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.
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: Ankit Sabharwal; Bibekananda Kar; Santiago Restrepo-Castillo; Shannon R Holmberg; Neal D Mathew; Benjamin Luke Kendall; Ryan P Cotter; Zachary WareJoncas; Christoph Seiler; Eiko Nakamaru-Ogiso; Karl J Clark; Stephen C Ekker Journal: CRISPR J Date: 2021-11-30