Allergology International (2002) 51: 121–129
`
`Original Article
`Anaphylaxis caused by the ingestion of cultivated
`mushroom (Agaricus bisporus): Identification of allergen
`as mannitol
`
`Venkatesh L Hegde, Jharna R Das and Yeldur P Venkatesh
`Department of Biochemistry and Nutrition, Central Food Technological Research Institute (CFTRI),
`Mysore, India
`
`ABSTRACT
`Background: The role of mushroom spores as
`inhalants in causing respiratory allergy has been well
`established. Although mushrooms are commonly used
`as food throughout the world, food allergy to mush-
`rooms is not very common. A severe case of anaphy-
`laxis in a 32-year-old woman who experienced facial
`edema and generalized urticaria minutes after eating
`mushroom curry is presented herein. The purpose of
`the present study was to identify the putative allergen
`in the cultivated mushroom Agaricus bisporus.
`Methods: A combination of biochemical fractionation/
`analytical techniques (gel filtration, ultrafiltration,
`ion-moderated cation-exchange chromatography,
`high-pressure
`liquid chromatography and gas
`chromatography–mass spectrometry (GC-MS)) and
`allergy diagnostic tests (skin prick test (SPT), allergen-
`specific IgE) were used.
`Results: The SPT with mushroom extract was strongly
`positive; however, allergen-specific IgE could not be
`detected by enzyme-linked immunosorbent assay. The
`SPT was also positive with cooked, steamed or dried
`mushroom extracts, suggesting the presence of a heat-
`stable allergen. Gel filtration of mushroom extract
`on Sephadex G-25, as analyzed by SPT, indicated
`the presence of a low molecular weight (< 1 kDa)
`
`Correspondence: Dr Yeldur P Venkatesh, Department of
`Biochemistry and Nutrition, Central Food Technological
`Research Institute (CFTRI), Mysore 570013, India.
`Email: ypv53@yahoo.co.in
`Received 19 July 2001. Accepted for publication 30 January
`2002.
`
`allergen. Using ion-moderated cation-exchange chrom-
`atography, the allergen was isolated and identified as
`mannitol based on skin reactivity. Mannitol was con-
`firmed by GC-MS analysis.
`Conclusions: This is the first report of food allergy to
`cultivated mushroom A. bisporus and also the first
`report describing a low molecular weight allergen
`(mannitol) in mushroom.
`
`Key words: Agaricus bisporus, anaphylaxis, cultivated
`mushroom, food allergy, low molecular weight aller-
`gen, mannitol.
`
`INTRODUCTION
`Mushrooms, although belonging to fungi, are commonly
`used as a vegetable in many parts of the world. The
`overall extent of mushroom allergy is not known; it may
`be very slight due to ingestion (1%), but could be as
`prevalent as pollen and mold allergy (10–30% of an
`allergic population).1 The importance of fungal spores in
`causing airborne respiratory allergies has been well
`established.2 Although edible mushrooms from the class
`Basidiomycetes are widely consumed as food throughout
`the world, food allergies caused by these mushrooms
`have not been reported, except for a couple of reports
`describing ingestive allergy to the common edible mush-
`room (Boletus edulis).3,4
`We report herein an interesting case of anaphylaxis
`caused by the ingestion of cultivated mushroom (Agaricus
`bisporus) in a 32-year-old woman who also had
`repeated history of anaphylaxis to the ingestion of pome-
`granate fruit. We had shown earlier that the allergen
`in pomegranate responsible for causing anaphylaxis in
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`122
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`VL HEGDE ET AL.
`
`this severely allergic individual was a small molecule
`(mannitol).5 Agaricus bisporus, popularly known as ‘white
`button mushroom’, is the major cultivated edible mush-
`room of economic importance.6 The aim of the present
`investigation was to identify the allergen in A. bisporus
`that was responsible for causing anaphylaxis. Attempts
`were made to determine the nature of the putative aller-
`gen by a combination of biochemical/analytical tech-
`niques involving molecular fractionation (ultrafiltration,
`gel filtration, ion-moderated cation-exchange chro-
`matography and high-pressure liquid chromatography
`(HPLC)), and allergy diagnostic tests (skin prick test (SPT),
`allergen-specific IgE by enzyme-linked immunosorbent
`assay (ELISA)). Gas chromatography–mass spectrometry
`(GC-MS) analysis was used to provide direct evidence for
`the identification of the allergen.
