Serum ferritin and transferrin levels as serologic markers of methylene diphenyl diisocyanate–induced occupational asthma

Serum ferritin and transferrin levels as serologic markers ofmethylene diphenyl diisocyanate–induced occupationalasthma

Gyu-Young Hur, MD, PhD,a Gil-Soon Choi, MD,a Seung-Soo Sheen, MD, PhD,b Hyun-Young Lee, MS,a Han-Jung Park,

MD,a Sung-Jin Choi, MD,a Young-Min Ye, MD,a and Hae-Sim Park, MD, PhDa Suwon, South Korea

Background: Although methylene diphenyl diisocyanate (MDI)may induce occupational asthma in the workplace, thepathogenic mechanisms are unclear.Objectives: By using bronchoalveolar lavage fluid, we sought toidentify proteins that were differentially expressed betweensubjects with MDI-induced occupational asthma (MDI-OA) andasymptomatic exposed controls (AECs).Methods: To find proteins that were differentially expressedbetween the MDI-OA and AEC groups, 2-dimensionalelectrophoresis was performed by using bronchoalveolar lavagefluid obtained from subjects after MDI-specific inhalationchallenge. The selected protein spots were then identified bymatrix-assisted laser desorption/ionization time-of-flight massspectrometry. The clinical relevance of the differentiallyexpressed spots was compared by ELISA using sera from theMDI-OA/eosinophilic bronchitis, AEC, and unexposed healthycontrol groups. Receiver operating characteristic curves werethen plotted, and the sensitivity and specificity were determined.Results: Twenty-three protein spots were identified thatdistinguished the subjects with MDI-OA from those in the AECgroup. Among them, ferritin expression was downregulatedwhereas transferrin expression was upregulated in subjects withMDI-OA compared with AEC; these results were validated byELISA using sera from the MDI-OA/EB and AEC groups. Toidentify subjects with MDI-OA, the optimal serum cutoff levelswere 69.84 ng/mL for ferritin and 2.48 mg/mL for transferrin.When these 2 parameters were combined, the sensitivity was71.43% and the specificity was 85.71%.Conclusion: Serum ferritin and transferrin levels are associatedwith the phenotype of MDI-OA. (J Allergy Clin Immunol2008;122:774-80.)

Key words: MDI, occupational asthma, ferritin, transferrin,proteome

From athe Department of Allergy and Rheumatology, and bPulmonary and Critical Care

Medicine, Ajou University School of Medicine.

Supported by a grant from the Korean Health 21 R&D Project of the Ministry of Health

and Welfare, Republic of Korea (A050571).

G.-Y. Hur is currently with the Department of Internal Medicine, Korea University

College of Medicine, Seoul, South Korea.

Disclosure of potential conflict of interest: The authors have declared that they have no

conflict of interest.

Received for publication February 19, 2008; revised July 28, 2008; accepted for publica-

tion July 29, 2008.

Reprint requests: Hae-Sim Park, MD, PhD, Department of Allergy and Rheumatology,

Ajou University School of Medicine, San-5, Wonchun-dong, Youngtong-gu, Suwon,

443-721, South Korea. E-mail: [email protected].

0091-6749/$34.00

� 2008 American Academy of Allergy, Asthma & Immunology

doi:10.1016/j.jaci.2008.07.034

774

Diisocyanates, which are widely used in the manufacture ofpolyurethane foams, elastomers, adhesives, coatings, insecti-cides, paints, plastics, and varnishes, may cause occupationalasthma as a low-molecular-weight antigen,1,2 and several effortshave been made to develop serologic markers for isocyanate-in-duced asthma. In the case of toluene diisocyanate–induced occu-pational asthma (TDI-OA), specific IgE and IgG antibodies totoluene diisocyanate (TDI)–human serum albumin conjugatewere found to be good diagnostic tools.3-5 In addition, serum-spe-cific IgG to cytokeratin 19 was suggested as a possible serologicmarker of TDI-OA, but the sensitivity of these 2 parameters wastoo low to be applied.3-6 In methylene diisocyanate–induced oc-cupational asthma (MDI-OA), although the level of serum spe-cific IgE and IgG antibodies was found to be increased in somepatients,7 it was not high enough to be used for diagnostic pur-poses. Considering that permanent impairment of lung functionwas reported in long-term follow-up studies of isocyanate-in-duced asthma,8 developing relevant serologic markers to identifysusceptible subjects among exposed workers is essential.

Proteomic analysis is a complex process involving the purifi-cation and identification of individual proteins from all of theproteins expressed in a cell or tissue.9 To clarify the pathogenicmechanisms of asthma with proteomic technology, many studieshave used human samples such as bronchoalveolar lavage fluid(BALF)10 and plasma.11,12

Abbreviations used

2-DE: 2-Dimensional electrophoresis

AEC: Asymptomatic exposed control

ANCOVA: Analysis of covariance

ARDS: Acute respiratory distress syndrome

AUC: Area under the curve

BALF: Bronchoalveolar lavage fluid

DBP: Vitamin D–binding protein precursor

EB: Eosinophilic bronchitis

MALDI-TOF/TOF MS: Matrix-assisted laser desorption ionization

time-of-flight/time-of-flight mass spectrometry

MDI: Methylene diisocyanate

MDI-EB: Methylene diisocyanate–induced

eosinophilic bronchitis

MDI-OA: Methylene diisocyanate–induced

occupational asthma

MS: Mass spectrometry

NC: Nonatopic healthy control

ROC: Receiver operating characteristic

TDI: Toluene diisocyanate

TDI-OA: Toluene diisocyanate–induced

occupational asthma

WRRS: Work-related lower respiratory symptom

J ALLERGY CLIN IMMUNOL

VOLUME 122, NUMBER 4

HUR ET AL 775

In this study, we sought to identify new serologic markers usinga proteomic approach and to validate them for screeningsuspected subjects from among MDI-exposed individuals.

