A study of the quantitative estimation of ethanolamine and serine in phospholipides.

Among the more commonly recognized constituents of naturally occurring phospholipides are phosphatidyl ethanolamine and phosphatidyl serine. The most feasible analytical procedures for these components have been based on the determination of ethanolamine and serine in a hydrolysate of the lipide. In general, assays for serine and ethanolamine have consisted of three main steps: (a) hydrolysis of the lipide, (b) separation of serine and ethanolamine or their derivatives, and (c) quantitative estimation of the serine and ethanolamine or their derivatives. An exception is the method of Burmaster (1) in which ethanolamine is determined as the difference in ammonia freed by periodate oxidation and carbon dioxide freed by ninhydrin oxidation. In a consideration of the hydrolytic release of these amino compounds from the intact lipide, basic hydrolysis, except as outlined by Axelrod et al. (2) and more recently Nojima and Utsugi (3), has not been in extensive use due to the well known instability of serine and ethanolamine under these conditions. Moreover, the difficulty of removing the base or, after neutralization, its salt from the hydrolysate has posed a problem. Although these two amine compounds are not completely stable to acid hydrolysis, this approach, especially with ease of removal of an acid such as HCl, has been the method of choice. Paper chromatography (4, 5), fractionation on ion exchangers such as Permutit (6, 7) or Zeo-Karb (8), and solvent fractionation of the DNPl derivatives (2, 3) have been used for the separation of the serine and ethanolamine .in hydrolysates. Subsequently, quantitative estimation has been performed through calorimetric assay (4, 7) and by direct photometry (5) after paper chromatography (5), by titrimetric assay of ammonia released by periodate oxidation of the bases after separation on ion exchangers (6), and by spectrophotometric assay of the DNP derivatives (2, 3). Other methods of analysis have employed fractional distillation of ethanolamine (9) and determination of amino acid nitrogen as a measure of serine (10). In the course of an investigation of the fractionation of phospholipides, it became apparent that many of the available methods for serine and ethanolamine were for one reason or another, cited below, unsatisfactory. In a reinvestigation of this problem, the stability of serine and ethanolamine under varying conditions of hydrolysis, and the hydrolysis products from a phosphatidyl ethanolamine-phosphatidyl serine fraction