`
`METHODS
`Case history
`
`A 32-year-old woman was evaluated in the allergy clinic
`for food allergy. She exhibited swelling and redness of the
`face (facial edema), severe skin rashes all over the body
`(generalized urticaria) and breathing difficulties within
`approximately 5 min after eating mushroom curry. The
`subject recollected having similar episodes to ingestion
`of mushroom curry on three to four previous occasions.
`She also had repeated history of generalized urticaria
`and angioedema to pomegranate fruit, sometimes
`needing emergency treatment due to giddiness and
`unconsciousness. On a few occasions, she had also
`experienced anaphylactic shock upon consuming cake
`icing and a chewable tablet (Cisapid MPS; Kopran,
`Mumbai, India; active ingredients cisapride, a peristaltic
`stimulant, and methyl polysiloxane, an antiflatulent). The
`patient was able to consume other fruits and vegetables
`without any adverse reactions. Informed consent was
`obtained from the subjects in the present study and diag-
`nostic tests were performed following approval by the
`Institutional Ethics Committee.
`
`Preparation of mushroom extract and
`3K-filtrate
`
`A 50% (w/v) aqueous extract of white button mushroom
`(A. bisporus; Premier Mushroom Farms, Secunderabad,
`India) was prepared by crushing the mushrooms in a
`blender for 5 min and filtering them through Whatman
`(Maidstone, England) 1 filter paper (mushroom extract).
`
`Mushroom 3K-filtrate was obtained by subjecting mush-
`room extract to ultrafiltration in an Amicon stirred cell
`using DIAFLO YM3 disc membrane (Millipore, Bedford,
`MA, USA) having a molecular weight cut-off (MWCO) of
`3000. Cooked extract was prepared by cooking mush-
`room pieces in boiling water for 20 min, homogenizing
`the mushroom pieces in the cooked water and clarifying
`the homogenate by filtration. Steamed extract was pre-
`pared by subjecting mushroom pieces to a pressure of
`103.5 kPa in an autoclave for 15 min and homogenizing
`and filtering as above. To prepare dried mushroom
`extract, a mushroom was cut into pieces and dried in an
`oven at 80°C for 16 h. Dried pieces were then powdered
`and reconstituted in water by stirring overnight at 4°C.
`Undissolved material was removed by centrifugation.
`
`Skin prick test
`
`The SPT was performed on the volar side of the forearm,
`as per the standard procedure,7 using a sterile prick
`lancetter (Bayer Pharmaceutical Division, Spokane, WA,
`USA). Wheal and flare diameters were measured after
`20 min. Histamine dihydrochloride (10 mg/mL) in 50%
`glycerol/phosphate-buffered saline (PBS) was the positive
`control. Glycerinated PBS was used as a negative control.
`
`Total IgE and allergen-specific IgE
`
`Total and allergen-specific IgE were determined by ELISA7
`using 96-well microtiter plates (Maxisorp; NUNC,
`Roskilde, Denmark). Horseradish peroxidase-conjugated
`goat antihuman IgE (Sigma Chemical, St Louis, MO,
`USA) was used as a secondary antibody (1 : 5000 dilu-
`tion). Sera from three subjects without any history of food
`allergy were taken as control sera.
`
`Gel filtration on Sephadex G-25
`
`A 250 µL sample of 50% (w/v) mushroom extract was
`loaded onto a Sephadex G-25 column (Pharmacia LKB
`Biotechnology, Uppsala, Sweden; 0.48 cm i.d. × 49 cm)
`equilibrated with water. The column was run at 25°C at a
`flow rate of 4 mL/h and 0.3 mL fractions were collected.
`Sephadex G-25 has a molecular weight fractionation
`range of 1–5 kDa for peptides and globular proteins.8
`The protein assay was performed on 50 µL fractions
`according to the method of Bradford9 using bovine serum
`albumin as the standard. The SPT was performed using
`alternate fractions, starting with fraction 10. As a marker
`for a molecule having a molecular weight of < 1 kDa,
`
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`

`D-glucose (4 mg/mL in water) was subjected to gel filtra-
`tion under identical conditions. Glucose was detected by
`phenol–sulfuric acid reagent.10
`
`Ion-moderated cation-exchange
`chromatography
`
`This was performed as described earlier11 using a cation-
`exchange resin (H+ form) after converting it to the Ca2+
`form.
