Alternate putrescine metabolites: quantitative analysis of delta'-pyrroline oxidation to 2-pyrrolidone in tissue homogenates by high-pressure liquid chromatography

Anal Biochem. 1985 Aug 1;148(2):461-70. doi: 10.1016/0003-2697(85)90253-2.


A sensitive analytical procedure for following the oxidation of delta'-pyrroline to 2-pyrrolidone in tissue homogenates is described. Homogenates are extracted with chloroform/acetonitrile and fractionated by high-performance liquid chromatography, and 2-pyrrolidone is quantitated by monitoring the column effluent at 200 nm. The lower limit of 2-pyrrolidone that can be accurately (+/- 5%) quantitated is approximately 100 pmol. Phenazine methosulfate significantly enhances the rate of 2-pyrrolidone biosynthesis from delta'-pyrroline. Phenazine methosulfate and reduced glutathione are required to obtain proportionality between 2-pyrrolidone formation and incubation time. Formation of 2-pyrrolidone as a function of protein concentration is linear and 2-pyrrolidone biosynthesis as a function of delta'-pyrroline concentration is characterized by hyperbolic kinetics. Based on analysis of enzyme activity in different tissues, liver appears to play the dominant role in 2-pyrrolidone biosynthesis. The metabolic step from delta'-pyrroline to 2-pyrrolidone was localized in the cellular cytosol. These results demonstrate that the oxidation of delta'-pyrroline to 2-pyrrolidone is enzyme mediated and provide a useful method for further characterization of this metabolic step.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Chromatography, High Pressure Liquid
  • Female
  • Kinetics
  • Liver / enzymology
  • Liver / metabolism*
  • Organ Specificity
  • Oxidation-Reduction
  • Pyrroles / metabolism*
  • Pyrrolidinones / biosynthesis*
  • Rats
  • Rats, Inbred Strains
  • Subcellular Fractions / enzymology


  • Pyrroles
  • Pyrrolidinones
  • delta(1)-pyrroline
  • 2-pyrrolidone