抄録
A chemiluminescence method for assay of polyamines in urine has been developed. Hydrolysis of conjugated polyamines in urine and extraction of polyamines from urine samples were carried out in a similar way to that reported by Okada et al.(Japan. J. Biochem., 53, 792 (1981)), as following.
0.5 ml of urine was treated with 20 U of acylpolyamine amidohydrase. After centrifugation the supernatant was applied to a Bio-Rex 70 column, which had been equilibrated with a 0.1 M tris buffer, pH 9.5 previously. Using 0.2 M HC1 as an eluant, polyamines were recovered complately in the eluate. The pH of the eluate was adjusted to 8.8 with 0.5 M NaOH and a 0.5 M tris buffer.
An aliquot of the eluate was used for polyamine assay by a chemiluminescence reaction.
The principle of the chemiluminescence reaction is as follows:
_??_
_??_It took about 120 mm to complete the whole procedures from hydrolysis of conjugated polyamines to the measurement of the intensity of luminescence. 40 samples could be assayed within a working day. Polyamine concentrations in a range of 1 to 600μM could be determined by this method without dilution of samples. The recovery of putrescine, cadaverine, spermidine, and spermine were 100%, 111%, 89%, and 44%, respectively by this method. The coefficient of variation between runs (10 runs) were 4.4% for a sample with 28.3μM of putrescine, and 2.5% for a sample with 58.0μM of putrescine.
The concentration of the total polyamines in 24 hour urine samples collected from 51 healthy subjects (29 female and 22 male) was 36.2+23.5 μmol/g of creatinine, mean+2SD, and that in random fresh urine samples collected from 44 persons of the same group was 32.8+24.8μmol/g of creatinine.
