High-throughput DL-amino acid analysis of deep sea water from Toyama Bay and anti-aging activity assessment using C. elegans

High-throughput DL-amino acid analysis of deep sea water from Toyama Bay and anti-aging activity assessment using C. elegans | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article High-throughput DL-amino acid analysis of deep sea water from Toyama Bay and anti-aging activity assessment using C. elegans Takahiro Takayama, Haruto Iwata, Reina Fujio, Ayaka Minamida, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7397638/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Deep sea water (DSW), defined as sea water deeper than 200 m, has notable applications in various fields such as energy, agriculture, food, cosmetics, and public health. Several studies have attributed its utility to mineral effects; however, its organic compounds have rarely been investigated. To emphasize the mechanistic evidence of DSW, a sensitive analytical method was developed for the individual analysis of D - and L -amino acids (AAs) using enantiochemical tagging–liquid chromatography–tandem mass spectrometry. A novel reagent, CMT-D-Leu, was developed to enable high-speed analysis of individual DL-AAs, achieving analysis of 19 DL -AAs within 17 min. A limit of detection of 10–100 pmol/L (in vial) was achieved, which was sufficient to reveal the DL-AA profiles in DSW. Three batches of DSW from Toyama Bay were subjected to quantitative analysis using the spiking standard method, detecting DL-AA concentrations of 10–100 nmol/L. Notably, D-Leu, D-Val, D-Ala, D-Ser, D-Thr, and D-Glu were detected at higher concentrations than other D-AAs. Finally, a lifespan assay using the C. elegans model showed that DSW exhibited a clear proliferative effect, similar to the positive control. Moreover, DL-AA kinetics analysis revealed a reduction in D-Asp, an aging marker, in the proliferated groups. Biological sciences/Biochemistry Biological sciences/Biological techniques Biological sciences/Biotechnology Physical sciences/Chemistry Earth and environmental sciences/Environmental sciences Enantiochemical tagging DL-amino acids Deep sea water C. elegans Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction In Japan, deep sea water (DSW) is defined as water deeper than 200 m below the sea surface. DSW is characterized by high purity, stability at low temperatures, high mineral concentrations, and the presence of bioactive nutritional species 1 . Consequently, its applications have expanded into the fields of energy, agriculture, food, cosmetics, and public health 2 . In human healthcare, several clinical trials have reported beneficial effects on hypercholesterolemia, allergy, and Helicobacter pylori infection 3 , 4 , 5 . Because the efficacy of DSW has only been partially established, mechanistic studies have attempted to identify which components in the “solution” are effective. Several studies have reported the relative efficacy of DSW in maintaining mineral balance 6 , 7 , 8 . For example, Lee et al. demonstrated that the hardness levels of DSW, particularly the Mg and Ca content ratios (Mg/Ca = 3/1), were critical for reducing hepatic cholesterol production 6 . Fukui et al. investigated the hardness of an adjusted solution containing DSW in relation to obesity in mice fed a high-fat diet 7 . They also analyzed the mineral composition of DSW using liquid chromatography–inductively coupled plasma mass spectrometry (LC–ICP–MS) and found that high levels of Mg and Ca may play a key role in its efficacy. Although reasonable explanations have been proposed regarding the role of minerals in these studies, almost no research has focused on amino acids (AAs) in DSW. A few studies have explored the contribution of AAs in DSW. For example, a previous study detected 17 types of AAs in DSW from the Gulf of Mexico, suggesting that organic compounds may contribute to its efficacy 9 . Ikari et al. analyzed the relationship between the kynurenine signal in DSW from Japan and the stress response in Paralichthys olivaceus 10 . As expected, these studies have detected extremely high amounts of minerals such as Mg in DSW, with AAs partially contributing as effective factors. However, to date, analytical results for AAs in DSW remain limited. AAs are generally proteinogenic L-AAs (20 types). Both D- and L-isomers exist, with high amounts of L-forms detected in biological specimens. Several studies have detected D-AAs in biological samples and reported various effects on living organisms 11 , 12 , 13 . For example, D -Ser acts as a neurotransmitter for the NMDA receptor 12 , while D-Ala is a precursor for neurotransmitters and osmotic compounds for marine life such as shrimp and crabs 13 . In addition, the utility of D -AAs, including D -Ala and D -Ser, has been recognized in recent biological research. D -AAs are produced and used by microorganisms such as bacteria and plankton 14 , 15 . Notably, one study suggested that D -AAs are selectively used by deep-sea microorganisms 14 , which may support the presence and role of D -AAs in living organisms in DSW. However, analytical research on AAs in DSW is limited, and even less research on D-AAs is available. Furthermore, because DSW is highly diluted, a sensitive and selective method is necessary to analyze DL -AAs in DSW. DL -AAs cannot be analyzed using conventional liquid chromatography–tandem mass spectrometry (LC-MS/MS) methods because enantiomers exhibit identical physical properties. Several methods employing chiral column technology or pre-column chiral derivatization have been reported. Chiral derivatization enables enantioseparation in conventional LC mode and highly sensitive detection by improving ionization efficiency for MS detection 16 , 17 , 18 . Several methods employing innovative derivatization reagents have been successfully used to analyze DL -AAs in various biological samples, including our methodologies 19 , 20 , 21 . Although these methods achieve rapid analysis (~ 20 min/measurement) of proteinogenic AAs with enantioseparation, higher-throughput analyses are required to meet the demands of big data generation. In this study, we screened novel reagents and developed methods that are more rapid than those used in previous studies. In addition, DSW from Toyama Bay, Japan was analyzed to profile DL -AA concentrations. In addition to this analysis, the effectiveness of DSW was evaluated using Caenorhabditis elegans ( C. elegans ) in a lifespan assay. The nematode C. elegans is approximately 1 mm in length (~ 1000 cells in the body) with a transparent body that facilitates the observation of organ tissues. It possesses muscles, a digestive tract, a nervous system, epithelium, and reproductive organs. Moreover, its short lifespan of approximately 20 d makes it suitable for lifespan assays to reduce generation time 22 , 23 , 24 . The life extension performance of DSW was assessed using C. elegans , and DL -AA kinetics were analyzed using the developed method. Experimental Reagents and chemicals DL -AA standards, including glycine (Gly, not optically active), alanine (Ala), serine (Ser), threonine (Thr), proline (Pro), valine (Val), leucine (Leu), isoleucine (Ile), aspartic acid (Asp), asparagine (Asn), glutamic acid (Glu), glutamine (Gln), methionine (Met), phenylalanine (Phe), tyrosine (Tyr), tryptophan (Trp), histidine (His), arginine (Arg), and lysine (Lys), were purchased from Tokyo Chemical Industry (Tokyo, Japan). Working solutions of each metabolite were prepared in acetonitrile/water (1:1) at a concentration of 10 mmol/L. For reagent synthesis, 2,4-dichloro-6-methoxy-1,3,5-triazine (DCMT) was also purchased from Tokyo Chemical Industry. Acetonitrile (CH 3 CN), methanol (CH 3 OH), ethyl acetate (EAAC), n -hexane (Hex), pyridine, triethylamine (TEA), hydrochloric acid (HCl), sodium bicarbonate (NaHCO 3 ), ammonium formate, sodium chloride (NaCl), and LC-MS grade formic acid (FA) were obtained from Fujifilm Wako Pure Chemical Co. (Osaka, Japan). Purified water was sourced from a Milli-Q EQ7000 system (Merck, Darmstadt, Germany). Screening of the ideal enantiochemical tagging reagent for DL-AA analysis DCMT powder (360 mg, 2.0 mmol) was dissolved in 20 mL CH 3 CN. In a separate flask, L-AAs (L-Phe, L-Pro, L-Val, or L-Leu; each 2.0 mmol) were dissolved in 20 mL of a mixture of H 2 O and MeOH (1:4 v/v). These solutions were mixed for 3 h at room temperature, with 35 µL of TEA added every 30 min. After the reaction was complete, the mixture was extracted into the aqueous layer of a water/Hex phase, and a small amount of HCl was added. It was then extracted with EAAC and Hex (1:1 v/v), washed with H 2 O- and NaCl-saturated solutions, and evaporated under reduced pressure. The resulting residues were purified by preparative TLC (silica gel 70 F 254 PLC, 1.0 mm, Fujifilm Wako Pure Chemical Co.) using an eluent of EAAC and Hex (1:1 v/v). Synthesize of reagents i.e., 2,4-Dichloro-6-methoxy-1,3,5-triazine-D-Leu DCMT powder (360 mg, 2.0 mmol) was dissolved in 20 mL of CH 3 CN. In another flask, D-Leu (262 mg, 2.0 mmol) was dissolved in 20 mL of