Absorbance measurements of oxidation of homogentisic acid accelerated by the addition of alkaline solution with sodium hypochlorite pentahydrate

www.nature.com/scientificreports OPEN Received: 23 February 2018 Accepted: 18 July 2018 Published: xx xx xxxx Absorbance measurements of oxidation of homogentisic acid accelerated by the addition of alkaline solution with sodium hypochlorite pentahydrate Yasunori Tokuhara1, Kenichi Shukuya2, Masami Tanaka2, Keisuke Sogabe3, Yasukazu Ejima4, Sho Hosokawa1, Hiroyuki Ohsaki5, Tatsuya Morinishi6, Eiichiro Hirakawa6, Yutaka Yatomi2 & Tatsuo Shimosawa 7 The urine of patients with alkaptonuria turns dark brown due to the oxidation of homogentisic acid (HGA) to benzoquinone acetic acid (BQA), and this is accelerated by the addition of alkali. We recently reported that alkaptonuric urine and HGA after the addition of alkali showed characteristic peaks at 406 and 430 nm. In order to improve the sensitivity of our spectrometric method for the detection of HGA, we accelerated the oxidation of HGA to BQA using sodium hypochlorite pentahydrate (NaOCl·5H2O), which is a strong oxidant. In the present study, we measured the absorption spectra of alkaptonuric urine and HGA solution after the addition of sodium hydroxide (NaOH) or NaOH with NaOCl·5H2O and analyzed the oxidation reaction of HGA after alkalization using a liquid chromatography time-of-flight mass spectrometer (LC/TOF-MS) and nuclear magnetic resonance (NMR) spectrometry. We accelerated the oxidation of HGA to BQA by adding NaOH with NaOCl·5H2O, and this absorbance measurement was useful for more sensitively observing the oxidation of HGA than LC/TOF-MS and NMR spectroscopy. This quick and easy screening method may be suitable for the diagnosis of alkaptonuria. Alkaptonuria, a hereditary metabolic disorder, occurs due to the absence of the enzyme homogentisic acid (HGA) oxidase. This defect leads to the accumulation of HGA, an intermediate in the catabolism of phenylalanine and tyrosine1,2. The urine of patients with alkaptonuria turns dark brown at room temperature after several hours to days; the oxidation of HGA to benzoquinone acetic acid (BQA) underlies this color change, which is accelerated by the addition of alkali3,4. The accumulation of HGA and its metabolites in tissues causes ochronosis, which is characterized by the darkening of cartilaginous tissues and bone, and may lead to arthritis, joint destruction, and the deterioration of cardiac valves5–7. In recent years, the measurement of HGA in urine has become possible using gas chromatography-mass spectrometry, a high-performance liquid-chromatographic (HPLC) method, and nuclear magnetic resonance (NMR) spectrometry8,9. However, these methods are very expensive and difficulties are associated with manipulating and maintaining machines. In an attempt to develop a quick and easy screening test for alkaptonuria using a spectrophotometer, we recently reported novel visible-light region absorbance peaks in the urine of patients with alkaptonuria after alkalization10. Alkaptonuric urine and HGA solution exhibit characteristic absorption spectra with peaks at 406 and 430 nm that appear one minute after alkalization. This method enables quick and easy screening to detect the oxidation of HGA to BQA in urine. 1 Department of Medical Technology, Ehime Prefectural University of Health Sciences, Ehime, Japan. 2Department of Clinical Laboratory, School of Medicine, the University of Tokyo, Tokyo, Japan. 3Kaneka Techno Research Corporation, Hyogo, Japan. 4Kaneka Corporation, Vinyls and Chlor-Alkali Solutions Vehicle, Osaka, Japan. 5 Department of Medical Biophysics, Kobe University Graduate School of Health Sciences, Kobe, Japan. 6Department of Medical Technology, Kagawa Prefectural University of Health Sciences, Kagawa, Japan. 7Clinical Laboratory Medicine, School of Medicine, International University of Health and Welfare, Chiba, Japan. Correspondence and requests for materials should be addressed to Y.T. (email: ) SCIEnTIfIC REPOrTS | (2018) 8:11364 | DOI:10.1038/s41598-018-29769-w 1 www.nature.com/scientificreports/ Figure 1. Color change and absorption spectra of alkaptonuric urine. (a) Alkaptonuric urine (HGA concentration of 271 mg/L) after the addition of NaOH (I), NaOH with NaOCl·5H2O (II), or NaOCl·5H2O (III). (b) Absorption spectra of alkaptonuric urine after the addition of NaOH, NaOH with NaOCl·5H2O, or NaOCl·5H2O. (c) Absorbance values at 430 and 406 nm of alkaptonuric urine after the treatment with NaOH or NaOH with NaOCl·5H2O. Data are the mean ± SD (n = 3). **P < 0.01. In the present study, in an attempt to improve the sensitivity of our spectrometric method for the detection of HGA, we accelerated the oxidation of HGA to BQA using sodium hypochlorite pentahydrate (NaOCl·5H2O), a strong oxidant with a solid (finely ground) form and an effective chlorine concentration of approximately 42%. Moreover, using a liquid chromatography time-of-flight mass spectrometer (LC/TOF-MS) and NMR spectrometry, we analyzed the oxidation reaction of HGA to BQA after alkalization and examined how interference substances affect the oxidation of HGA. The results obtained suggest that the addition of alkaline solution with NaOCl·5H2O to alkaptonuric urine accelerates the oxidation of HGA to BQA, and the measurement of the absorption spectrum in the visible region is useful for observing a urine color change due to the oxidation of HGA to BQA. Results Color change and absorption spectra in the visible region. We initially added NaOH, NaOH with NaOCl·5H2O, or NaOCl·5H2O to the urine sample collected from the alkaptonuria patient and observed changes in its color. Urine became a darker brown following the addition of NaOH with NaOCl·5H2O than with the addition of NaOH (Fig. 1a). However, when the urine sample was incubated with NaOCl·5H2O, it did not show a color change (Fig. 1a). We then conducted a spectrophotometric analysis in the visible region (380–500 nm) to detect the absorption curve of the urine sample. The absorbance curve of the urine sample showed sharper peaks following the addition of NaOH with NaOCl·5H2O than with the addition of NaOH (Fig. 1b). Furthermore, the absorbance values at 406 and 430 nm of the urine sample were significantly higher following the addition of NaOH with NaOCl·5H2O than with the addition of NaOH (Fig. 1c). Similar results were obtained for the HGA solution (Fig. 2). Absorption spectra in the UV region. We then examined absorption spectra at 200–500 nm covering the UV region obtained following the addition of NaOH with NaOCl·5H2O to HGA solution. The shift in the absorbance peak from 290 to 250 nm after the addition of NaOH with NaOCl·5H2O to HGA solution was identical to that following the addition of NaOH (Fig. 3a). However, a significant difference was not observed between the absorbance values at 250 nm after the addition of NaOH with NaOCl·5H2O and after that of NaOH (Fig. 3a). Two characteristic peaks at 406 and 430 nm of HGA after the addition of NaOH or NaOH with NaOCl·5H2O disappeared at the time of the measurement in the UV (...truncated)