Abstract for Poster 30

 

 

Assessment of Acrylamide Exposure by Measuring of Hemoglobin Adducts

M. Ospina*, H. Vesper, T. Meyers, A. Smith, L. Ingham, G. Gray, G Myers
Centers for Disease Control and Prevention, Atlanta, United States

Background

Acrylamide (AA) is a chemical used in a variety of applications ranging from the production of grouting agents for the construction bussiness to production of polyacrylamide for clarification of drinking waters. Occupational exposure to acrylamide mainly occurs through skin and inhalation. Some cosmetic preparations contain polyacrylamides due to their function as stabilisers, foam builders, binders, film formers, antistatic agents and hair fixatives. These are also souces of acrylamide exposure due to acrylamide monomer residues present in these preaprations. AA is a known neurotoxicant and has been classified as a potential human carcinogen. Acrylamide can be absorbed through skin, mucous membranes, lungs and the gastrointestinal tract. Acrylamide can cause skin irritation characterized by a rash or burning feeling on contact. Sensitivity can build up with repeated exposure. AA is metabolized to Glycidamide (GA), assumed to be the genotoxic metabolite of acrylamide. AA and GA react with hemoglobin to form hemoglobin adducts. Hemoglobin adduct analysis is used to assess and estimate the dose associated with exposure to chemicals, as well as the potential effects of these exposures.

Methods

Globin is derivatized with pentafluorophenylisothiocyanate (PFPITC) for 3h at 45C. The derivatives are extracted with liquid-liquid extraction procedure using isopropyl ether as extractant. Octapeptides (VHLTPEEK) with acrylamide or glycidamide added to the N-terminal valine were synthesized by Bachem Bioscience (King of Prusia, PA) and used as calibrators. The same peptides with a 13C5, 15N-labeled valine were used as internal standards. Samples are analyzed by isotope dilution LC/MS/MS with atmospheric pressure chemical ionization. Separation of the adducts was obtained with a Luna C18 (2) 100 x 2.1 mm column using a methanol gradient at 300 uL/min. A Finnigan TSQ Quantum Ultra mass spectrometer equipped with atmospheric pressure chemical ionization and operated in positive-ion mode and single reaction monitoring (SRM) was used for analysis of samples. Concentrations of samples were calculated from the slope and intercept obtained from the linear regresion of the calibration curves.

Results

Calibration curves were linear for both adducts from 2-100 nM, with r2 higher than 0.99. Using 50 mg globin, the detection limits for these hemoglobin adducts were 7 pmol /g globin for GA adducts and 3 pmol/g globin for AA-adducts. Intra-day and interassay variability were determined using QC batches of three different levels over 20 different days. 

Conclusions

A method to measure acrylamide and glycidamide hemoglobin adducts was developed. The use of isotopically labeled standards corrects for the losses occurred throughout the process, improving the precision. The method was characterized using globin isolated from smokers and non-mokers. The use of atmospheric pressure chemical ionization in the positive ion mode for the mass spectrometric analysis of hemoglobin adducts minimized ion suppression effects observed with electrospray ionization. The use of liquid chromatography for separation instead of gas chromatography used in traditional methods of analysis for these adducts, eliminated the need for one more derivatization step required for determination of glycidamide. The LOD`s indicate that this method can be used for the measuring acrylamide and glycidamide hemoglobin adducts not only in exposed populations but also for the determination of background levels in the general population. 

 

Content last modified: 17 May 2005