Aldehyde fixatives: Quantification of acid‐producing reactions

Abstract Under conditions commonly used for the preservation of tissue for electron microscopy, substantial amounts of hydrogen ions were produced when either formaldehyde or glutaraldehyde was added to solutions of amines or proteins, or to tissue homogenates. In glycine‐aldehyde reactions, hydrogen ions were generated in linear proportion to the glycine concentration over the range of 0.001 M to 0.100 M glycine. In the presence of 1% formaldehyde or 1% glutaraldehyde, 0.3–0.9 equivalents of acid were produced per mole of primary amine. This variation is a function of the specific primary amine used. Acid production increased with increasing formaldehyde concentration but did not increase appreciably with increasing glutaraldehyde concentration. The concentration of amines in tissue is high. These amines are probably responsible for much of the acid generated when aldehydes react with tissue homogenates. In amine‐aldehyde reactions, the positive charge originally associated with the parent amine is simultaneously neutralized with the formation of hydrogen ions. Thus, in tissues exposed to aldehydes, there is a decrease in the overall positive charge which is related to the amount of acid generated. The inherent buffering capacity of liver and muscle was not sufficient to prevent large pH decreases when aldehydes were added to homogenates of these tissues. These data suggest that significant but transient pH decreases may occur within cells exposed to aldehydes. To minimize the aldehyde‐induced pH decreases, a buffer should be chosen so that its pKa is 0.2–0.3 pH units less than the pH desired for a particular experiment. In addition, buffer concentrations of at least 0.10 M are suggested for fixatives in order to provide adequate buffering capacity during tissue preservation.