METHODSThe protocol for this study was reviewed and approved by the Ajou

University Institute Review Board, and informed consent was obtained from

each study subject.

Study subjectsSeven subjects with MDI-OA and/or eosinophilic bronchitis (EB), 51

asymptomatic exposed controls (AECs), and 74 nonatopic healthy controls

(NCs) were enrolled in this study. Among 7 subjects of the MDI-OA/EB

group, 5 had MDI-OA and 2 had MDI-induced EB (MDI-EB). All subjects

with MDI-OA/EB and AECs were selected from among 58 previously studied

MDI-exposed workers in a single car upholstery factory.7 First, 58 subjects

were screened by respiratory questionnaire, and 26 subjects who had com-

plained of work-related lower respiratory symptoms (WRRSs) underwent

methacholine challenge test. Then, 11 subjects showing a positive response

to methacholine challenge test underwent the MDI-specific inhalation tests

subsequently, except 2 subjects who refused the MDI-specific inhalation

test. Five symptomatic workers who had positive responses to both methacho-

line challenge test and MDI-specific inhalation challenge were defined as hav-

ing MDI-OA, and their clinical features are summarized in Table I. WRRS was

defined as lower respiratory symptoms aggravated during the work, but im-

proved after the work or during holidays, including cough, sputum, wheezing

episodes, or shortness of breath.7,13 The subjects with MDI-EB were defined

as complaining of WRRS with increased sputum eosinophilia (>3%) after

MDI bronchial challenges, but having negative responses to the methacholine

bronchial challenge and MDI-specific inhalation challenge tests.14 Subjects

with AEC were defined as subjects who did not have MDI-OA/EB although

they had worked in the same workplace. Two subjects who had positive

responses to methacholine challenge tests but refused further studies were

excluded from the AEC group. The NCs were healthy nonatopic volunteers

who had never been exposed to MDI. The detailed results of MDI-specific in-

halation challenge were reported in our previous article.7

Skin prick test and bronchial challenge testingThe skin prick test, which included 9 common inhalant allergens (tree

mixture, grass mixture, mugwort, ragweed, cat fur, dog fur, Dermatophagoides

pteronyssinus, Dermatophagoides farinae, and Alternaria; Bencard, Bradford,

United Kingdom), was performed on all subjects. The result of each test was

reported as the ratio of the mean wheal diameter of the allergen to histamine.

For values above 1, the reaction was defined as positive. Atopy was defined

as more than 1 positive response to the common inhalant allergens on the

skin prick test.

Airway responsiveness to methacholine was tested in those 26 subjects who

had WRRS by using the 5-breath dosimeter protocol.15 MDI-specific inhala-

tion challenge testing was performed on 15 subjects with definite WRRS or

positive responses to the methacholine challenge test by using a modified ver-

sion of a previously described method.7,16 In brief, 20 mL polymeric MDI

(Desmodur 44V20; Bayer, Leverkusen, Germany) was heated to 1808C in a

beaker on a hot plate to approach the optimal MDI exposure conditions for

bronchial provocation. The atmospheric concentration of MDI was measured

serially by using personal samplers (GilAir; Sensidyne Inc, Clearwater, Fla)

placed 60 cm away from the hot plate. The initial exposure lasted 20 minutes,

and another was performed after a 1-hour break. During the break, the booth

was thoroughly ventilated. Spirometric measurements were recorded before

the challenge, at 10-minute intervals for 1 hour after the challenge, and subse-

quently at 1-hour intervals for the next 4 hours. Peak flow measurements were

collected at 2-hour intervals for 24 hours. The test was performed in a booth

equipped with a ventilating fan and transparent windows through which the

subjects were observed. All subjects with MDI-OA had positive results of

methacholine challenge, and their PC20 values ranged from 2.76 to 21.93

mg/mL.

Bronchoscopy and bronchoalveolar lavageFiber optic bronchoscopy was performed 24 hours after the MDI-specific

challenge test. Bronchoalveolar lavage was performed through a flexible

bronchoscope (Olympus BF-P240; Olympus Corp, Tokyo, Japan), which was

wedged into the subsegmental bronchus of the right middle lobe. Three 30-mL

aliquots of sterile saline solution were instilled through the bronchoscope. The

fluid was immediately recovered by gentle suction after each instillation. The

harvested BALF was centrifuged at 1000 rpm for 10 minutes, and the

supernatant was collected for proteomic analysis according to the MDI-

specific inhalation challenge test result. Serum was obtained from each subject

after MDI-specific inhalation challenge testing.

