A sensitive colorimetric assay for polyamines in erythrocytes using oat seedling polyamine oxidase.
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.
Takagi
Kenji
K
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
Tatsumi
Yasuaki
Y
Kitaichi
Kiyoyuki
K
Iwase
Mitsunori
M
Shibata
Eiji
E
Nakao
Makoto
M
Matsumoto
Takatoshi
T
Takagi
Kenzo
K
Hasegawa
Takaaki
T
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
D000587:Oxidoreductases Acting on CH-NH Group Donors; D010545:Peroxides; D011073:Polyamines; D000097667:Polyamine Oxidase