an H 2 O–MeOH mixture (1:4 v/v). These solutions were mixed for 3 h at room temperature, with 35 µL of TEA added every 30 min. After the reaction was complete, the mixture was extracted into the aqueous layer of a water/Hex phase, and a small amount of HCl was added. Subsequently, it was extracted with EAAC and Hex (1:1 v/v), washed with H 2 O- and NaCl-saturated solutions, and evaporated under reduced pressure. The resulting residue was redissolved in 4 mL of H 2 O/MeOH (1:1 v/v) and purified using a PU 714M pump, SC 762 system controller, and PLC 761 fraction collector (GL Sciences, Tokyo, Japan). The preparative column used was a TSKgel ODS-80Ts column (5 µm, 25 cm × 20.0 mm i.d., Tosoh Corporation). The elution conditions were as follows: mobile phase, H 2 O/MeOH mixture containing 0.1% (v/v) FA; gradient elution (MeOH% (min)): 20 (0–1), 70 (1–200), 95 (200–205), and 5 (205–210). The flow rate of the mobile phase was 5.0 mL/min. Purity (HPLC%) was determined as > 99% by photo diode array (190 nm–600 nm, ACQUITY PDA, Waters). The m/z of the peak was 275.0904 (theoretical value; 275.0910 [M + H + ]) by high-resolution mass spectrometry (Xevo G2-XS QTOF, Waters). Analytical conditions and results of these purity tests were shown in Supporting Information (Figure S1 ). Instrument operating conditions Ultraperformance liquid chromatography–electrospray ionization–tandem mass spectrometry (UPLC-ESI-MS/MS) was performed using an ACQUITY UPLC H-class instrument (Waters, Milford, MA, USA) connected to a Xevo TQ-XS mass spectrometer (Waters, Milford, MA, USA). For MS detection of the derivatives, positive ESI and multiple reaction monitoring (MRM) modes were used. The analytical columns used were an ACQUITY™ Premier BEH C18 column (1.7 µm, 100 × 2.1 mm i.d., Waters) or an ADME-HR column (2.0 µm, 100 × 2.1 mm i.d., Osaka Soda). A shorter ADME-HR column (2.0 µm, 50 × 2.1 mm i.d., Osaka Soda) was employed under optimal conditions. The elution conditions for the ACQUITY™ Premier BEH C18 column were as follows: mobile phase, a CH 3 CN/H 2 O mixture containing 0.1% (v/v) FA; gradient elution (CH 3 CN% (min)): 5 (0–0.5), 42 (0.5–16), 98 (16–18), and 5 (18–20). The flow rate of the mobile phase was 0.4 mL/min. The elution conditions for the ADME-HR column were as follows: mobile phase, a CH 3 CN/H 2 O mixture containing 10 mmol/L ammonium formate; gradient elution (CH 3 CN% (min)): 2 (0–0.3), 25 (0.3–7.5), 98 (7.5–8.5), and 2 (8.5–10). The flow rate of the mobile phase was 0.5 mL/min. The elution conditions for the shorter ADME-HR column were as follows: mobile phase, a CH 3 CN/H 2 O mixture containing 10 mmol/L ammonium formate; gradient elution (CH 3 CN% (min)): 2 (0–0.2), 39 (0.2–4.5), 98 (4.5–5.5), and 2 (5.5–7). Another set of conditions was also evaluated as follows: mobile phase, a CH 3 CN/H 2 O mixture containing 0.1% (v/v) FA; gradient elution (CH 3 CN% (min)): 2 (0–0.2), 25 (0.2–9), 98 (9–10), and 2 (10–11). The flow rate of the mobile phase was 0.5 mL/min. The MS/MS conditions are described in detail in the Supporting Information (Table S1 ). Derivatization conditions of AAs An aliquot of 10 µL of a 1 µmol/L DL -AA mixture was added to 10 µL of 20 mmol/L CMT-D-Leu in CH 3 CN, and 10 µL of 100 mmol/L NaHCO 3 in H 2 O. These mixtures were reacted for 90 min at 60°C. After the reaction was complete, the mixture was evaporated under reduced pressure and redissolved in 100 µL of 1% FA in CH 3 CN/H 2 O (2:98, v/v). Validation of DL -AA quantification analysis of DSW A mixture of 1900 µL DSW and 100 µL of DL -AA solutions at various concentrations (0–50 µM) was desalinated by adding it to 9 mL of MeOH. These mixtures were centrifuged at 4000 × g for 5 min at 4°C. The supernatant (1 mL) was then added to 10 µL of CMT-D-Leu in CH 3 CN and 50 µL of NaHCO 3 in H 2 O. The resulting mixtures were subsequently used for derivatization. Calibration curves were constructed by plotting the peak area against the sample concentration. These mixtures were reacted for 120 min at 60°C. After the reaction was complete, the mixture was evaporated under reduced pressure and redissolved in 100 µL of 2% FA in CH 3 CN/H 2 O (2:98, v/v). A recovery test from DSW was performed at three concentrations. Intra- and inter-day precisions were assessed by quantifying DL -AAs in DSW over 3 d (n = 3 each day). Lifespan assay using C. elegans model The lifespan assay was conducted following procedures described in a previous study. The biomaterials used in this study are described in the Supplementary Data. Briefly, the wild-type