Two-dimensional electrophoresis and image

analysisTo evaluate differential protein expression in the MDI-OA and AEC

groups, we chose 2 subjects with a more than 25% decrease in FEV1 after the

MDI-specific inhalation challenge test (typical MDI-OA) and 2 subjects with

no decrease in FEV1 after the MDI-specific inhalation challenge test (AECs),

and collected BALF for 2-DE.

Each sample was prepared by trichloroacetic acid/acetone precipitation

for desalting and concentrating. Immubilized pH gradient (IPG) strips (Immobi-

line DryStrip, pH 3-10 NL, Amersham Pharmacia Biotech, Uppsala, Sweden)

and Pharmalytes (pH 3-10; Amersham Pharmacia Biotech) were used for

isoelectric focusing. For the first-dimensional analysis, 1 mg protein was

focused on IPG strips as described except that a total of 80,000 voltage-hours

was applied.17,18 For second-dimensional separation, electrophoresis was per-

formed by using 9% to 16% gradient polyacrylamide gels until the dye front

reached the lower end of the gel. To quantify the relative abundance of the pro-

teins, the gels were stained with Coomassie Brilliant Blue G-250 (Bio-Rad, Her-

cules, Calif). The stained gels were then scanned using a GS-710 imaging

densitometer (Bio-Rad) and analyzed with Image Master Platinum 5 (GE

Healthcare, Piscataway, NJ).

In-gel digestion and mass spectrometric analysisProtein spots that increased or decreased more than 2-fold between the

positive and negative groups were selected for mass spectrometry (MS)

TABLE I. Clinical features of the study subjects

MDI-OA/EB

(n 5 7)

AEC

(n 5 49)

NC

(n 5 74)

P

value

Age (y) 46.43 6 4.43 39.94 6 9.49 29.34 6 6.63 <.001

Sex

(male/female)

2/5 26/23 22/52 .056

Working duration

(y)

5.56 6 1.28 4.02 6 2.43 NA .205

% Predicted

FEV1*

105.17 6 13.56 99.92 6 17.78 99.91 6 10.16 .778

PC20 (mg/mL) 15.05 6 9.82 22.83 6 7.77� ND NA

Log total IgE

(IU/mL)

2.13 6 0.63 2.00 6 0.65 1.80 6 0.53 .513

Geometric mean 134.52 99.41 62.79

95% CI 34.9-518.3 64.7-152.7 27.7-142.2

Atopy (%) 0 (0) 15 (30.6) 0 (0) NA

Smoking history

(%)

1 (14.3) 21 (41.2) 16 (21.6) .150

NA, Not applicable; ND, not done.

*Performed before methacholine challenge test.

�Derived from 17 individuals taking the test.

J ALLERGY CLIN IMMUNOL

OCTOBER 2008

776 HUR ET AL

FIG 1. 2-DE separation of BALF proteins from AECs (A) and patients with MDI-OA (B). The proteins identified

by MALDI-TOF MS (arrows) are marked by their spot numbers. Arrows indicate the spots corresponding to

ferritin (8) and transferrin (1).

analysis. The spots were excised from the gels with a sterile scalpel and placed

into Eppendorf tubes. The proteins were then digested by using trypsin

(Promega, Madison, Wis) as previously described.19 For MALDI-TOF/TOF

MS, the tryptic peptides were concentrated using a POROS R2, Oligo R3 col-

umn (Applied Biosystems, Foster City, Calif). After washing the column con-

secutively with 70% acetonitrile, 100% acetonitrile, and 0.05 M ammonium

bicarbonate, the samples were applied to the R2, R3 column and eluted

with cyano-4-hydroxycinamic acid (Sigma, St Louis, Mo). The samples

were dissolved in 70% acetonitrile and 2% formic acid before MALDI-

TOF/TOF MS.18 Mass spectra were acquired by using a 4800 Proteomics An-

alyzer (Applied Biosystems) operated in the MS and MS/MS modes. Peptide

fragmentation in the MS/MS mode was achieved by collision-induced disso-

ciation by using atmospheric air as the collision gas. The instrument was op-

erated in reflectron mode and calibrated by using the 4800 calibration mixture

(Applied Biosystems); each sample spectrum was further calibrated by using

trypsin autolysis peaks. Peptide matching and protein searches were per-

formed by using Mascot and the Swiss-Prot and National Center for Biotech-

nology Information databases (http://www.matrixscience.com).

Measurement of ferritin and transferrin in seraThe serum levels of ferritin (Alpha Diagnostic International, San Antonio,

Tex) and transferrin (Alpha Diagnostic International) in the subjects from the

MDI-OA/EB, AEC, and NC groups were measured by using an ELISA kit

according to the manufacturer’s instructions.

Statistical analysesLog-transformed serum ferritin and transferrin values were compared by

ANOVA with Bonferroni correction for multiple testing. All values were

adjusted by age and sex by using analysis of covariance (ANCOVA). Receiver

operating characteristic (ROC) curves were used to evaluate the validity of the

serum ferritin and transferrin levels for discriminating between MDI-OA and

AEC, and the area under the curve (AUC) with a 95% CI was computed.20

Sensitivity and specificity were calculated according to the identified optimal

cutoffs, and P values were adjusted by age and sex. All computations were per-

formed by using SPSS Version 12.0 (SPSS Inc, Chicago, Ill) and MedCalc

(Mariakerke, Belgium).

