Hemoglobin adducts in workers exposed to 1,6-hexamethylene diisocyanate

Flack, Sheila L.; Fent, Kenneth W.; Gaines, Linda G. Trelles; Thomasen, Jennifer M.; Whittaker, Stephen G.; Ball, Louise M.; Nylander‐French, Leena A.

doi:10.3109/1354750x.2010.549242

Cited by 18 publications

(13 citation statements)

References 37 publications

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“…While measurement of the diamine based hydrolysis product has been utilized for bio-monitoring, only one study has reportedly applied this approach to characterize in vitro prepared HSA conjugates (37). Several solvents are commercially available that have been used for the extraction of diamines derived from dNCO hydrolysis; these include toluene, dichloromethane, and ethyl acetate (38;39). Toluene is the most common extraction solvent reported in the literature; however, MDA has reduced solubility in toluene.…”

Section: Discussionmentioning

confidence: 99%

Characterization of methylene diphenyl diisocyanate-haptenated human serum albumin and hemoglobin

Mhike

Chipinda

Hettick

et al. 2013

Analytical Biochemistry

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Protein haptenation by polyurethane industrial intermediate methylene diphenyl diisocyanate (MDI) is thought to be an important step in the development of diisocyanate (dNCO)-specific allergic sensitization; however, MDI haptenated albumins used to screen specific antibody are often poorly characterized. Recently, the need to develop standardized immunoassays using a consistent, well characterized dNCO-haptenated protein to screen for the presence of MDI-specific IgE and IgG from workers’ sera has been emphasized and recognized. This has been challenging to achieve due to the bivalent, electrophilic nature of dNCO leading to the capability to produce multiple cross-linked protein species and polymeric additions to proteins. In the present study, MDI was reacted with human serum albumin (HSA) and hemoglobin (Hb) at molar ratios ranging from 1:1 to 40:1 MDI: protein. Adducts were characterized by (1) loss of available trinitrobenzene sulfonic acid (TNBS) binding to primary amines, (2) electrophoretic migration in polyacrylamide gels, (3) quantification of methylene diphenyl diamine following acid hydrolysis and (4) immunoassay. Concentration dependent changes in all the above noted parameters were observed demonstrating increase in both number and complexity of conjugates formed with increasing MDI concentration. In conclusion, a series of bio-analytical assays should be performed to standardize MDI-antigen preparations across lots and laboratories for measurement of specific antibody in exposed workers which in total indicate degree of intra- and inter-molecular cross-linking, number of dNCO bound, number of different specific binding sites on the protein and degree of immuno-reactivity.

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Section: Discussionmentioning

confidence: 99%

Characterization of methylene diphenyl diisocyanate-haptenated human serum albumin and hemoglobin

Mhike

Chipinda

Hettick

et al. 2013

Analytical Biochemistry

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“…Samples were immediately analyzed by GC-MS (Thermo Trace GC Ultra interfaced with a PolarisQ ion trap mass spectrometer and AI/AS 3000 injector, and Xcalibur 1.4 SR1 software, Thermo Electron Corporation, Austin, TX) in negative chemical ionization mode, according to Flack and colleagues [8], in scan mode ( m/z 200-600) using methane as the reagent gas. Injections (1 μl) were made under splitless mode of 30 s with injector temperature of 220°C.…”

Section: Methodsmentioning

confidence: 99%

“…In addition, workers receive dermal and inhalation exposure to different forms of diisocyanates (e.g., monomers, oligomers, and polymers) simultaneously, further complicating attempts to investigate the relationship between exposure and dose. Nevertheless, several volunteer and occupational studies have demonstrated a good association between 1,6-hexamethylene diamine (HDA), in urine or plasma, and inhalation exposure to HDI monomer [2-8]. …”

Section: Introductionmentioning

confidence: 99%

“…The HDA detection limits in urine or plasma as reported in various publications are in the range of 0.02 – 3.0 μg/l (summarized in Table 1). The high detection limit of HDA in samples, resulting in low analytical sensitivity, has hindered quantification of plasma HDA in several studies [3,6] and resulted in relatively large number of non-detectable levels in both plasma and urine [4,8]. However, measurement of HDA in acid-hydrolyzed plasma among workers exposed to HDI allows for further characterization of blood biomarkers and evaluation of their usefulness as markers of HDI exposure [8].…”