nematode C. elegans (Bristol strain N2) was used as a model organism. C. elegans stocks were maintained on nematode growth medium (NGM) plates seeded with 50 µL E. coli (DH5αFT) at 20°C as a food source. Eggs were collected from C. elegans by lysing them using KOH and NaClO. These eggs were left in Milli-Q water overnight to synchronize C. elegans at the L1 stage. After the worms reached L1, they were transferred onto NGM plates with E. coli until the L4 stage. On the third day, 50 µM 5-Fluoro-2’-deoxyuridine (FUDR; FUJIFILM, Miyazaki, Japan) was added to suppress reproduction. On the fourth day, C. elegans were exposed to liquid culture medium composed of 75 µL FUDR, 550 µL S-basal, 1.5 µL 2% cholesterol, 75 µL solvent (DSW, 25% DSW, 500 mM metformin, or Milli-Q water as control), and 50 µL Milli-Q water containing C. elegans . Next, 750 µL of this liquid culture was added to each well of a 24-well plate containing a cell culture insert with a 0.4 µm translucent polyethylene terephthalate (PET) membrane (ThinCerts; Greiner Bio-One, Kremsmünster, Austria). The membrane served as a net to contain the worms while allowing diffusion of liquid culture components across it. Each well contained approximately 30 worms. In addition, 500 µL of liquid culture outside the insert was replaced three times per week. The number of live worms was counted three times per week. The worms were considered dead if they showed no movement. Samples of C. elegans were collected on days 0, 7, and 14 after exposure. C. elegans were collected from the well and diluted to 400 µL. The samples were crushed with beads (3 g of 3 mm beads and 0.7 g of 1 mm beads) using a Multi-beads Shocker (YASUI KIKAI, Osaka, Japan). Subsequently, 200 µL of supernatant was added to 800 µL of CH 3 CN for deproteinization by centrifugation at 10000 × g for 5 min at 4°C. Next, 800 µL of the resulting supernatant was evaporated under reduced pressure and redissolved in 100 µL of CH 3 CN/H 2 O (1:1, v/v). Then, 50 µL of the redissolved solution was reacted with 10 µL of CMT-D-Leu in CH 3 CN and 10 µL of NaHCO 3 for 1 h at 60°C. After the reaction was complete, the mixture was evaporated under reduced pressure and redissolved in 2% FA in CH 3 CN/H 2 O (2:98, v/v). Results and discussion Screening of reagent structure for DL-AA separation Chiral derivatization reagents were synthesized and evaluated to develop a rapid method for screening DL -AAs. DMT-( S )-Pro-OSu is a reagent that enables selective detection of DL-AAs with high sensitivity, owing to ESI efficiency enhancement by the triazine moiety 25 . However, this method requires five different LC modes and a total of 40 min to complete detection. In this study, we designed and developed a novel reagent. Bhushan et al. previously developed several chlorotriazine-type chiral derivatization reagents for DL-AA separation 26 , enabling a moderate separation speed using the standard LC mode. Therefore, we aimed to enhance separation efficiency by modifying the optically active sites in the reagent. Figure 1 shows the screened structures of the candidate reagents. All the reagents possess an asymmetric carbon at the α-AA moiety. Semi-purified reagents obtained using pTLC were employed to evaluate the separation efficacy for all the DL -AAs. Table 1 compares the chromatographic resolutions between enantiomers for all the target compounds. The best separation was achieved using derivatives of the CMT-L-Leu reagent. The L-AAs eluted earlier than the D-AAs, which is undesirable because the detection sensitivity of D-AAs is thought to be lower than that of L-AAs. In principle, the retention order can be reversed by inverting the asymmetric carbon in the reagent moiety; therefore, CMT-D-Leu was selected as the optimal reagent. Table 1 Resolutions of all target DL-AAs. DL-AA Structure of enantio-chemical-tag site L-Leu L-Phe L-Pro L-Val Ala Ser Pro Val Thr Ile Leu Asn Asp Gln Lys Glu Met His Phe Arg Tyr Trp 5.98 2.45 1.44 6.87 6.91 6.36 7.62 0.64 1.54 1.10 0.66 2.44 6.17 0.64 8.26 1.69 3.79 4.54 6.99 2.72 3.12 0.00 7.89 0.00 0.00 0.17 0.78 1.94 0.44 4.07 6.21 0.29 3.12 1.69 4.99 5.68 0.83 1.05 1.39 2.73 2.49 2.61 0.35 1.77 0.33 2.99 2.26 0.91 1.99 0.45 2.08 2.40 1.40 0.16 4.90 2.27 1.86 5.38 5.27 4.59 5.01 0.19 1.81 1.27 0.00 2.99 4.88 0.54 3.83 1.60 5.63 3.46 Resolutions were calculated by the following equation: Rs = 1.18×(t RD -t RL )/(W 0.5D +W 0.5L ), where t RD and t RL indicated retention time of D- and L- form peak and W 0.5D and W 0.5L indicated half time of peak of D- and L- form. Figure S2A shows