensitization was most prominent. These patients were between 26 and 37 years old and there is a broad age distribution in the sensitized group, thus the degree of sensitization to lactase seemed not to be influenced by age. Due to incomplete information about smoking habits and the tasks each worker performed within the plant, we cannot estimate the concrete influence of these factors relating to the degree of sensitization. However, we assume an occupational sensitization to lactase in all the cases because none of the employees reported to use lactase due to lactase intolerance. It is probable that sensitization was mediated via the respiratory system, but also the dermal exposure might be relevant for the induction of lactase-s.-IgE. All but one lactase-sensitized employee reported to suffer from work related inhalative symptoms, especially at the time when lactase was manufactured; this is in accordance with the
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ACCEPTED MANUSCRIPT literature.7-9 Allergic reactions due to the consumption of lactase, here swelling of the throat, reported one of our patients (no. 05). This person had a history of hay fever due to grass, weeds and birch pollen allergy, and suffered from food allergy to nuts, celery and fruits. If sensitization in this patient should be induced due to aerogen or oral exposure is not clear. In the literature, allergic swelling of the throat due to the ingestion of lactase tablets has been reported in a single case .18 The patient was not exposed to airborne lactase and the author proposed an inhalant
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Aspergillus allergy as the cause of sensitization. Sensitization to commercial Aspergillus extracts was found in only 2 of the lactase sensitized employees (no. 09 and 12). Both revealed weak skin prick test reactions to A. fumigatus and one of them had significant s.-IgE levels to Aspergillus-mix, A. flavus and even α-amylase (no. 09). It is worth mentioning relating to Aspergillus allergens that A. flavus is genetically almost identical to A. oryzae,4 this may explain the ImmunoCAP reactivity to Aspergillus-mix and A. flavus. By means of incubation the serum with 20 µg heat-inactivated lactase, IgE-binding to Aspergillusmix and α-amylase could be completely inhibited (< 0.01 k/UL) (Data not shown). This indicates rather a cross-reaction between lactase and α-amylase than a co-sensitization. A contamination of the lactase extract with other components of A. oryzea could be excluded by a native polyacrylamide gel electrophoresis, which revealed a single protein band for the enzyme extract (Data not shown). In a study of Muir and colleagues, only 6 of 42 lactase skin test positive individuals were further positive to Aspergillus.7 Our data suggest that lactase sensitization is not associated with a sensitization to Aspergillus or α-amylase.
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ACCEPTED MANUSCRIPT Most lactase allergic patients were atopic, which is in line with former observations.7,8 A similar correlation was also demonstrated for other occupational allergens like α-amylase,2 lipase and cellulase.19 The combination of atopy and a relevant lactase exposure seems be a main risk factor for the development of sensitization and allergy to this allergen. In conclusion, the results of our study demonstrates, that lactase has a relevant sensitizing
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potential. Occupational exposure to lactase powder can induce IgE-mediated sensitization leading to rhinoconjunctivits, asthma and even to oral allergy symptoms, especially in individuals with an atopic predisposition. Exposure should be minimised to reduce the risk for occupational sensitization and the development of allergic symptoms. In Germany suspicious work-related diseases have to be reported to the occupational insurance association, and may be financially compensated in the case of officially accepted occupational diseases. Our test results were reported to the responsible occupational health physician, and the results were discussed with the company to introduce protection measures against airborne lactase exposure. Regulations for the avoidance of lactase exposure are needed for an improved employment protection. Literature data and our results indicate that fungal lactase should be classified with the risk phrase 42 (may cause sensitization by inhalation) by the European Union Directive. Statement of funding No specific funding was given to finance this study. All investigations were performed as part of the regular procedure to diagnose potential allergy in occupationally exposed workers at the University Hospital in Münster, Germany.
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ACCEPTED MANUSCRIPT Acknowledgements The authors would like to thank MTA M. Behring and H. Stuckmann from the Department of
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Dermatology for performing the skin and lung function tests.
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ACCEPTED MANUSCRIPT References 1
Raulf-Heimsoth M, van Kampen V, Kespohl S, et al. Workplace-related respiratory allergies. Current developments. Bundesgesundheitsbl 2012;55:363-372.
2
Green BJ, Beezhold DH. Industrial fungal enzymes: an occupational allergen perspective. J Allergy Published Online First: 21 June 2011;doi:10.1155/2011/682574.
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3
Baur X. Enzymes as occupational and environmental respiratory sensitisers. Int Arch Occup Environ Health 2005;78:279-286.
4
Hedayati MT, Pasqualotto AC, Warn PA, et al. Aspergillus flavus: human pathogen, allergen and mycotoxin producer. Microbiology 2007;153:1677-1692.
5
Todorova-Balvay D, Stoilova I, Gargova S, et al. An efficient two step purification and molecular characterization of β-galactosidases from Aspergillus oryzae. J Mol Recognit 2006;19:299-304.
