title: A sensitive colorimetric assay for polyamines in erythrocytes using oat seedling polyamine oxidase.
abstract: BACKGROUND
  Most of the polyamines circulating in blood are spermidine (Spd) and spermine (Spm) with only trace amounts of putrescine (Put), and they are mainly localized in erythrocytes. We developed a simple and sensitive colorimetric assay for polyamines in erythrocytes using oat seedling polyamine oxidase (OSPO). The method is based on the unique substrate specificity of OSPO, which is active toward Spd and Spm, but not toward diamines such as Put and cadaverine and monoamines such as histamine.
  
  
  METHODS
  The polyamines, which were purified from packed erythrocytes by weak cation-exchange chromatography, were incubated with OSPO at 37 degrees C for 15 min. In the presence of the H(2)O(2) produced by this polyamine oxidase reaction and a new chromogen, N-(carboxymethylaminocarbonyl)-4,4'-bis(dimethylamino)-diphenylamine sodium salt (DA-64), peroxidase (POD) catalyzes the formation of N-[4-[[4-(dimethylamino)phenyl]imino]-2,5-cyclohexadien-1-ylidene]-N-methylmethanaminium chloride (Bindschedler's Green) having an absorption maximum at 727 nm.
  
  
  RESULTS
  The detection limit was 0.2 microM/l for packed erythrocytes. The within-run and between-run precisions (coefficient of variation, CVs) were 5.6-15.2% and 6.5-16.4%, respectively. Analytical recoveries were 93.3-97.4%. Polyamine values obtained by this assay correlated well with those obtained by an HPLC (y=0.948x + 1.912; r=0.944; n=46).
  
  
  CONCLUSIONS
  This colorimetric assay is simple and highly sensitive and practical for clinical use.
authors.affiliation: Department of Medical Technology, Nagoya University School of Health Sciences, 1-1-20 Daikominami, Nagoya 461-8673, Higashi, Japan. ktakagi@met.nagoya-u.ac.jp
authors.forename: Kenji
authors.initials: K
authors.lastname: Takagi
authors.forename: Yasuaki
authors.initials: Y
authors.lastname: Tatsumi
authors.forename: Kiyoyuki
authors.initials: K
authors.lastname: Kitaichi
authors.forename: Mitsunori
authors.initials: M
authors.lastname: Iwase
authors.forename: Eiji
authors.initials: E
authors.lastname: Shibata
authors.forename: Makoto
authors.initials: M
authors.lastname: Nakao
authors.forename: Takatoshi
authors.initials: T
authors.lastname: Matsumoto
authors.forename: Kenzo
authors.initials: K
authors.lastname: Takagi
authors.forename: Takaaki
authors.initials: T
authors.lastname: Hasegawa
mesh_terms: D000328:Adult; D018554:Avena / Q000201:enzymology*; D003124:Colorimetry / Q000379:methods*; D004912:Erythrocytes / Q000737:chemistry*; D005260:Female; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008297:Male; D008875:Middle Aged; D015394:Molecular Structure; D010084:Oxidation-Reduction; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000302:isolation & purification / Q000378:metabolism*; D010545:Peroxides / Q000378:metabolism; D011073:Polyamines / Q000097:blood*; D036226:Seedlings / Q000201:enzymology*; D012680:Sensitivity and Specificity; D013053:Spectrophotometry; D013997:Time Factors; D000097667:Polyamine Oxidase
chemical_list: D000587:Oxidoreductases Acting on CH-NH Group Donors; D010545:Peroxides; D011073:Polyamines; D000097667:Polyamine Oxidase