the MRM chromatograms of DL-AA separation using CMT-D-Leu under conventional octadecylsilane-type columns and FA acidic conditions. All the targeted DL-AAs were separated, except for Asn and His. Increasing the basicity of the mobile phase partially resolved this issue; however, Asp and Glu remained unseparated (Figure S2B). Therefore, further separation was performed by varying the column type. In a previous study on DMT-( S )-Pro-OSu, an ADME column was employed, showing an improved retention pattern compared with the conventional column. Similarly, in this study, an ADME column was evaluated for DL-AA separation. Figure S3A shows the MRM chromatograms obtained under acidic conditions, indicating insufficient separation of Asn and His. However, as shown in Figure S3B, good separation was achieved for all other targets in 10 mmol/L ammonium formate condition. Although all the targeted DL-AAs were completely separated within 10 min per run, the detection sensitivities of Asp and Glu were more than 50 times lower than those of the other compounds. These derivatives include tricarboxylic acids, thus suggesting that significantly stronger interactions affected peak performance and/or detection sensitivities. Therefore, a shorter ADME column (50 mm) was employed to improve throughput, and divided identical methods were developed within a single analytical system (Fig. 2 ). The limit of quantification (LOQ), defined as the concentration with S/N = 10, was determined. Values of 0.33–54.9 pmol/L on column (in vial) were obtained under the final conditions (Table S2). These sensitivities were sufficient to detect rare AAs in DSW. Based on these results, a high-throughput and highly sensitive method using the optimal reagent, CMT-D-Leu, was developed. Optimization of the reaction conditions and validation The reaction conditions for DL -AAs were optimized in high concentrations of mineral water for DSW analysis. Initially, interference from the minerals in the DSW hindered the reaction (data not shown). Therefore, a method was developed to reduce mineral content in DSW. We considered the differences in solubility between the minerals and AAs. When nine times the sample volume of methanol was added, significant precipitation was observed in the sample tube. Figure 3 shows the reaction time course between 10 and 120 min for five representative AAs. A 100 mmol/L NaHCO 3 solution was used as the basic catalyst. The maximum and plateau responses were reached after 60 min. Under these reaction conditions, the recovery rate of DSW was examined. Table S3 shows the results for the recovery rate and precision. The spiking concentrations were set at 1.0, 0.5, and 0.2 µmol/L for L -AAs and 0.2, 0.1, and 0.04 µmol/L for D -AAs. The results indicate that the spiked concentrations were fully recovered using the standard spiking calibration method. Quantification results of DSW Based on the above results, a quantitative analysis of DSW was performed using the established method. Table 2 shows the quantification results for the DSW samples from Toyama Bay. The samples were analyzed three times over the course of a year to determine the stability of the DSW content. Owing to the low rate of ocean currents in DSW, stable concentrations were observed throughout the year. Relatively high concentrations of D -Leu, D -Val, D -Ala, D -Thr, and D -Ser were quantified. Lower concentrations of D -Glu, D -Asn, and D -Pro were also detected in DSW. These results suggest that the AA content profile varies with ocean water depth. To the best of our knowledge, this is the first study to quantify DL-AAs in DSW from Japan. Different effects of D-AAs have been reported; therefore, these results provide an interesting perspective to explain the efficacy of DSW and/or its concentration. The higher concentration of D-AAs in the DSW is suggested to result from marine snow 27 . Marine snow forms during the death cycle of plankton, marine bacteria, and living organisms such as fish. Most marine snow is thought to contain the remains of bacteria and plankton, suggesting that its composition includes various types of prokaryotes 28 . Prokaryotes generally use D-AAs for initiation and production. One study suggested that D-AAs are selectively used by deep-sea microorganisms. In addition, D-Asp, D-Glu, and D-Ala have been detected in seabed hydrothermal sediments from the Izena and Yoron Cauldrons, Okinawa Trough 29 . These observations and our results suggest that DL-AA profiles may be suitable for analyzing marine areas, and their measurement