RESULTS

Characteristics of the study subjectsAll subjects were classified into 2 groups, MDI-OA/EB and

AEC, on the basis of the results of a methacholine challenge testand MDI-specific inhalation challenge test; their clinical featuresare summarized in Table I. The NCs were significantly youngerthan the MDI-exposed workers, including those in the MDI-OA/EB and AEC groups (P < .001), which have been attributableto selection bias. No significant difference was observed in termsof sex, FEV1(%), or log total IgE among the MDI-OA/EB, AEC,and NC groups, and no difference in work duration was detectedamong the MDI-exposed subjects.

2-DE analysis of BALFTo evaluate differential protein expression in the MDI-OA

and AEC groups, we chose 2 subjects with a more than 25%decrease in FEV1 after the MDI-specific inhalation challengetest (typical MDI-OA) and 2 subjects with no decrease inFEV1 after the MDI-specific inhalation challenge test (AECs),and collected BALF for 2-DE. Five hundred five spots wereidentified in the gels, of which 28 increased and 89 decreasedmore than 2-fold on the basis of a comparison of the mean ex-pression level in each group (Fig 1, A and B). Among them, 12increased and 25 decreased spots were selected for further MSanalysis.

Identification of the proteins by MALDI-TOF MSAmong the 37 protein spots selected for MS analysis, 23 were

identified by MALDI-TOF/TOF MS (Table II). Of these, 7 upre-gulated and 16 downregulated proteins were identified in thesubjects with MDI-OA.

J ALLERGY CLIN IMMUNOL

VOLUME 122, NUMBER 4

HUR ET AL 777

TABLE II. List of proteins identified by MALDI-TOF MS analysis

No. Protein Exp gi No. Score Peptide match Theory-mass (kD) pI Cov %

1 Transferrin, human serum [ 7245524 58 7 37241 6.49 20

2 Immunoglobulin heavy chain [ 3819788 92 9 24238 5.56 30

3 Human FcaRI [ 31615936 96 8 23642 7.12 37

4 Unnamed protein product [ 34530195 70 15 87805 9.56 20

5 Human hemoglobin [ 56967333 86 6 15018 8.07 46

6 Hemoglobin a1 globin chain [ 13650074 66 9 15292 7.96 66

7 DBP [ 139641 68 13 52929 5.40 35

8 Ferritin light subunit Y 182516 79 8 16441 5.65 49

9 Human salivary amylase Y 15988375 94 12 56411 6.21 27

10 Aldehyde dehydrogenase [NAD(P)] Y 283971 127 13 50703 5.99 27

11 Aldo-keto reductase family 7 Y 19343681 102 11 37553 6.50 45

12 Gelsolinlike capping protein Y 63252913 47 8 38474 5.82 31

13 Fructose-1,6-bisphosphatase 1 Y 57165050 121 16 37190 6.54 27

14 Annexin A1 Y 119582952 74 12 40057 7.60 38

15 Annexin A2 Y 4757756 65 8 38580 7.57 37

16 Rho guanine nucleotide exchange factor 4 Y 15011981 65 22 76372 6.00 34

17 Hypothetical protein Y 119613232 65 14 37160 6.17 40

18 Immunoglobulin heavy chain Y 112700812 53 6 10870 6.34 82

19 Bullous pemphigoid antigen Y 2134838 64 15 76452 6.15 23

20 hCG2042874 Y 119605532 61 15 90369 5.86 17

21 Pulmonary surfactant apoprotein precursor Y 190565 83 6 26836 5.17 29

22 Chain H, chrystal structure of the MRP14 (S100A9) Y 20150236 72 6 13159 5.71 74

23 Annexin V Y 809185 154 20 35783 4.94 56

Cov, Coverage; gi No., genInfo identifier; pI, isoelectric point.

Measurement of the serum ferritin and transferrin

levels by ELISATo validate the proteins identified by MALDI-TOF/TOF MS,

we measured the serum ferritin and transferrin levels in the MDI-OA/EB, AEC, and NC groups (Fig 2), which was adjusted by ageand sex. Table III showed both unadjusted and adjusted P valuesof serum ferritin and transferrin, which indicated that both ferritinand transferrin levels had significant difference between thegroups after age and sex adjustment (P 5 .005 and <.001,respectively).

Determination of the optimal cutoff levels for serum

ferritin and transferrinWe next evaluated the diagnostic value of serum ferritin (Fig 3,

A) and transferrin (Fig 3, B) in discriminating between subjectswith MDI-OA/EB and the AECs using the AUC, which was com-puted from a ROC curve. We then selected the appropriate cutoffvalues for each ROC curve. Using the cutoff for ferritin (69.84 ng/mL; Table III), the sensitivity and specificity were 85.71% and71.48%, respectively, with 0.786 (95% CI, 0.614-0.957; P 5

.053) of the AUC value. When we applied the transferrin cutoffvalue (�2.48 mg/mL), the sensitivity and specificity decreasedto 71.43% and 51.02%, with 0.612 (95% CI, 0.396-0.829; P 5

.400) of the AUC value. When both parameters were combinedto improve the discriminating power,21 the AUC value increasedto 0.789 (95% CI, 0.580-0.991; P 5 .020), whereas the sensitivityand specificity increased to 71.43% and 85.71%, respectively(Table IV).

