Quantification of protein adducts of hexahydrophthalic anhydride and methylhexahydrophthalic anhydride in human plasma

Abstract

Hexahydrophthalic anhydride (HHPA) and methylhexahydrophthalic anhydride (MHHPA) are two highly allergenic compounds used in the chemical industry. A method was developed for quantification of protein adducts of HHPA and MHHPA in human plasma. The plasma was dialysed and the anhydrides were hydrolysed from the proteins at mild acidic conditions. The released hexahydrophthalic acid (HHP acid) and methylhexahydrophthalic acid (MHHP acid) were purified by reversed solid phase extraction followed by derivatisation with pentafluorobenzyl bromide. The derivatives were analysed using GC-MS in negative ion chemical ionisation mode with ammonia as moderating gas. As internal standards, deuterium labelled HHP and MHHP acids were used. The detection limits were 0.06 pmol mL(-1) plasma for HHP acid and 0.03 pmol mL(-1) plasma for MHHP acid. The between-day precisions for HHP acid were 18% at 0.3 pmol mL(-1) and 8% at 4 pmol mL(-1). For MHHP acid, the precisions were 13% at 0.3 pmol mL(-1) and 9% at 4 pmol mL(-1). There were strong correlations (r=0.94 for HHPA and 0.99 for MHHPA) between total plasma protein adduct concentrations and serum albumin adduct levels. Workers exposed to time-weighted average air levels of HHPA between < 1 and 340 microg m(-3) and between 2 and 160 microg m(-3) for MHHPA had plasma adduct levels between the detection limits of the methods and 8.40 and 19.0 pmol mL(-1), respectively.

Total plasma protein adducts of allergenic hexahydrophthalic and methylhexahydrophthalic anhydrides as biomarkers of long-term exposure

“…In fact, the obtained correlations were far better than any previously reported associations between exposure and adduct levels in general. In addition, as reported in an earlier study, we found high correlations between the TPPA levels and the adducts with merely the HSA in 10 workers with differing protein levels (22). Furthermore, in this study, we found a close correlation also between TPPA and the Hb adducts of HHPA.…”

“…In this report we have used a recently developed method to quantify the levels of TPPA (22). This method is easy and convenient, and it dispenses with tedious, labor-intensive protein isolation steps.…”

“…The levels of HHPA and MHHPA bound to plasma proteins were determined as published recently (22). Thus aliquots of plasma were dialyzed, and the bound HHPA and MHHPA were hydrolyzed from the proteins to the corresponding acids.…”

Exposure-response relationships for hexahydrophthalic and methylhexahydrophthalic anhydrides with total plasma protein adducts as biomarkers

“…The total plasma protein adducts of HHPA and MHHPA were quantified using a recently published gas chromatographic-mass spectrometric (GC-MS) method (9). Briefly, aliquots of plasma were dialyzed, and the adducted HHPA and MHHPA were hydrolyzed from the proteins to the corresponding acids.…”

“…However, despite the potential of protein adducts as biomarkers of exposure, development in the area has been remarkably slow, probably because of the rather laborious laboratory procedures coupled with the high costs of the equipment needed for analyzing the samples. Recently, we reported a convenient method for analyzing the total plasma protein adducts of HHPA and MHHPA (9) and showed that these adducts are excellent biomarkers of long-term exposure to the anhydrides (10).…”