Shpend Dragusha1, Takayuki Shibata1, Sheng Yin1, Jun-ya Fujita1, Tsutomu Kabashima1, Masaaki Kai2. 1. Faculty of Pharmaceutical Sciences, Graduate School of Biomedical Sciences, Nagasaki University, 1-14 Bunkyo-Machi, Nagasaki 852-8521, Japan. 2. Faculty of Pharmaceutical Sciences, Graduate School of Biomedical Sciences, Nagasaki University, 1-14 Bunkyo-Machi, Nagasaki 852-8521, Japan. Electronic address: ms-kai@nagasaki-u.ac.jp.
Abstract
BACKGROUND: Cytosine in urine is one of the biomarkers for the diagnosis of metabolic immunodeficiency. It has been mentioned that a high level of cytosine is found in urine of children having immunodeficiency. In this study, we have developed a fluorescence (fluorescence) derivatization reaction of cytosine using 4-trifluoromethylbenzamidoxime (4-TFMBAO) as a fluorogenic reagent. METHODS: In this reaction, cytosine was mixed with 4-TFMBAO, K3[Fe(CN)6], N,N-dimethylformamide (DMF) and KOH in an aqueous solution. The mixture was heated at 100°C for 20 min. The fluorescence intensity of the mixture was measured with a spectrofluorometer. RESULTS: Under the optimized reaction conditions, a strong fluorescence was produced only from cytosine amongst 62 compounds including structurally related bio-substances. The selectivity and sensitivity of this method were compared with a conventional fluorescence one using 2-bromoacetophenone that reacts with cytosine, adenine and their related substances. The present method was sufficiently selective toward cytosine, and approximately 50 times more sensitive than the conventional one. CONCLUSIONS: Our method permitted the quantitative determination of cytosine in human urines without any pretreatment for a primary screening test of inborn disorder in pyrimidine metabolism with immunodeficiency, and indicated the lower detection limit of 0.1 μmol/l cytosine which gave 3 times greater fluorescence intensity than that observed for the reagent blank.
BACKGROUND:Cytosine in urine is one of the biomarkers for the diagnosis of metabolic immunodeficiency. It has been mentioned that a high level of cytosine is found in urine of children having immunodeficiency. In this study, we have developed a fluorescence (fluorescence) derivatization reaction of cytosine using 4-trifluoromethylbenzamidoxime (4-TFMBAO) as a fluorogenic reagent. METHODS: In this reaction, cytosine was mixed with 4-TFMBAO, K3[Fe(CN)6], N,N-dimethylformamide (DMF) and KOH in an aqueous solution. The mixture was heated at 100°C for 20 min. The fluorescence intensity of the mixture was measured with a spectrofluorometer. RESULTS: Under the optimized reaction conditions, a strong fluorescence was produced only from cytosine amongst 62 compounds including structurally related bio-substances. The selectivity and sensitivity of this method were compared with a conventional fluorescence one using 2-bromoacetophenone that reacts with cytosine, adenine and their related substances. The present method was sufficiently selective toward cytosine, and approximately 50 times more sensitive than the conventional one. CONCLUSIONS: Our method permitted the quantitative determination of cytosine in human urines without any pretreatment for a primary screening test of inborn disorder in pyrimidine metabolism with immunodeficiency, and indicated the lower detection limit of 0.1 μmol/l cytosine which gave 3 times greater fluorescence intensity than that observed for the reagent blank.