LC-UV-MS and MS/MS Characterize Glutathione Reactivity with Different Isomers (2,2' and 2,4' vs. 4,4') of Methylene Diphenyl-Diisocyanate.

Abstract

Methylene diphenyl diisocyanate (MDI), the most abundantly produced diisocyanate worldwide, is among the best recognized chemical causes of occupational asthma. The bulk of synthesized MDI, the 4,4' isomer, has been the focus of most biochemical research to date. The biological reactivity of other MDI isomers (2,2' and 2,4'), present at concentrations approaching 50% in some commercial products, remains less clear. We hypothesized 2,2' and 2,4' MDI react with glutathione (GSH), a major anti-oxidant of the lower airways, similarly to 4,4' MDI, and that the products could be characterized using a combination of LC-UV-MS and MS/MS. Purified 2,2' and 2,4' MDI isomers were mixed with GSH in pH-buffered aqueous phase at 37°C and reaction products were analyzed at varying time points. Within minutes, S-linked bis(GSH)-MDI conjugates were detectable as the dominant [M+H]⁺ ion, with an 865.25 m/z and more intense [M+2H]²⁺ ions of the same nominal mass. Upon longer reaction, [M+H]+ ions with greater retention times and the 558.17 m/z expected for mono(GSH)-MDI reaction products were observed, and exhibited MS/MS collision-induced dissociation (CID)-fragmentation patterns consistent with cyclized structures. Compared with 4,4' MDI, 2,2' and 2,4' isomers exhibit similar rapid reactivity with GSH and formation of bis(GSH)-MDI conjugates, but greater formation of cyclized mono(GSH) conjugates following extended reaction times (10 minutes to 2 hours). Further translational studies will be required to determine if the present in vitro findings extend to the complex lower airway microenvironment in vivo.