Human hemoglobin adducts following exposure to hexahydrophthalic anhydride and methylhexahydrophthalic anhydride

Hexahydrophthalic anhydride (HHPA) and methylhexahydrophthalic anhydride (MHHPA) are highly allergenic compounds used in the chemical industry. The aim of this study was to characterize the protein adducts in erythrocytes following exposure to HHPA and MHHPA. Blood and urine samples were obtained from 51 HHPA- and MHHPA-exposed workers. Erythrocytic proteins from HHPA- and MHHPA-exposed workers were fractionated by gel filtration and ion exchange chromatography. In vitro synthesized conjugates between tritium-labeled and unlabeled HHPA and hemoglobin (Hb) were hydrolyzed by acid or digested by Pronase E. Levels of in vivo formed anhydride-Hb adducts and urinary/plasma levels of the corresponding acids were analyzed by gas chromatography-mass spectrometry (GC-MS) and correlated. The decay of adducts was studied in workers leaving employment or during vacation. More than 85% of the adduct forming protein in vivo coeluted with Hb in gel filtration and ion exchange chromatography. At least 70% of the HHPA in the in vitro formed adducts was found on lysine by GC-MS. Similar findings were obtained using Pronase E-digested tritium-labeled Hb-HHPA. The adduct levels in workers ranged 0-26 pmol/g Hb (mean 2. 7 pmol/g Hb) for HHPA, and the range for MHHPA was 0-55 pmol/g Hb (mean 4.1 pmol/g Hb). The Spearman's rank correlation coefficient between urine data and adducts was for HHPA rs = 0.80 and for MHHPA, rs = 0.78. For the plasma, the correlation using HHPA data was rs = 0.80 and for MHHPA, rs = 0.69. The adducts seemed to be stable in vivo. The adduct levels may be used as biomarkers of exposure to HHPA and MHHPA.