Shinya Fujii1, Toshio Maeda2, Ichiro Noge3, Yutaka Kitagawa4, Kenichiro Todoroki1, Koichi Inoue1, Jun Zhe Min1, Toshimasa Toyo'oka5. 1. Laboratory of Analytical and Bio-Analytical Chemistry, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan. 2. Laboratory of Clinical Pharmaceutics and Pharmacy Practice, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan. 3. Department of Pharmaceutical, Numazu City Hospital, Harunoki Higashi-Shiiji Numazu 410-0302, Japan. 4. Department of Clinical and Molecular Endocrinology, Numazu City Hospital, Harunoki Higashi-Shiiji Numazu 401-0302, Japan. 5. Laboratory of Analytical and Bio-Analytical Chemistry, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan. Electronic address: toyooka@u-shizuoka-ken.ac.jp.
Abstract
BACKGROUND: In diabetes mellitus (DM) patients with ketoacidosis, ketone bodies, i.e., acetone, acetoacetic acid (AA) and β-hydroxybutyric acid (HA), are increased in the blood and urine. Acetone is also excreted by breathing due to the spontaneous decomposition of AA. Thus, the increase in acetone has been considered as one of the biomarkers for the diagnosis of DM. However, the determination of acetone in one's breath is not recommended because of the sample handling difficulty. We measured acetone in saliva by reversed-phase liquid chromatography (LC) with fluorescence (FL) detection. The proposed method was applied to the determination of acetone in the saliva of healthy volunteers and DM patients with and without ketoacidosis. METHODS: 3-Pentanone (I.S.) and DBD-H in acetonitrile were added to freshly collected saliva and reacted at room temperature for 20 min in the presence of trifluoroacetic acid. After the reaction, the solution was centrifuged at 10,000 × g and 4 °C for 5 min. The supernatant was separated by reversed-phase LC and the FL detected at 550 nm (excitation at 460 nm). RESULTS: The concentrations of acetone in the DM patients with ketoacidosis were significantly higher than those of the normal subjects and DM patients without ketoacidosis. Furthermore, the total contents of the ketone bodies in the blood correlated with acetone in the saliva of the DM patients. The concentrations of acetone in the saliva of an emergency patient also correlated with the ketone bodies in the blood at each sampling time. CONCLUSION: The proposed method using LC-FL seems to be useful for the determination of acetone in the saliva of DM patients with ketoacidosis. The method offers a new option for the diagnosis and monitoring of DM patients with ketoacidosis.
BACKGROUND: In diabetes mellitus (DM) patients with ketoacidosis, ketone bodies, i.e., acetone, acetoacetic acid (AA) and β-hydroxybutyric acid (HA), are increased in the blood and urine. Acetone is also excreted by breathing due to the spontaneous decomposition of AA. Thus, the increase in acetone has been considered as one of the biomarkers for the diagnosis of DM. However, the determination of acetone in one's breath is not recommended because of the sample handling difficulty. We measured acetone in saliva by reversed-phase liquid chromatography (LC) with fluorescence (FL) detection. The proposed method was applied to the determination of acetone in the saliva of healthy volunteers and DMpatients with and without ketoacidosis. METHODS:3-Pentanone (I.S.) and DBD-H in acetonitrile were added to freshly collected saliva and reacted at room temperature for 20 min in the presence of trifluoroacetic acid. After the reaction, the solution was centrifuged at 10,000 × g and 4 °C for 5 min. The supernatant was separated by reversed-phase LC and the FL detected at 550 nm (excitation at 460 nm). RESULTS: The concentrations of acetone in the DMpatients with ketoacidosis were significantly higher than those of the normal subjects and DMpatients without ketoacidosis. Furthermore, the total contents of the ketone bodies in the blood correlated with acetone in the saliva of the DMpatients. The concentrations of acetone in the saliva of an emergency patient also correlated with the ketone bodies in the blood at each sampling time. CONCLUSION: The proposed method using LC-FL seems to be useful for the determination of acetone in the saliva of DMpatients with ketoacidosis. The method offers a new option for the diagnosis and monitoring of DMpatients with ketoacidosis.