Abstract
3-Hydroxykynurenine (3-OHKY) and 3-hydroxyanthranilic acid (3-OHAA) are metabolites of tryptophan which have been described as “endogenous carcinogens”, and the relation between these substances in urine and bladder cancer has been discussed. In this work, the method of determination of 3-OHKY, 3-OHAA, 3-OHAA glucuronide (3-OGAA) and 3-OHAA sulfuric ester (3-OSAA) were devised and urinary excretion of these substances in humans and animals was estimated.
3-OHKY was determined as follows. To 12ml of diluted urine, 16ml of p-tolue-nesulfonylchloride solution in acetone (0.5g in 100ml of acetone) and 4ml of NaHCO3 aq.(5g in 100ml of water) were added. The mixture was allowed to stand for 30 min and 4ml of (NH4)2 CO3 aq.(10g in 100ml of water) was added. After 30 min, the mixture was acidified with 4m1 of 4N H2SO4. The fluorescent product was extracted with 40ml of ether, separated on alumina G thin layer with the lower layer of a mixture of CHCl3: acetone: EtOH: 1.5% NH4OH (2:2:1:1) and determined fluorometrically (ex. 360mμ, fl. 465mμ) in a mixture of acetorle: water (1:1).
3-OHAA was extracted with ether at pH 3, Separated on cellulose powder thin layer with a mixture of benzene: EtOH: water (50:10:1) and determined fluorometrically (ex. 348mμ, fl. 410mμ) in a mixture of MeOH: dioxane (1:9).
3-OGAA and 3-OSAA were determined as follows. The urine was desalted by acetone and MeOH and adsorbed on a column of DEAE Sephadex CO3 (1×20cm). After the column was washed with 125ml of dil.(NH4)2CO3 (1g in 100ml of water), 3-OGAA was eluted with 80ml of conc.(NH4)2CO3 (15g in 100 ml of water), separated on cellulose powder thin layer with a mixture of n-BuOH: n-PrOH: EtOH: water (3:6:4:2) and determined fluorometrically (ex. 340mμ, fl. 420mμ) in MeOH. 3-OSAA was eluted with further 80ml of conc.(NH4)2CO3 from the column, separated on silica gel H thin layer with a mixture of n-BuOH: n-PrOH: EtOH: water (6:3:21) and determined fluorometrically (ex. 340mμ, fl. 420mμ) in MeOH.
The results showed that 3-OHAA in human urine increased in patients with bladder cancer. The principal urinary metabolite of tryptophan determined by these methods was 3-OHAA in humans, 3-OSAA in rats and 3-OGAA in guinea pigs.