DISCUSSIONTo elucidate the pathogenic mechanisms of MDI-OA, we

adopted a proteomic approach as a screening tool. First, 2-DE was

used to screen for target proteins in BALF collected from subjectswith MDI-OA and the AECs, and 23 protein spots that wereupregulated or downregulated more than 2-fold were selected.Among those spots, transferrin (spot 1), immunoglobin (spot 2),hemoglobin (spots 5 and 6), and vitamin D–binding proteinprecursor (DBP; spot 7) were upregulated, whereas ferritin lightchain (spot 8), gelsolinlike capping protein (spot 12), and multipleannexins (spots 14, 15, and 23) were downregulated.

We found that the ferritin light chain was downregulatedwhereas transferrin was upregulated in the MDI-OA groupcompared with the AEC group by using a proteomic approach.Both ferritin and transferrin are regarded as markers of ironmetabolism, and they are fully associated with one another.Ferritin is an iron storage protein consisting of 2 subunits, a heavyand light chain, encoded by separate genes that sequester iron inthe ferric (Fe31) state. Ferritin is an acute-phase reactant withclinical implications as a serologic marker of acute and chronicinflammation. It often increases during periods of oxidative stresssuch as acute respiratory distress syndrome (ARDS)22,23 or cysticfibrosis24 for the detoxification of free iron, because free iron fa-cilitates the formation of highly toxic hydroxyl radicals from hy-drogen peroxide.25,26 In addition, polymorphisms within the genethat encodes the ferritin light chain are associated with the devel-opment of ARDS.27 Thus, altered intracellular and extracellularferritin expression may have implications in the development oflung inflammation, including ARDS. Consistent with our results,a recent study reported that epithelial cells treated with carbonmonoxide (CO) gas, which has a high affinity for ferrous ironand can alter iron metabolism in bronchial epithelial cells,28 ex-hibited a decreased ferritin concentration. Some studies haveshown reduced ferritin levels29 and increased transferrin levels30

in the sera of patients with asthma. Transferrin is an antioxidantthat binds iron in the plasma. The iron delivered by transferrinmay be associated with increased oxidative cell injury,31 because

J ALLERGY CLIN IMMUNOL

OCTOBER 2008

778 HUR ET AL

FIG 2. Comparison of the serum concentrations of log ferritin (A) and log transferrin (B) in subjects with

MDI-OA/EB, AECs, and NCs. *All data are adjusted by age and sex. Dotted line indicates the mean of

each value.

hypotransferrinemic mice are resistant to injury by hyperoxia andmetal particles.32,33 Although the mechanisms have not been fullyclarified, our results indicate that ferritin was downregulatedwhereas transferrin was upregulated in the BALF of the patientswith MDI-OA/EB. These findings were validated by our ELISAresults, which showed a significant decrease in serum ferritin inthe MDI-OA/EB group compared with the AEC and NC groups,and a higher transferrin level in the MDI-OA/EB group comparedwith the AEC and NC groups. Serum ferritin and transferrin levelswere negatively correlated with one another (r 5 –0.786; P 5

.036).Increased epithelial production of reactive oxygen species is

accepted as a major pathogenic mechanism of isocyanate-inducedOA. It was previously shown in vitro study that exposure to isocy-anates, including TDI, hexamethylene diisocyanate, and MDI, in-creased the level of intracellular peroxide,34 whereas in vivoantioxidants were found to decrease TDI-induced airway inflam-mation.35 On the basis of these findings, reactive oxygen speciesproduction may be one pathogenic mechanism of MDI-OA. Fer-ritin and transferrin are both antioxidants, but they have differentroles; ferritin is used for detoxification during oxidative stress–in-duced inflammations, whereas hypotransferrinemia is associatedwith resistance to oxidative injury. Therefore, some susceptiblesubjects with defects in iron metabolism and lower serum ferritinlevels may develop MDI-OA after MDI exposure, whereas some

TABLE III. Comparisons of log ferritin and transferrin values

between study groups

Log ferritin P value Log transferrin P value

ANOVA* .065 ANOVA* <.001

MDI-OA/EB vs AEC .059 MDI-OA/EB vs AEC .028

MDI-OA/EB vs NC .104 MDI-OA/EB vs NC 1.000

AEC vs NC 1.000 AEC vs NC <.001

ANCOVA� .007 ANCOVA� .001

MDI-OA/EB vs AEC .022 MDI-OA/EB vs AEC .009

MDI-OA/EB vs NC .002 MDI-OA/EB vs NC .458

AEC vs NC .064 AEC vs NC .001

*ANOVA with Bonferroni correction.

�ANCOVA with Bonferroni correction to adjust for age and sex.

subjects with lower serum transferrin levels may be resistant toMDI exposure. We speculate that ferritin acts defensively againstMDI, and that some subjects with an impaired ferritin level maybe more susceptible to MDI-OA than those with a normal ironmetabolism when exposed to MDI.

