Conversions of Formaldehyde-modified 2-Deoxyadenosine 5'-Monophosphate in Conditions Modeling Formalin-fixed Tissue Dehydration

Vladimir K. Rait ¹, Qingrong Zhang ¹, Daniele Fabris ¹, Jeffrey T. Mason ¹ and Timothy J. O’Leary ^1*

¹ Department of Biophysics (VKR,JTM,TJO), Armed Forces Institute of Pathology, Rockville, Maryland; Department of Chemistry and Biochemistry (QZ,DF), University of Maryland, Baltimore County, Baltimore, Maryland; and Biomedical Laboratory Research and Development Service (TJO), Veteran Health Administration, Washington, District of Columbia

^* To whom correspondence should be addressed. E-mail: timothy.oleary{at}va.gov.

Submitted on April 26, 2005
Accepted on 4 August 2005

	Abstract

Formalin-fixed, paraffin-embedded specimens typically provide molecular biologists with low yields of extractable nucleic acids that exhibit extensive strand cleavage and covalent modification of nucleobases. This study supports the idea that these deleterious effects are promoted by the first step in formalin-fixed tissue processing--i.e., tissue dehydration with a graded series of alcohols. We analyzed the conversions of formaldehyde-modified 2'-deoxyadenosine 5'-monophosphate (dAMP) by reverse-phase ion-pair high performance liquid chromatography and found that dehydration does not stabilize N-methylol groups in the modified nucleotide. Furthermore, spontaneous demodification in a dry state or in anhydrous ethanol can be as fast as it is in aqueous solutions, if the preparation is contaminated with salts of orthophosphoric acid. In ethanol, orthophosphates also catalyze formation of abundant N⁶-ethoxymethyl-dAMP, as well as cross-linking and depurination of nucleotides present in the mixture. Identification of the products was performed using UV absorbance spectroscopy and electrospray ionization Fourier-transform ion cyclotron resonance mass spectrometry. Alternatives to the traditional processing of formalin-fixed tissues are discussed.

Key Words: formalin fixation, dAMP, mixed acetals, crosslinking, depurination, HPLC, ESI FTICR MS

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