`
`Preparation of resin
`
`A 75 g sample of ion-exchange resin Dowex-50 W
`(Sigma Chemical; 200–400 dry mesh; 8% cross-linked)
`was washed three times with 1 L water, decanting each
`time to remove fines. Then, 1 L of 1 mol/L HCl was
`added to the resin and heated to boiling on a hot
`plate. After cooling and filtering through a fine porosity
`sintered-glass funnel under vacuum, the resin was
`washed twice with 250 mL water. Then, 1 L of 1 mol/L
`CaCl2 was added and heated to boiling on a hot plate.
`The resin was cooled, filtered and washed with water, as
`above. Finally, the resin was made into slurry with 200 mL
`water and packed into a glass column.
`
`Chromatographic procedure
`
`A 500 µL aliquot of 10× concentrated mushroom 3K-
`filtrate was loaded onto a calcium form of the Dowex-
`50 W column (1 × 60 cm) equilibrated with water and
`run at 25°C at a flow rate of 15 mL/h; 2.0 mL fractions
`were collected. Standard mixture (1 mL) containing 2 mg
`each of D-glucose, D-fructose, D-mannitol and sorbitol
`was also chromatographed under identical conditions.
`Each of these standards was also chromatographed
`separately under identical conditions.
`
`Assays for sugars and sugar alcohols
`
`Detection of reducing sugars
`
`Reducing sugars were detected by phenol–sulfuric acid
`reagent.10 Aliquots (10 µL) of each fraction were diluted
`to 0.5 mL with water and were mixed with 0.3 mL of 5%
`(v/v) phenol. Sulfuric acid (1.8 mL) was added to this
`from a burette and the mixture was vortexed immediately.
`Tubes were allowed to cool and absorbance was read
`at 490 nm. D-Glucose was used as a representative
`reducing sugar.
`
`ANAPHYLAXIS CAUSED BY MANNITOL
`
`123
`
`Detection of fructose
`
`Fructose was detected using cold anthrone reagent.12 This
`reagent was prepared fresh by dissolving 150 mg anthrone
`(Sigma Chemical) in 100 mL of 71.7% sulfuric acid. A
`10 µL aliquot of each fraction diluted to 50 µL with water
`was mixed with 1.5 mL cold anthrone reagent; after
`1–1.5 h at 25°C, the absorbance was measured at 620 nm.
`
`Detection of sugar alcohols
`
`Sugar alcohols were detected by the polyol assay,13 which
`involves periodate oxidation followed by estimation of
`the formaldehyde formed. Other than sugar alcohols,
`fructose is the only sugar that is reactive in this assay.13
`A 50 µL sample of each fraction was diluted to 1 mL
`with water and to this was added 0.5 mL of 10 mmol/L
`sodium metaperiodate in 0.5 mol/L sulfuric acid. After
`mixing, the solutions were allowed to stand for at least
`10 min at 25°C. Next, 0.2 mL of 10% (w/v) sodium
`bisulfite was added, with immediate mixing, followed by
`0.2 mL of 2% (w/v) aqueous chromotropic acid solution.
`Finally, 3 mL concentrated sulfuric acid was added from
`a burette. The solutions were mixed well by vortexing
`and the tubes were placed in a boiling water bath for
`30 min. After the tubes were cooled, the absorbance
`was read at 570 nm. As representative of model sugar
`alcohols, D-mannitol and sorbitol were used.
`
`HPLC analysis
`
`Mushroom 3K-filtrate, and fractions from Dowex-50 W
`chromatography were analyzed by a Shimadzu HPLC
`system (Shimadzu, Kyoto, Japan) on a Supelcosil LC-NH2
`column (Supelco, Sigma–Aldrich, St Louis, MO, USA;
`4.6 × 250 mm, 5 µm aminopropyl-bonded silica) with
`acetonitrile : water (85 : 15) as the mobile phase. The
`column was run at 25°C at a flow rate of 1 mL/min.
`Sugars and sugar derivatives were detected using a
`refractive index (RI) detector (RID-6A; Shimadzu). Sugar
`standards were run separately to determine the elution
`profile and a mixture of sugar standards (such as
`D-glucose, D-fructose and D-mannitol) was also run to
`determine the chromatographic resolution.
