We are exposed to many chemicals in our everyday life. Some of these chemicals could pose risks for our health. To reduce such risks, it is important to know what we are exposed to, how much, and how toxic the chemicals are.

This thesis focuses on development and novel applications of a method for identifying and quantifying adducts as biomarkers of exposure to electrophiles. Electrophiles are reactive and can be measured as their stable reaction products (adducts) with haemoglobin (Hb) using the FIRE procedure™. This method utilizes Fluorescein Isothiocyanate to selectively cleave the adducted (R) N-terminal valine in human Hb, with a modified Edman procedure.

The primary aim was to further develop the FIRE procedure for application to a larger number of human blood samples, and to decrease the amount of sample needed. To achieve this, the method was adapted to 96-well plates. Further improvements resulted in a method that uses ca. half of the material used in the original method and can be used to analyse 1000 samples in one to two months.

The newly developed version of the FIRE procedure was applied to analyse blood from 144 Norwegian adults (Paper I) and 600 Swedish adolescents (Paper II), to estimate exposure to the electrophile acrylamide. IARC has classified acrylamide as a probable human carcinogen. Diet is a major source of exposure to acrylamide in the general population, as it is formed in carbohydrate-rich food during high temperature processing. Acrylamide intake was calculated from Hb adduct levels and compared with intake estimates calculated from self-reported food consumption data obtained from dietary records and food frequency questionnaires, combined with data for acrylamide content in food. In the Norwegian study, acrylamide intake was estimated by probabilistic calculations of the two types of food consumption data, which resulted in that no large difference in the median estimate obtained by these methods was observed in comparison of estimated daily intake from Hb adduct data (0.24-0.30 µg/kg body weight). In both studies (Papers I, II), the calculated margin of exposure with regard to risk for cancer indicates that acrylamide intake from food is of concern in the studied populations.

In Paper III, an unknown adduct, observed in earlier FIRE adductomics work, was identified with the help of high resolution accurate mass spectrometry, a synthesized standard, and nuclear magnetic resonance spectroscopy. A strategy to trace the source of the adduct was evaluated, and the epoxide glycidic acid was confirmed as a possible precursor, by measurement of adduct formation rate in vitro in human blood.

Finally, in Paper IV the FIRE procedure was compared to bottom-up proteomics to study Hb adducts from acrylamide, acrylic acid, glycidic acid, 2,3-epoxypropyl phenyl ether, 2-methyleneglutaronitrile, and 1-chloro-2,4-dinitrobenzene (DNCB). Adducts from all electrophiles were identified with bottom-up proteomics, with Cys93 in the beta chain of Hb as the most reactive side chain. The FIRE procedure was inefficient to detect bulkier/electron-withdrawing adducts, as from the contact allergen DNCB.

In conclusion, FIRE has high detectability for Hb adducts, although it is not suitable for adducts from all electrophiles. It is a valuable tool to identify and measure adducts, and to estimate exposure/intake of electrophiles. The new version of FIRE would facilitate the application for exposure measurement in epidemiological studies.