In an effort to minimize artifacts created during biochemical tissue staining procedures, spermine, a naturally occurring, non-toxic and relatively odorless polyamine, was added to the aqueous solutions used in standard staining procedures.  Elimination of these artifacts increases the accuracy of subsequent disease diagnosis. The purpose of the polyamine pretreatment in this novel approach to tissue staining is to react this polyamine with residual formaldehyde to prevent artifacts that result from the occurrence of methylene bridges. Incorporating spermine into this procedure assists in the overall effort to enhance photographic detail. <br/>Because the subject polyamine contains both primary and secondary amino groups, two possible reaction mechanisms were investigated: (1) imine formation via reaction between formaldehyde and the primary amino groups, and (2) ‘methylene bridge’ formation via the reaction between formaldehyde and the secondary amino groups.  Reactions between most aldehydes and primary or secondary amines are well known that result in the formation of imines and enamines respectively.  Imine formation from the reaction of aldehydes and primary amines is a known dehydration reaction.  Because of this, spermine and formaldehyde were reacted in the lab under non-aqueous conditions in order to shift the equilibrium towards the formation of the imine and sequester water molecules released in the reaction.<br/>Because of the unique structure of formaldehyde having no α-carbons, it forms ‘methylene bridges’ between secondary amino groups.  Ordinarily, this reaction would form the expected enamines.  In an effort to avoid polymerization between juxtapositional spermine molecules via ‘methylene bridges,’ spermine was reacted with only two equivalents of formaldehyde that would sterically favor the formation of the imine rather than the enamine. <br/>The current research focuses on limiting the stoichiometry of this reaction to facilitate the exact characterization of our product and prove the validity of the presupposed reaction, that imines are indeed formed.  Its validity will allow for additional investigation of reaction mechanisms that occur during tissue staining that ultimately facilitate disease diagnosis.<br/>

Spermine: Its biochemical use in tissue processing

251st National Meeting (2016)

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