`
`GC-MS analysis
`Acetylation14
`
`Purified sample (2–3 mg) and standard D-mannitol
`(2 mg) were taken in separate tubes (in 0.5 mL deionized
`
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`124
`
`VL HEGDE ET AL.
`
`Results of the skin prick test
`
`Table 1
`Sample
`
`Histamine dihydrochloride (10 mg/mL)
`Glycerinated phosphate-buffered saline
`Agaricus bisporus
`Fresh extract 50% (w/v)
`Mushroom 3K-filtrate
`Fresh extract 33.3% (w/v)
`Cooked extract 33.3% (w/v)
`Steamed extract 33.3% (w/v)
`Dried extract 33.3% (w/v)
`D-Mannitol
`0.001% (w/v)
`0.01% (w/v)
`0.1% (w/v)
`1.0% (w/v)
`
`Wheal/flare
`diameter (mm)
`8/30+
`1/0
`
`5/25
`5/25
`4/25
`3/25
`4/20
`4/25
`
`3/0
`4/0
`5/25
`6/30
`
`water). Dry and distilled acetic anhydride and pyridine
`(0.5 mL each) were added and kept in a boiling water
`bath for 2 h after tightly stoppering the tubes. Excess
`reagents were removed by codistilling with water (1 mL,
`three times) and toluene (1 mL, three times). After
`thorough drying, the contents were dissolved in chloro-
`form and filtered through glass-wool and dried by
`passage through nitrogen gas. The residues were dis-
`solved in chloroform for analysis.
`
`GC-MS conditions15
`
`Analyses were performed on a Shimadzu QP 5000 system
`using an SP 2330 capillary column (30 m × 0.25 mm
`i.d.). Helium was used as the carrier gas with a flow of
`2 mL/min. The oven temperature was programmed
`between 180 and 200°C with an increase of 4°C/min.
`Injector
`interface
`temperatures were approximately
`250°C. The MS conditions used were: ionization poten-
`tial 70 eV, mass range 40–400 (m/z).
`
`RESULTS
`Results of SPT with various samples are shown in Table 1.
`The SPT with mushroom extract was positive. The SPT of
`mushroom extract in 12 healthy, non-allergic individuals
`was negative (data not shown). The SPT using extracts of
`cooked, steamed or dried mushroom was also positive.
`Total IgE in the subject’s serum was 200 IU/mL com-
`pared with a normal value of < 120 IU/mL. Allergen-
`specific IgE in the subject’s serum could not be detected
`by ELISA using mushroom extract.
`
`Fig. 1 Size exclusion chromatography of 50% (w/v) Agaricus
`bisporus extract (0.25 mL) on a Sephadex G-25 column
`(0.48 × 49 cm). The eluent was water; the flow rate was
`4 mL/h; protein detection by Bradford’s dye-binding assay,
`A595 (—䊊—); skin prick test (SPT), wheal diameter (—䊐—). The
`arrow indicates the elution position of D-glucose (G) under
`identical conditions.
`
`The gel filtration pattern of mushroom extract on a
`Sephadex G-25 column is shown in Fig. 1. Protein assay
`(dye-binding) on the column fractions showed two peaks,
`one at the void volume of the column (fractions 12–22
`containing molecules > 5 kDa) and the other at the
`column volume (fractions 26–34 containing molecules
`< 1 kDa). The SPT results of alternate fractions starting
`with fraction 10 show that allergenic activity is associated
`with fractions 28 and 30. Fractions containing proteins
`eluting at the void volume, as detected using Bradford’s
`reagent, did not show any skin reactivity. The SPT with
`mushroom 3K-filtrate gave a wheal/flare diameter of
`5/25 mm (Table 1), similar to fresh mushroom extract,
`50% w/v.