Section: Introductionmentioning

confidence: 99%

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Occupational exposure to HDI: Progress and challenges in biomarker analysis

Flack

Ball

Nylander‐French

2010

Journal of Chromatography B

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Abstract1,6-hexamethylene diisocyanate (HDI) is extensively used in the automotive repair industry and is a commonly reported cause of occupational asthma in industrialized populations. However, the exact pathological mechanism remains uncertain. Characterization and quantification of biomarkers resulting from HDI exposure can fill important knowledge gaps between exposure, susceptibility, and the rise of immunological reactions and sensitization leading to asthma. Here, we discuss existing challenges in HDI biomarker analysis including the quantification of N-acetyl-1,6-hexamethylene diamine (monoacetyl-HDA) and N,N′-diacetyl-1,6-hexamethylene diamine (diacetyl-HDA) in urine samples based on previously established methods for HDA analysis. In addition, we describe the optimization of reaction conditions for the synthesis of monoacetyl-HDA and diacetyl-HDA, and utilize these standards for the quantification of these metabolites in the urine of three occupationally exposed workers. Diacetyl-HDA was present in untreated urine at 0.015 -0.060 μg/l. Using base hydrolysis, the concentration range of monoacetyl-HDA in urine was 0.19 -2.2 μg/l, 60-fold higher than in the untreated samples on average. HDA was detected only in one sample after base hydrolysis (0.026 μg/l). In contrast, acid hydrolysis yielded HDA concentrations ranging from 0.36 to 10.1 μg/ l in these three samples. These findings demonstrate HDI metabolism via N-acetylation metabolic pathway and protein adduct formation resulting from occupational exposure to HDI.

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“…120 days in humans). Peptide mapping and tandem mass spectrometry, coupled with classical biochemical strategies, as enzymatic digestions and the modified Edman degradation, have been extensively applied to investigate protein adducts and to evaluate human exposure to toxicants (18,(27)(28)(29). Another method, based on the quantification of modified hemoglobin peptides, has been proposed, and in some cases successfully applied, for the biological monitoring of workers occupationally exposed to different classes of toxicants such as epichlorohydrin, methyl bromide and butadiene (30)(31)(32), putting in evidence the potentiality of this strategy.…”

Section: Introductionmentioning

confidence: 99%

New perspectives in hemoglobin adducts analysis: selective digestion with Calpain I

Pieri¹

2014

PER

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Background: hemoglobin adducts are considered suitable biomarkers to evaluate human exposure to electrophile agents. One of the procedures used for the molecular dosimetry of hemoglobin adducts is the enzymatic digestion of the protein and the subsequent quantification of modified peptides by mass spectrometry. The aim of the study is to evaluate the ability of Calpain to discriminate between normal and alkylated globin chains. Methods: digestions of pure hemoglobin sample and of an alkylated one with Calpain I at 200:1 and 25:1 weight ratios, for 1, 5 and 18 hrs were carried out. Only the standard deviations (SD) of the ratios between alkylated globins with respect to the unmodified ones (αECH/α and bECH/b) were estimated because the aim of the study is to assess the ability of Calpain I to selectively digest the samples and, above all, to find the optimal digestion conditions and characterize obtained peptides. Results: by using a 200:1 weight ratio only peptides α(1-137), α(1-138) and b(1-142) were produced, pointing out that the first proteolytic sites are located at the C-terminus ends of both α-and b-chains. The identification of peptides obtained from the 25:1 digestions allowed the determination of internal secondary proteolytic sites, not reported in literature. In both cases, a non-negligible proteolysis was observed only when a reaction time of 18 hrs was used. Discussion: results showed a progressive enrichment of modified hemoglobin with respect to the unmodified one, particularly for the α-chain and represent a promising initial step in the development of a selective purification procedure to be applied to protein adducts analysis.

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Hemoglobin adducts in workers exposed to 1,6-hexamethylene diisocyanate

Cited by 18 publications

References 37 publications

Characterization of methylene diphenyl diisocyanate-haptenated human serum albumin and hemoglobin

Characterization of methylene diphenyl diisocyanate-haptenated human serum albumin and hemoglobin

Occupational exposure to HDI: Progress and challenges in biomarker analysis

New perspectives in hemoglobin adducts analysis: selective digestion with Calpain I

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