is valuable not only for mechanistic research on DSW but also for discussing the specificity of the ecosystem. Table 2 Quantification results of DSW from Toyama Bay. Sample Lot 240415 250109 250519 Average SE Gly Null 153 372 115 213 80 Ala D 14.3 36.3 6.85 19.1 8.8 L 213 491 155 286 104 Ser D 10.1 30.3 19.6 20.0 5.8 L 464 630 329 474 87 Thr D 3.4 74.8 7.0 28.4 23.2 L 128 150 98.0 125 15 Pro D 2.16 7.85 7.95 5.99 1.91 L 104 104 48.4 85.3 18.5 Val D 11.0 27.0 4.51 14.2 6.7 L 69.0 129 46.5 81.5 24.6 Leu D 935 162 51.0 383 278 L 439 192 404 345 77.2 Ile D 3.58 7.65 NC 3.83 2.13 L 22.9 54.5 13.5 30.3 12.4 Met D ND 2.07 ND 2.07 NA L 6.25 29.4 30.7 22.1 7.93 Glu D 8.65 12.1 2.82 7.86 2.71 L 91.5 108 114 105 7 Gln D 6.90 9.85 NC 5.77 2.74 L 27.2 28.9 28.4 28.1 0.5 Asp D 5.00 1.44 2.95 3.13 1.03 L 690 2775 945 1470 657 Asn D 8.70 15.0 1.71 8.45 3.83 L 58.0 40.5 30.4 42.9 8.1 Lys D 20.6 24.9 ND 22.7 1.7 L 121 69.5 ND 95.0 20.8 Arg D 3.19 ND 3.26 3.21 0.04 L 179 182 208 189 9 His D 2.55 3.69 8.05 4.76 1.68 L 79.5 108 85.5 90.8 8.5 Tyr D 6.4 4.57 NC 3.91 1.66 L 39.3 103 43.0 61.8 20.7 Phe D 1.42 3.34 ND 2.38 0.78 L 39.9 62.5 23.4 41.9 11.3 Trp D 1.55 7.8 4.19 4.51 1.81 L ND 16.4 1.62 9.01 6.04 These concentrations were indicated as nmol/L of DSW. NC: Not calculated because the value showed under LLOQ, ND: Not detected. Lifespan assay of C. elegans with DSW addition and DL-AA kinetics Finally, we investigated the mechanistic contribution of DSW using the C. elegans model. Several studies have indicated that the benefits of DSW utility require continuous intake 30 . An animal model, C. elegans , was used in this study to evaluate lifespan during screening. The nematode C. elegans is a small organism with a shorter lifespan than higher-order animals such as mice. In addition, it has few organs and a genome similar to that of humans. Therefore, this microorganism is considered suitable for use in lifespan assays employing a simple experimental system, such as a well chamber. In this study, previous reports using the Transwell port system to facilitate daytime treatments during the experimental schedule were referenced 31 , 32 . Figure 4 A shows a microscopic image, and Fig. 4 B shows the resulting lifespan analysis curve. The movements of living organisms were clearly observed under microscopy. Observations throughout the lifespan revealed a clear prolongation of lifespan in the positive control and DSW-added samples (see also Table S4 as significant test). These results suggest that DSW demonstrates anti-aging efficacy in the C. elegans model. Given that the AA concentrations were lower than those of the mineral components, their contribution to the prolonged effect may be limited. However, AAs are suspected to serve as synergistic factors alongside mineral effects. In addition, several D-AAs are present at low concentrations (~ 10 nmol/L); therefore, they may contribute to the proliferation mechanism. Further studies, such as additional experiments with DL-AAs in DSW and/or desalinated DSW samples, are needed to elucidate the contribution of DL-AAs to the anti-aging effect. Furthermore, Fig. 4 C shows the kinetics results for DL-Asp (days 7 and 14), which was the only AA exhibiting significant changes. The D% significantly decreased in the treated groups on day 14. A decrease in D% was also observed on day 7. D-Asp is attracting attention as an aging marker derived from the aging reaction of the proteogenic L-Asp moiety 33 , 34 . These results suggest that D-Asp may be a suitable rapid biomarker for aging in C. elegans . In addition, fluctuations in D-Asp could potentially serve as a faster marker for lifespan assays. Further validation studies may establish D -Asp as a rapid biomarker for lifespan assays. Conclusions In this study, a rapid DL -AA screening method was developed using a novel enantiochemical tagging reagent, CMT-D-Leu. Screening of reagent columns enabled a 14 min gradient elution with good separation of 19 AAs for quantitative analysis. The detection sensitivity ranged from 10 to 100 pmol/L (LOD). Based on previous reports, this sensitivity level is sufficient to detect DL-AAs in sea water. Sea water samples were successfully analyzed, and several DL-AAs were quantified at 10–100 nmol/L. Although these concentrations may be insufficient to directly account for the efficacy, the synergistic effects with minerals and higher activity of D-AAs compared to L-AAs suggest their potential contribution to the mechanism of DSW. The content profiles of DL-AAs in DSW can be used for mechanistic