`The Ca2+ ion-moderated cation-exchange chromato-
`graphy profile of mushroom 3K-filtrate on Dowex-50 W
`is shown in Fig. 2. Detection of reducing sugars in the
`fractions by phenol-sulfuric acid yielded two peaks (peaks
`1 and 2; Fig. 2a), the elution positions of which co-
`incided with those of D-glucose and D-fructose standards,
`respectively. Peak 2 was also identified by cold anthrone
`assay as fructose. When the fractions were assayed for
`polyol, a major peak, peak 3, was obtained at fractions
`24–32 (Fig. 2b). The elution position of the polyol
`
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`ANAPHYLAXIS CAUSED BY MANNITOL
`
`125
`
`High-pressure liquid chromatography analysis of the
`mushroom 3K-filtrate and certain fractions from Dowex-
`50 W chromatography on the Supelcosil LC-NH2 column
`are shown in Fig. 3. Mushroom 3K-filtrate showed three
`peaks (Fig. 3a), the retention times of which are compa-
`rable to those of D-fructose, D-glucose and D-mannitol
`standards run under identical conditions. The HPLC
`pattern of fractions 16, 22 and 28 from the Dowex
`column is shown in Fig. 3b. Components from peak frac-
`tions of peak 1 and 2 (fractions 16 and 22) had retention
`times similar to those of standard glucose and fructose,
`respectively, whereas peak fraction (fraction 28) of peak
`3 showed a component eluting at the position of stan-
`dard mannitol.
`The SPT with various concentrations of commercial
`D-mannitol (analytical grade) is shown in Table 1.
`D-Mannitol, at concentrations of 0.1 and 1% (w/v),
`showed strong positive skin reactivity in the allergic
`subject. The subject felt intense itching for these samples
`during skin testing. However, D-mannitol (1%) did not
`produce any positive SPT when tested on 12 healthy
`volunteers (data not shown).
`In GC-MS analysis, standard mannitol showed a
`retention time of 4.675 min, whereas the allergenic com-
`ponent purified from A. bisporus by Dowex-50 W chrom-
`atography (peak 3 in Fig. 2b) had a retention time of
`4.683 min (data not shown). Figure 4 shows the mass
`spectral matching of allergenic component isolated from
`A. bisporus (Fig. 4c), with those of standard D-mannitol
`(Fig. 4b) and library spectrum for hexitol hexaacetate15
`(Fig. 4a). As can be seen, the sample spectrum is a fairly
`good match with the library spectrum for hexitol hexa-
`acetate and the spectrum for D-mannitol.
`
`DISCUSSION
`The present study describes a case of anaphylaxis to the
`ingestion of cultivated white button mushroom A. bisporus.
`The case history of the subject and SPT with mushroom
`extract indicated an IgE-mediated (type I hypersensitivity)
`reaction. The same subject was also severely allergic
`to pomegranate fruit and it was shown earlier5 that this
`was due to sensitization to a low molecular weight
`(LMW) allergen (mannitol) present naturally at a con-
`centration of 0.25% (0.25 g/100 g edible portion).
`Mushrooms contain mannitol, pentosans, hexosans and
`α,α-trehalose, along with traces of glucose.6 Mannitol is
`the major sugar component in fungi and it helps to main-
`tain the osmotic concentration in the fruit body, which
`
`Fig. 2 Ca2+ ion-moderated cation-exchange chromato-
`graphy of Agaricus bisporus 3K-filtrate (0.5 mL, 10× concen-
`trated) on a Dowex 50 W column (1 × 60 cm, 8% cross-linked,
`200–400 dry mesh). The eluent was water; the flow rate
`was 15 mL/ h. (a) Detection by phenol-sulfuric acid (—䉭—).
`(b) Polyol detection, A570 (—䊉—); Bradford’s protein assay, A595
`(—䊊—). The numbers above peak 3 indicate wheal/flare
`diameter (mm) produced by the peak fractions 26 and 28.
`Other fractions tested (6, 16, 22, 24, 30 and 32) did not
`produce a wheal/flare reaction. Arrows indicate elution
`positions of standards under identical conditions: D-glucose
`(G), D-fructose (F), D-mannitol (M), sorbitol (S).
`
`component (peak 3) coincided with that of the D-mannitol
`standard run under identical conditions. The SPT results
`of fractions 6, 16, 22, 24, 26, 28, 30 and 32 showed
`that allergenic activity is associated only with fractions
`from peak 3 (fractions 26 and 28), which gave a
`wheal/flare diameter of 5/25 mm. Bradford’s assay on
`the column fractions (Fig. 2b) did not show any dye-
`binding component.
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`126
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`VL HEGDE ET AL.
