A spectrophotometric method for the determination of tryptophan following oxidation by the addition of sodium hypochlorite pentahydrate

PLOS ONE RESEARCH ARTICLE A spectrophotometric method for the determination of tryptophan following oxidation by the addition of sodium hypochlorite pentahydrate Sho Hosokawa1, Tatsuya Morinishi2, Kazuaki Ohara3, Kentaro Yamaguchi3, Satoshi Tada2, Yasunori Tokuhara ID2* a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 1 Department of Medical Technology, Ehime Prefectural University of Health Sciences, Ehime, Japan, 2 Department of Medical Technology, Kagawa Prefectural University of Health Sciences, Kagawa, Japan, 3 Faculty of Pharmaceutical Sciences at Kagawa Campus, Tokushima Bunri University, Kagawa, Japan * Abstract OPEN ACCESS Citation: Hosokawa S, Morinishi T, Ohara K, Yamaguchi K, Tada S, Tokuhara Y (2023) A spectrophotometric method for the determination of tryptophan following oxidation by the addition of sodium hypochlorite pentahydrate. PLoS ONE 18(1): e0279547. https://doi.org/10.1371/journal. pone.0279547 Editor: Ajaya Bhattarai, Tribhuvan University, NEPAL Received: July 20, 2022 Accepted: December 9, 2022 Published: January 26, 2023 Copyright: © 2023 Hosokawa et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: Y.T. - This work was supported by JSPS KAKENHI Grant Number JP21K07394. The funder had no role, and we included the statement in our cover letter. Competing interests: The authors have declared that no competing interests exist. Tryptophan (Trp) is an essential amino acid that functions in various biological processes and human daily health. As the significant functions of Trp become more apparent, its measurement is becoming increasingly important in various situations. Herein, we improved the Trp color reaction based on the Hopkins-Cole reaction and established a simple colorimetric method for Trp determination using several different reagents, including sodium hypochlorite pentahydrate and monosodium glutamate. The detection method can be performed using safe materials, rather than conventional toxic substances, and induces a crimson color change with an absorption peak at 525 nm, enabling the quantification of Trp by simple spectrophotometry in just 10 min. This assay exhibited a linear detection range from 10 to 100 mg/L (R2 = 0.9996). The average recoveries in the spiked cerebrospinal fluid ranged from 90.5% to 104.3%, with a relative standard deviation of 0.27% (n = 3, 29.40 mg/L Trp) to 1.19% (n = 3, 72.90 mg/L Trp). This novel spectrophotometric method may enable many researchers and laboratory technicians to detect Trp in various sample solutions without expensive analytical instruments or complicated operations. Introduction Tryptophan (Trp) is one of the essential amino acids that must be obtained through the diet. Beyond its role in protein synthesis, Trp and its metabolites serve functions in daily health such as sleep, circadian rhythm, and cognition [1–3]. Furthermore, changes in the body concentration and metabolism of Trp are associated with cancer, infection, stress, and depression [4–7]. As the significant functions of Trp become more apparent, its measurement technique is becoming increasingly important in various situations [8]. Although multiple sophisticated techniques are available for the determination of Trp and their metabolites, such as high-performance liquid chromatography (HPLC) with mass PLOS ONE | https://doi.org/10.1371/journal.pone.0279547 January 26, 2023 1 / 12 PLOS ONE A spectrophotometric method for the determination of tryptophan spectrometry and fluorescence detection, these methods require expensive analytical equipment, complicated operation, and a lot of time for preparation and maintenance [9–12]. Conversely, spectrophotometry involves easy handling and inexpensive equipment that requires almost no maintenance; therefore, it remains a popular method and has been installed in many automated biochemical analyzers. Moreover, most of the colorimetric detection methods involve oxidation of the Trp condensation products with various aldehydes and have long been used for the determination of Trp, such as the Hopkins-Cole reaction [13, 14]. However, the previously established methods may still be time-consuming, require inconvenient sample treatments, rely on unstable colored reactions, or use a hazardous chemical such as concentrated sulfuric acid [15–18]. Thus, more rapid, simple, safe, and accurate methods for Trp detection should be developed. Sodium hypochlorite pentahydrate (NaOCl�5H2O) is a novel oxidant, often provided in a pure solid form (finely ground) [19]. In previous experiments, we have developed spectrophotometric detection methods of amino acid metabolites using NaOCl�5H2O and clarified its usefulness as an oxidizing agent in terms of high stability, quick reaction, and ease of adjustment [20, 21]. Therefore, we attempted to improve the Trp color reaction based on the Hopkins-Cole reaction using NaOCl�5H2O as the oxidizing agent. In the present study, we evaluated the color reaction of Trp solution using NaOCl�5H2O and determined the optimized condition for establishing the practical Trp detection method. Using NaOCl�5H2O and monosodium glutamate (MSG), a stable crimson color change of Trp solution was observed at 525 nm by spectrophotometry. The optimized Trp colorimetric method was employed for interference and recovery tests of pool specimens, which detected varying Trp concentrations in cerebrospinal fluid (CSF) with little interference. The results may contribute to the development of an accurate, rapid, and stable Trp detection method. Materials and methods Reagents and apparatus Asparagine, glycine, alanine, arginine, aspartic acid, cysteine, glutamine, glutamic acid, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tyrosine, valine, Trp, 10% hydrochloric acid (HCl), sodium hydroxide (NaOH), ascorbic acid (AA), sodium chloride (NaCl), D (+)-glucose, lysozyme, and glyoxylic acid monohydrate were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). MSG monohydrate and NaOCl�5H2O were purchased from Nacalai Tesque (Kyoto, Japan). Pooled Human Cerebrospinal Fluid (CSF) was purchased from Lee Biosolutions, Inc. (USA, Missouri). A block incubator, WSC-2620 PowerBLOCK (ATTO Corporation, Tokyo, Japan), was used to set and maintain the temperature (10, 20, 30, 40, 50, 60, and 70˚C) of sample solutions. A double-beam spectrophotometer U-2900 (Hitachi High-Technology Co., Ltd., Tokyo, Japan) with microcells and a 10-mm path length was used to measure the absorption spectra of sample solutions. Reagents and standards The solutions were prepared from high-purity analytical reagents and distilled water. Trp was prep (...truncated)