6
Pribila BA, Hertzler SR, Martin BR, et al. Improved lactose digestion and intolerance among African-American adolescent girls fed a dairy-rich diet. J Am Diet Assoc 2000;100(5):524-528.
7
Muir DCF, Verrall AB, Julian JA, et al. Occupational sensitization to lactase. Am J Ind Med 1997;31:570-571.
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Bernstein JA, Bernstein DI, Stauder T, et al. A cross-sectional survey of sensitization to Aspergillus oryzae-derived lactase in pharmaceutical workers. J Allergy Clin Immunol 1999;103:1153-1157.
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Laukkanen A, Ruoppi P, Remes S, et al. Lactase-induced occupational protein contact dermatitis and allergic rhinoconjunctivitis. Contact Dermatitis 2007;57:89-93.
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10
Mertens M, Amler S, Moerschbacher BM, et al. Cross-reactive carbohydrate determinants strongly affect the results of the basophil activation test in hymenopteravenom allergy. Clin Exp Allergy 2010;40:1333-1345.
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Baur X, Barbinova L. Occupational airborne exposure, specific sensitization and the atopic status: evidence of a complex interrelationship. J Occup Med Toxicol Published Online First: 13 Febuary 2013;doi:10.1186/1745-6673-8-2.
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Table 1. Clinical characteristics of lactase exposed workers Case no.
Age
Smoki FEV1 ng pred. (%)
Job Exposure Workcatego to lactase related ry (years) symptoms
SPT common allergens
Atop y
01
37
Y
109.3
1
5
R, S, Co
G, W
Y
02
42
N
158.7
NI
1
SOB
N
03
43
NI
117.2
3
6
-
N
04
32
N
206.7
1
6
R, C, S
G, W, B
Y
05
26
NI
111.9
2
8
OAS
G, W, B
Y
06
45
N
109.1
1
2
R
N
07
37
N
109.8
2
6
R, C, S
N
08
52
NI
97.6
NI
1
R, P
G, W, B, D
Y
09
32
NI
118
1
5
R, C, S, SOB, Co
G, W, B, D, A
Y
10
59
NI
81.9
NI
6
-
D, A
Y
11
47
Y
123.3
2
NI
-
B
Y
12
42
Y
122.3
1
1
C, P
A
N
13
41
NI
129.2
4
9
C, S
B
Y
Job category: 1, production; 2, quality control; 3, production manager; 4, product development. Work-related symptoms: R, rhinitis; S, sneeze; Co, cough; SOB, shortness of breath; C, conjunctivitis; OAS, oral allergy symptoms; P, pruritus; -, no Symptoms. SPT common allergens: G, grass pollen; W, weed pollen; B, birch pollen; D, house dust mite; A, Aspergillus fumigatus. Atopy: Status was defined as positive, if the worker stated a known allergy and/or SPT was positive for at least one common allergen. FEV1, forced expiratory volume in 1 second; pred., predicted; SPT, skin prick test; Y, yes; N, no; NI, no information.
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Table 2. Results of lactase skin-prick testing and total and specific IgE measurements Specific IgE (kU/L)
SPT lactase (mm wheal diameter)
αamylase
A. flavus
Aspergillu s mix
0.38+
0.02
0.07
0.03
23.9
0.14
< 0.01
0.08
< 0.01
NP
114
0.41+
0.04
0.08
0.06
5*
NP
14.1
0.35+
0.06
0.09
0.03
3*
4*
NP
17.9
0.01
< 0.01
0.06
0.03
09
6*
NP
NP
125
3.73+
1.32+
1.46+
0.69+
11
-
2
4*
8.17
0.12
0.01
0.03
0.04
13
2
2
3*
58.9
0.07
< 0.01
0.02
0.04
02
-
-
-
22.9
0.01
< 0.01
0.07
0.02
03
-
-
-
28
0.01
< 0.01
0.07
0.03
06
-
-
-
2.95
0.01
< 0.01
0.02
0.04
10
-
-
-
306
0.04
< 0.01
0.05
0.05
12
-
-
-
55.4
0.09
< 0.01
0.04
0.28
Case no.
0.01 %
0.1 %
1%
01
4*
NP
04
3*
05
Total IgE
Lactase
NP
43.7
4*
NP
5*
NP
07
3*
08
a
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Lactase SPT positive
Lactase SPT negative
*A wheal diameter of at least 3 mm was considered positive when there was no reaction with + the negative control. Values ≥ 0.35 kU/l were defined as positive. # Lactase used for in-house-made ImmunoCAPs was heat inactivated at 65°C for 10 minutes. NP, not performed; -; no wheal.
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Table 3. Association between lactase SPT reactivity and BAT reactivity and presence of lactase-s.-IgE antibodies, respectively Lactase SPT reactivity +
-
P value
+
7
1
.032*
-
1
4
+
8
0
-
0
5
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Lactase BAT reactivity
Lactase-s.-IgE .001**
* significant (P < 0.05), ** highly significant (P < 0.01)
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Figure 1
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Figure 2
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Figure 3
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