`
`Fig. 3 High-pressure liquid chromatography analysis of Agaricus bisporus on a Supelcosil LC-NH2 column (4.6 × 250 mm, 5 µm
`aminopropyl-bonded silica). The mobile phase consisted of acetonitrile : water (85 : 15); the flow rate was 1 mL/min; the column
`temperature was 25°C; detection: refractive index (RI). The dotted line indicates the elution profile of the standards: D-fructose (F),
`D-glucose (G), D-mannitol (M); retention time (RT in min) for each standard is indicated above its respective peak. (a) 3K-filtrate:
`RT (min) of peaks from the sample are 6.37, 7.49 and 8.26. (b) fractions from Dowex-50 W chromatography: RT (min) of peaks from
`fractions 16, 22 and 28 are 7.42, 6.71 and 8.22, respectively.
`
`is required to maintain a water content of as high as
`90%.16–18 The present study was performed in order to
`investigate whether the allergen in A. bisporus causing
`anaphylaxis after ingestion in the present case is the
`same as in the case of pomegranate (mannitol) or a dif-
`ferent allergen, which can be either a protein or a LMW
`component.
`The SPT with extracts from cooked, steamed or dried
`mushrooms gave a positive result, indicating that the
`allergen is heat-stable. This heat-stability of the allergen
`appears to be responsible for the anaphylaxis seen in the
`present case following ingestion of mushroom curry. A
`positive skin test with the 3K-filtrate of mushroom extract
`indicated the presence of a LMW allergen of < 3 kDa.
`Sephadex G-25 has a molecular fractionation range
`of 1–5 kDa for globular proteins and peptides. Based
`on gel filtration, it was found that the allergenic skin
`reactivity is associated with fractions eluting at the column
`volume, which contain LMW components of < 1 kDa.
`Fractions at the column volume also indicated the pres-
`ence of dye-binding components; this may be due to the
`high content of polyphenols known to be present in
`mushroom, which interfere in the dye-binding protein
`assay.19
`Among many sugars and sugar derivatives (D-glucose,
`D-fructose, D-mannose, D-galactose, sucrose, maltose,
`lactose, mannitol, galactitol, sorbitol) tested by SPT, only
`
`Fig. 4 Mass spectral matching of mannitol isolated from
`Agaricus bisporus by ion-moderated cation-exchange chromato-
`graphy (c), with the library spectrum (a) and D-mannitol standard
`(b). All mass spectra were taken using acetylated derivatives.
`
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`D-mannitol gave a positive response. Sugars and sugar
`alcohols can be separated by ion-moderated cation-
`exchange chromatography on a column of Dowex-50 W
`resin in the Ca2+ form. Mushroom 3K-filtrate was
`chromatographed on this column using water as the
`eluent and the fractions were tested for allergenic activity
`by SPT. It was found that the allergenic activity was asso-
`ciated with peak fractions from peak 3 corresponding to
`mannitol. These fractions were further analyzed by HPLC
`and were found to contain only mannitol. The allergenic
`component corresponding to peak 3 of the Dowex-50 W
`column had a retention time identical to that of D-mannitol
`in GC analysis. The high degree of similarity of acetylated
`sample mass spectrum to both the library (hexitol hexa-
`acetate) and standard (acetylated D-mannitol) mass
`spectra shows the likelihood of correct identification.
`Bradford’s assay on the Dowex fractions did not reveal
`any dye-binding component, indicating that the mannitol-
`containing fractions that produced a positive SPT do not
`contain any peptides or LMW dye-binding components
`seen in Sephadex G-25 chromatography.
`Although most of the allergens so far identified in foods
`are proteins, small molecules have also been shown as
`potential allergens in some cases.20–25 Chlorogenic acid
`has been identified as an important allergen from green
`coffee beans in workers in the coffee-processing industry
`who developed occupational asthma and rhinitis.20–22
`Ethanol has been identified as a LMW allergen in over-
`ripe rock melon (Cucumis melo)23 and as a possible
`allergen in some other cases.24 Sensitization to acetic
`acid, the main metabolite of ethanol, has been reported
`in a 22-year-old woman with immediate type I allergy to
`some alcoholic beverages and vinegar.25 During the early
`part of the present study, we tested these LMW chemicals
`on the allergic subject by SPT and the results were
`negative.