Several upregulated and downregulated proteins were identi-fied in the BALF of the subjects with MDI-OA and the AECs. Forexample, vitamin DBP was upregulated in the MDI-OA group.Vitamin D metabolites are known to be associated with immunemodulation of TH2 inflammation,36 and genetic polymorphismsof vitamin D receptor are associated with an individual’s suscep-tibility to asthma and atopy.37-39 Therefore, consistent with ourresults, vitamin D metabolism seems to contribute to the patho-genesis of asthma. In comparison, gelsolin, a calcium-activatedactin filament, was downregulated in the MDI-OA group. Gelso-lin has been linked to several pathologic conditions, including in-flammation, cancer, and amyloidosis40; it is secreted frombronchial epithelial cells and is related to increased mucus pro-duction in patients with asthma.41,42 Finally, multiple annexinswere downregulated in the MDI-OA group. Annexins are water-soluble proteins that form voltage-dependent calcium channelswithin planar lipid bilayers,43 and they are expressed at reducedlevels in asthmatic mice.42 Additional studies are needed to eval-uate the clinical relevance of each protein for MDI-exposedworkers.

Developing a useful serologic marker for identifying suscep-tible patients from among isocyanate-exposed workers is essen-tial. In terms of the long-term prognosis of TDI-OA, it waspreviously shown that more than 50% of patients with TDI-OAhad persistent asthmatic symptoms in spite of complete avoid-ance.8 Therefore, the early detection of susceptible workers iscritical to prevent permanent impairment of lung function in pa-tients with isocyanate occupational asthma. Several serologicmarkers have been considered in an effort to improve the predict-ability of isocyanate-induced asthma. In the case of TDI-OA,specific IgE and IgG antibodies to TDI–human serum albuminconjugate were suggested as a useful diagnostic marker, but thesensitivity was only around 20% to 30%.3,44 Recently, a volatileform of TDI with 43.9% diagnostic sensitivity was used to dis-criminate between TDI-OA and AEC.5 However, in the case ofMDI-OA, only 1 study has shown that serum specific IgG

J ALLERGY CLIN IMMUNOL

VOLUME 122, NUMBER 4

HUR ET AL 779

FIG 3. ROC curves for the serum levels of ferritin (A) and transferrin (B), as well as the combined value (C)

used to determine the optimal cutoffs for MDI-OA. Arrows indicate the optimal value for ferritin, transferrin,

and the combined value (ferritin � 69.84 ng/mL and transferrin � 2.48 mg/mL). Gray regions represent the

sensitivity range (60-100) and specificity range (50-100).

antibodies may be a useful predictive factor (57.1% sensitivity).7

In this study, we evaluated the diagnostic value of serum ferritinand transferrin measurements in identifying MDI-OA/EB. We ap-plied ROC curves to compute the optimal values and selectedthose areas with more than 60% sensitivity and more than 50%specificity (Fig 3). We obtained the optimal AUC values basedon those values, although the serum ferritin cutoff value affordedsuperior sensitivity and specificity compared with the serumtransferrin cutoff value (sensitivity, 85.71% vs 71.43%; specific-ity, 71.48% vs 51.02%). When we combined ferritin with transfer-rin, however, the P value improved (P value, ferritin only, 0.053;vs ferritin with transferrin, .020). Although a follow-up longitudi-nal study will be essential to confirm whether these 2 markers willbe useful for predicting development of MDI-induced asthma in alarger cohort, on the basis of these findings, we suggest that the 2combined serologic markers (serum ferritin �69.84 ng/mL andserum transferrin �2.48 mg/mL) can be used to discriminate be-tween subjects with MDI-OA/EB and the AECs from amongMDI-exposed workers with 71.43% sensitivity and 85.71%specificity.

In conclusion, this study is the first to consider differentialprotein expression in the BALF of subjects with MDI-OA andAECs. We found 23 differentially expressed (more than 2-fold)protein spots after a MDI-specific inhalation challenge test inpatients with MDI-OA. Among them, the ferritin light chain wasdownregulated and whereas transferrin was upregulated, andthese results were validated by using sera from exposed workers.Our findings suggest that combined serum ferritin and transferrin(ferritin�69.84 ng/mL and transferrin�2.48 mg/mL) testing canbe used to identify cases of MDI-OA from among the MDI-exposed workers.

TABLE IV. Sensitivity, specificity, and the values of the area

under the ROC with 95% CI and P values

Cutoffs

Sensitivity

(%)

Specificity

(%) AUC (95% CI)

P

value*

Ferritin

� 69.84 ng/mL

85.71 71.48 0.786 (0.614-0.957) .053

Transferrin � 2.48

mg/mL

71.43 51.02 0.612 (0.396-0.829) .400

Ferritin � 69.84 ng/

mL and transferrin

� 2.48 mg/mL

71.43 85.71 0.786 (0.580-0.991) .020

*Values are adjusted by age and sex.

We thank Dr Eun-Young Lee (Yonsei Proteome Research Center, Seoul,

Korea) for assistance with the proteomic analysis.

Clinical implications: Serum ferritin and transferrin can beused as serologic markers for identifying MDI-OA in MDI-ex-posed workers.

REFERENCES

1. Mapp CE. Agents, old and new, causing occupational asthma. Occup Environ Med

2001;58:354-60.

2. Mapp CE, Boschetto P, Maestrelli P, Fabbri LM. Occupational asthma. Am J Re-

spir Crit Care Med 2005;172:280-305.

3. Park HS, Kim HY, Nahm DH, Son JW, Kim YY. Specific IgG, but not specific IgE,

antibodies to toluene diisocyanate-human serum albumin conjugate are associated

with toluene diisocyanate bronchoprovocation test results. J Allergy Clin Immunol

1999;104:847-51.

4. Park HS, Lee SK, Kim HY, Nahm DH, Kim SS. Specific immunoglobulin E and

immunoglobulin G antibodies to toluene diisocyanate-human serum albumin con-

jugate: useful markers for predicting long-term prognosis in toluene diisocyanate-

induced asthma. Clin Exp Allergy 2002;32:551-5.

5. Ye YM, Kim CW, Kim HR, Kim HM, Suh CH, Nahm DH, et al. Biophysical

determinants of toluene diisocyanate antigenicity associated with exposure and

asthma. J Allergy Clin Immunol 2006;118:885-91.

6. Choi JH, Nahm DH, Kim SH, Kim YS, Suh CH, Park HS, et al. Increased levels of

IgG to cytokeratin 19 in sera of patients with toluene diisocyanate-induced asthma.

Ann Allergy Asthma Immunol 2004;93:293-8.

7. Hur GY, Koh DH, Choi GS, Park HJ, Choi SJ, Ye YM, et al. Clinical and immu-

nologic findings of methylene diphenyl diisocyanate-induced occupational asthma

in a car upholstery factory. Clin Exp Allergy 2008;38:586-93.

8. Park HS, Cho SH, Hong CS, Kim YY. Isocyanate-induced occupational asthma in

far-east Asia: pathogenesis to prognosis. Clin Exp Allergy 2002;32:198-204.

9. Crameri R. The potential of proteomics and peptidomics for allergy and asthma

research. Allergy 2005;60:1227-37.

10. Wu J, Kobayashi M, Sousa EA, Liu W, Cai J, Goldman SJ, et al. Differential pro-

teomic analysis of bronchoalveolar lavage fluid in asthmatics following segmental

antigen challenge. Mol Cell Proteomics 2005;4:1251-64.

11. Lee SH, Rhim T, Choi YS, Min JW, Kim SH, Cho SY, et al. Complement C3a and

C4a increased in plasma of patients with aspirin-induced asthma. Am J Respir Crit

Care Med 2006;173:370-8.

12. Murphy VE, Johnson RF, Wang YC, Akinsanya K, Gibson PG, Smith R, et al. Pro-

teomic study of plasma proteins in pregnant women with asthma. Respirology

2006;11:41-8.

13. Hur GY, Koh DH, Kim HA, Park HJ, Ye YM, Kim KS, et al. Prevalence of work-

related symptoms and serum-specific antibodies to wheat flour in exposed workers

in the bakery industry. Respir Med 2008;102:548-55.

14. Quirce S. Eosinophilic bronchitis in the workplace. Curr Opin Allergy Clin Immu-

nol 2004;4:87-91.

15. Crapo RO, Casaburi R, Coates AL, Enright PL, Hankinson JL, Irvin CG, et al.

Guidelines for methacholine and exercise challenge testing-1999. This official

statement of the American Thoracic Society was adopted by the ATS Board of Di-

rectors, July 1999. Am J Respir Crit Care Med 2000;161:309-29.

J ALLERGY CLIN IMMUNOL

OCTOBER 2008

780 HUR ET AL

16. Zammit-Tabona M, Sherkin M, Kijek K, Chan H, Chan-Yeung M. Asthma caused

by diphenylmethane diisocyanate in foundry workers: clinical, bronchial provoca-

tion, and immunologic studies. Am Rev Respir Dis 1983;128:226-30.

17. Cho YM, Bae SH, Choi BK, Cho SY, Song CW, Yoo JK, et al. Differential expres-

sion of the liver proteome in senescence accelerated mice. Proteomics 2003;3:

1883-94.

18. Choi BK, Cho YM, Bae SH, Zoubaulis CC, Paik YK. Single-step perfusion

chromatography with a throughput potential for enhanced peptide detection by

matrix-assisted laser desorption/ ionization-mass spectrometry. Proteomics 2003;

3:1955-61.

19. Shevchenko A, Wilm M, Vorm O, Mann M. Mass spectrometric sequencing of pro-

teins silver-stained polyacrylamide gels. Anal Chem 1996;68:850-8.

20. Hanley JA, McNeil BJ. The meaning and use of the area under a receiver operating

characteristic (ROC) curve. Radiology 1982;143:29-36.

21. Grube E, Bootsveld A, Buellesfeld L, Yuecel S, Shen JT, Imhoff M. Computerized

two-lead resting ECG analysis for the detection of coronary artery stenosis after

coronary revascularization. Int J Med Sci 2008;5:50-61.