`Hypersensitivity reactions to intravenous infusions of
`mannitol (10 or 20%),26–31 dextrose (50%)32 and galac-
`tose (30%)33 have been reported. These are ‘anaphylac-
`toid’ reactions caused by hyperosmolar concentration of
`sugars or mannitol (> 100 mmol/L) and are clinically
`indistinguishable from IgE-mediated allergic or anaphy-
`lactic reactions in vivo. The mannitol concentration in
`A. bisporus is 1.15% based on fresh weight.16 Other
`foods that contain mannitol in significant amounts34 are
`celery stem (Apium graveolens L.; 1–2%) and pumpkin
`(Cucurbita pepo L.; 15–20%). Vegetables, such as carrot
`and onion, parsley and strawberry fruit have only trace
`amounts of mannitol34 and its quantity in the edible
`
`ANAPHYLAXIS CAUSED BY MANNITOL
`
`127
`
`portions has not been listed in the available databases
`(Dr Duke’s Phytochemical & Ethnobotanical Databases;
`http://www.ars-grin.gov/duke/).
`In the present case, the allergic reaction is caused by
`the ingestion of very low amounts of mannitol in the
`mushroom curry and, hence, appears to be IgE-mediated
`anaphylaxis. However, allergen-specific IgE could not be
`detected in the allergic subject’s serum by ELISA. It
`appears that the negative result with ELISA may not be
`due to the absence of allergen-specific IgE per se, but
`may be because of the non-binding of the LMW allergen
`(mannitol) in mushroom extract/mushroom 3K-filtrate to
`the polystyrene ELISA plates. Because mannitol is a LMW
`allergen, the initial sensitization could have occurred due
`to the presence of its conjugate with a high molecular
`weight substance. In order to prove the haptenic nature
`of mannitol, attempts are being made to prepare a
`conjugate of mannitol with carrier protein.
`Intestinal permeability represents a state of intestinal
`mucosa that permits molecules or compounds (such as
`mannitol, insulin, lactose or polyethylene glycols) to
`diffuse across the membrane. In the normal state, these
`molecules do not cross the intestinal barrier because they
`have no active transport system. Derangements of the
`intestinal epithelium (secondary to mediators of allergic
`inflammation in food-induced allergy) may be respon-
`sible
`for abnormalities
`in
`intestinal permeability.35
`Although an intestinal permeability test was not per-
`formed in the present case, it appears likely that mannitol
`is absorbed in the gastrointestinal system due to derange-
`ment of the intestinal epithelium following release of
`mediators of allergic inflammation.
`Protein allergens have been identified only from the
`spores of some edible mushrooms responsible for causing
`inhalative allergy;1,2,36 none has so far been identified
`from edible mushrooms causing ingestive allergy.3,4 The
`present report describes the identification of the allergen
`as mannitol in a severe case of allergy (anaphylaxis)
`following mushroom ingestion. Anaphylaxis experienced
`by the allergic subject following consumption of cake
`icing (which contains mannitol as a nutritive sweetener
`and stabilizer/thickener) and the chewable tablet Cisapid
`MPS appears to have been caused by mannitol. This has
`been experimentally tested by isolating mannitol from
`aqueous extracts of Cisapid MPS and showing it to be
`allergenically active in SPT (VL Hegde and YP Venkatesh,
`unpubl. obs., 2001). This is the first report of food allergy
`to Agaricus bisporus and also the first describing a LMW
`allergen (mannitol) from mushroom.
`
`MYLAN INST. EXHIBIT 1089 PAGE 7
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`MYLAN INST. EXHIBIT 1089 PAGE 7
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`

`128
`
`VL HEGDE ET AL.
`
`ACKNOWLEDGMENTS
`We thank all the participants in this study. We thank Dr PA
`Mahesh (Allergy, Asthma, & Chest Center, Mysore) for his
`assistance in performing the skin prick tests. We thank
`Dr V Prakash (Director, CFTRI, Mysore) for his keen inter-
`est and encouragement in this project. This work was
`supported by the Department of Science & Technology,
`New Delhi (grant SP/SO/B48/97) for project on ‘Food
`allergens from plant sources’ (YPV) and Council of
`Scientific & Industrial Research, New Delhi (junior
`research fellowship award to VLH).
`
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