22. Connelly KG, Moss M, Parsons PE, Moore EE, Moore FA, Giclas PC, et al. Serum

ferritin as a predictor of the acute respiratory distress syndrome. Am J Respir Crit

Care Med 1997;155:21-5.

23. Ghio AJ, Carter JD, Richards JH, Richer LD, Grissom CK, Elstad MR. Iron and

iron-related proteins in the lower respiratory tract of patients with acute respiratory

distress syndrome. Crit Care Med 2003;31:395-400.

24. Reid DW, Carroll V, O’May C, Champion A, Kirov SM. Increased airway iron as a

potential factor in the persistence of Pseudomonas aeruginosa infection in cystic

fibrosis. Eur Respir J 2007;30:286-92.

25. Ghio AJ, Turi JL, Yang F, Garrick LM, Garrick MD. Iron homeostasis in the lung.

Biol Res 2006;39:67-77.

26. Turi JL, Yang F, Garrick MD, Piantadosi CA, Ghio AJ. The iron cycle and oxida-

tive stress in the lung. Free Radic Biol Med 2004;36:850-7.

27. Lagan AL, Quinlan GJ, Mumby S, Melley DD, Goldstraw P, Bellingan GJ, et al.

Variation in iron homeostasis genes between patients with ARDS and healthy con-

trol subjects. Chest 2008;133:1302-11.

28. Ghio AJ, Stonehuerner JG, Dailey LA, Richards JH, Madden MD, Deng Z, et al.

Carbon monoxide reversibly alters iron homeostasis and respiratory epithelial cell

function. Am J Respir Cell Mol Biol 2008;38:715-23.

29. Vural H, Uzun K, Uz E, Kocyigit A, Cigli A, Akyol O. Concentrations of copper,

zinc and various elements in serum of patients with bronchial asthma. J Trace Elem

Med Biol 2000;14:88-91.

30. Brogan TD, Ryley HC, Neale L, Yassa J. Soluble proteins of bronchopulmonary secre-

tions from patients with cystic fibrosis, asthma, and bronchitis. Thorax 1975;30:72-9.

31. Breuer W, Greenberg E, Cabantchik ZI. Newly delivered transferrin iron and oxi-

dative cell injury. FEBS Lett 1997;403:213-9.

32. Ghio AJ, Carter JD, Richards JH, Crissman KM, Bobb HH, Yang F. Diminished

injury in hypotransferrinemic mice after exposure to a metal-rich particle. Am J

Physiol Lung Cell Mol Physiol 2000;278:L1051-61.

33. Yang F, Coalson JJ, Bobb HH, Carter JD, Banu J, Ghio AJ. Resistance of hypotrans-

ferrinemic mice to hyperoxia-induced lung injury. Am J Physiol 1999;277:L1214-23.

34. Elms J, Beckett PN, Griffin P, Curran AD. Mechanisms of isocyanate sensitisation:

an in vitro approach. Toxicol In Vitro 2001;15:631-4.

35. Kim SH, Park HJ, Lee CM, Choi IW, Moon DO, Roh HJ, et al. Epigallocatechin-3-

gallate protects toluene diisocyanate-induced airway inflammation in a murine

model of asthma. FEBS Lett 2006;580:1883-90.

36. Wittke A, Weaver V, Mahon BD, August A, Cantorna MT. Vitamin D receptor-

deficient mice fail to develop experimental allergic asthma. J Immunol 2004;

173:3432-6.

37. Poon AH, Laprise C, Lemire M, Montpetit A, Sinnett D, Schurr E, et al. Associ-

ation of vitamin D receptor genetic variants with susceptibility to asthma and at-

opy. Am J Respir Crit Care Med 2004;170:967-73.

38. Raby BA, Lazarus R, Silverman EK, Lake S, Lange C, Wjst M, et al. Association

of vitamin D receptor gene polymorphisms with childhood and adult asthma. Am J

Respir Crit Care Med 2004;170:1057-65.

39. Vollmert C, Illig T, Altmuller J, Klugbauer S, Loesgen S, Dumitrescu L, et al.

Single nucleotide polymorphism screening and association analysis: exclusion of

integrin beta 7 and vitamin D receptor (chromosome 12q) as candidate genes for

asthma. Clin Exp Allergy 2004;34:1841-50.

40. Spinardi L, Witke W. Gelsolin and diseases. Subcell Biochem 2007;45:55-69.

41. Candiano G, Bruschi M, Pedemonte N, Caci E, Liberatori S, Bini L, et al. Gelsolin

secretion in interleukin-4-treated bronchial epithelia and in asthmatic airways. Am

J Respir Crit Care Med 2005;172:1090-6.

42. Houtman R, Krijgsveld J, Kool M, Romijn EP, Redegeld FA, Nijkamp FP, et al.

Lung proteome alterations in a mouse model for nonallergic asthma. Proteomics

2003;3:2008-18.

43. Kourie JI, Wood HB. Biophysical and molecular properties of annexin-formed

channels. Prog Biophys Mol Biol 2000;73:91-134.

44. Cartier A, Grammer L, Malo JL, Lagier F, Ghezzo H, Harris K, et al. Specific

serum antibodies against isocyanates: association with occupational asthma. J

Allergy Clin Immunol 1989;84:507-14.