analysis and environmental research in marine studies. Furthermore, although further improvements to the method are needed, such as target expansion (DL-kynurenine, citrulline, allothreonine, and highly reactive thiol AAs such as cysteine), this novel tagging reagent shows promise for the rapid separation of additional targets. In addition, biological analysis using C. elegans revealed the proliferative effects of DSW. Although concentrated D-AAs in DSW were not detected in the C. elegans body, the aging marker D-Asp significantly decreased in the proliferated groups. This suggests that DSW reduced aging in C. elegans , similar to the positive control. An anti-aging mechanistic study will be conducted in the future to determine the contribution of DL-AAs to DSW. Declarations Acknowledgements We thank Airi Minami (Master’s student) for technical advice and experimental support. We also thank Yukinobu Saeki and Sunao Fujii (GOSHU, Toyama, Japan) for preparing the DSW used in this study. This study was supported by a Sasakawa Scientific Research Grant from the Japan Science Society. Author Contributions T. T. and H. I. contributed equally to this study. T. T. and K. I. conceived the study. H. I. and T. T. performed the experiments, data analysis, interpretation, and writing of the original draft. R. F., A. M., Y. S., and T. T. collected, adjusted, and analyzed C. elegans samples and conducted observations. Data Availability The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request. Additional Information The additional methods and data include the detailed LC-MS(/MS) conditions, purity test results, MRM chromatograms, LLOQ and validation results, and significant tests of lifespan assay were available in Supporting Information. The authors declare no competing interests. Fundings This study was supported by a Sasakawa Scientific Research Grant from the Japan Science Society (No. 2024-6007). References C. Gao, Y. Zhang, D. Wu, L. Ma, Y. Zhang, Q. Zhang, X. Huang, IOP Conf. Ser. Earth Environ. Sci. 2019 , 384 , 012030. S. Z. M. Nani, F. A. A. Majid, A.B. Jaafar, A. Mahdzir, M. N. Musa, Evid. Based Complementary Altern. Med. 2016 , 2016 , 6520475. Z-Y. Fu, F.L. Yang, H-W. Hsu, Y-F. Lu, J. Med. Food , 2012 , 15 , 535-541 Y. Hataguchi, H. Tai, H. Nakajima and H. Kimata, Eur. J. Clin. Nutr. , 2005 , 59 , 1093-1096 M. Kawada, H. Takeuchi, S.A. Con, E. Yamamoto, T. Yasukawa, K. Nakagawa, Y. Ikegami and T. Sugiura, J. Medical Microbiol. Diagnosis , 2012 , 2012 , 1-7 K. S. Lee, Y. S. Kwon, S. Kim, D. S. Moon, H. J. Kim, K. S. Nam, Biomed Pharmacother , 2017 , 86 , 405-413. K. Fukui, Y. Suzuki, Y. Kato, N. Takeuchi, H. Takenaka, M. Kohno, Nutrients , 2022 , 25 , 14. E. Proksch, H. P. Nissen, M. Bremgartner, C. Urquhart, Int. J. Dermatol. , 2005 , 44 , 151-157. K. Park, W. T. Williams, J. M. Prescott, D. W. Hood, Science. 1962 , 26 , 531-532. T. Ikari, Y. Furusawa, Y. Tabuchi, Y. Maruyama, A. Hattori, Y. Kitani, K. Toyota, A. Nagami, J. Hirayama, K. Watanabe, A. Shigematsu, M. A. Rafiuddin, S. Ogiso, K. Fukushi, K. Kuroda, K. Hatano, T. Sekiguchi, R. Kawashima, A. K. Srivastav, T. Nishiuchi, A. Sakatoku, M. A. Yoshida, H. Matsubara, N. Suzuki, Sci Rep. , 2023 , 29 , 8700. S. Du, M. Wey, D. W. Armstrong, Chirality, 2023 , 35 , 508-534. A. D. Ivanov, J. P. Mothet, Neurosci. Lett. , 2019 , 10 , 21-25. T. Fujimori, H. Abe, Comp. Biochem. Physiol. A Mol. Integr. Physiol. , 2002 , 131 , 893-900. T. Kubota, T. Kobayashi, T. Nunoura, F. Maruyama, S. Deguchi, Front Microbiol. , 2016 , 19 , 511. J. Kobayashi J. 2019 , 12 , 690. Q. Y. Cheng, J. Xiong, W. Huang, Q. Ma, W. Ci, Y. Q. Feng, B. F. Yuan, Sci. Rep., 2015 , 13 , 15217. C. Zhang, Y. Liu, R. Liu, W. Li, C. Liu, L. He, Chirality, 2022 , 34 , 955-967. J. A. Weiß, S. Mohr, M. G. Schmid, Chirality , 2015 , 27 , 211-215. T. Sakamoto, M. Onozato, S. Uekusa, H. Ichiba, M. Umino, M. Shirao, T. Fukushima, J Chromatogr. A , 2021 , 30 , 462341. X. Wang, X. Sun, Y. Jin, S. Cheng, Y. Han, M. Zhang, L. Zhang, X. L. Li, C. Y. Xu, J. Z. Min, Anal. Methods , 2023 , 16 , 884-895. C. Lella, L. Nestor, D. D. Bundel, Y. H. Vander, A. E. Van, Int J Mol Sci. 2024 , 19 , 12410. Z. Qi, H. Ji, M. Le, H. Li, A. Wieland, S. Bauer, L. Liu, M. Wink, I. Herr, Aging, 2021 , 20 , 1649-1670. S. Yanase, K. Yasuda, N. Ishii, Methods Mol. Biol. , 2019 , 1916 , 123-132. F. R. Amrit, R. Ratnappan, S. A. Keith, A. Ghazi, Methods , 2014 , 68 , 465-75. T. Mochizuki, T. Takayama, K. Todoroki, K. Inoue K, J. Z. Min, T. Toyo'oka, Anal. Chim. Acta , 2015 , 22 , 73-82. R. Bhushan, V. Kumar, J. Chromatogr. A , 2008 , 1201 , 35-42. P. A. Steiner, E. Sintes, R. Simó, D. De Corte, D. M. Pfannkuchen, I. Ivančić, M. Najdek, G. J. Herndl, Environ. Microbiol. Rep. , 2019 , 11 , 699-707. B. E. Clifton, U. Alcolombri, G. I. Uechi, C. J. Jackson, P. Laurino, Nature , 2024 , 634, 721-728. S. Fuchida, H. Masuda, R. Fukuchi, T. Yamanaka, Geochemical Journal, 2015 , 49 , 295-307. H. Takeuchi, Y. Yoshikane, H. Takenaka, A. Kimura, J. M. Islam, R. Matsuda, A. Okamoto, Y. Hashimoto, R. Yano, K. Yamaguchi, S. Sato, S. Ishizuka, Nutrients , 2022 , 28 , 581. V. Fitzgerald, M. Mensack, P. Wolfe, H. Thompson, Biotechniques , 2009, 47, ix-xv. K. J. Helmcke, D. S. Avila, M. Aschner, Neurotoxicol. Teratol. , 2010 , 32 , 62-67. S. Ha, T. Kinouchi, N. Fujii, Biochim. Biophys. Acta Proteins Proteom. , 2020 , 1868 , 140410. H. Mizuno, Y. Miyazaki, K. Ito, K. Todoroki, J. Z. Min, T. Toyo'oka, J. Chromatogr. A , 2016 , 1467 , 318-325. Additional Declarations No competing interests reported. Supplementary Files SupportingInformationSFR2025.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7397638","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":509253714,"identity":"c784444f-d34d-4b6a-8b78-d3bab366d43a","order_by":0,"name":"Takahiro Takayama","email":"data:image/png;base64,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","orcid":"","institution":"Ritsumeikan University","correspondingAuthor":true,"prefix":"","firstName":"Takahiro","middleName":"","lastName":"Takayama","suffix":""},{"id":509253715,"identity":"3f86d209-47a0-4503-ab65-bd5c8459bbfa","order_by":1,"name":"Haruto Iwata","email":"","orcid":"","institution":"Ritsumeikan University","correspondingAuthor":false,"prefix":"","firstName":"Haruto","middleName":"","lastName":"Iwata","suffix":""},{"id":509253716,"identity":"36f17dea-34e7-4c1d-8f3a-0f76cbe3f240","order_by":2,"name":"Reina Fujio","email":"","orcid":"","institution":"Ritsumeikan University","correspondingAuthor":false,"prefix":"","firstName":"Reina","middleName":"","lastName":"Fujio","suffix":""},{"id":509253717,"identity":"a0dec0fa-d35b-4e1f-954a-68a44edfb5b3","order_by":3,"name":"Ayaka Minamida","email":"","orcid":"","institution":"Ritsumeikan University","correspondingAuthor":false,"prefix":"","firstName":"Ayaka","middleName":"","lastName":"Minamida","suffix":""},{"id":509253718,"identity":"d351d119-3603-4062-a656-6ba80f90b912","order_by":4,"name":"Yuko Sakaguchi","email":"","orcid":"","institution":"Ritsumeikan University","correspondingAuthor":false,"prefix":"","firstName":"Yuko","middleName":"","lastName":"Sakaguchi","suffix":""},{"id":509253719,"identity":"5d72319a-c311-48a5-88ae-50027d12288d","order_by":5,"name":"Koichi Inoue","email":"","orcid":"","institution":"Ritsumeikan University","correspondingAuthor":false,"prefix":"","firstName":"Koichi","middleName":"","lastName":"Inoue","suffix":""}],"badges":[],"createdAt":"2025-08-18 08:53:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7397638/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7397638/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":90583467,"identity":"0d425dc2-b0aa-485e-a30c-6e68ed9bdd69","added_by":"auto","created_at":"2025-09-04 10:51:37","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":106813,"visible":true,"origin":"","legend":"\u003cp\u003eScreened structure of reagent candidates and their reaction.\u003c/p\u003e\n\u003cp\u003eA: candidate structures, B: reaction scheme\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7397638/v1/741b099b3d4a8040f5208338.png"},{"id":90583468,"identity":"d46eebb2-cbed-461b-91c6-23afa29602c6","added_by":"auto","created_at":"2025-09-04 10:51:37","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":943656,"visible":true,"origin":"","legend":"\u003cp\u003eMS chromatograms of optimized condition for 19 amino acids including enantiomers.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7397638/v1/dc036b1f3b50427b6de1c935.png"},{"id":90583260,"identity":"4738c457-2666-4dc3-a5d3-cebd135221ec","added_by":"auto","created_at":"2025-09-04 10:43:37","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":139720,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative reaction time courses of derivatization.\u003c/p\u003e\n\u003cp\u003eFour series of representative Aas were shown in the figure. The circle symbols and triangle symbols indicated the L- and D-form results. The error bars indicated standard error (N=3).\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7397638/v1/cd8957aa0af6489f1219bf6b.png"},{"id":90583246,"identity":"dfc8e6ce-9b24-4f68-8ab5-d66dc3d3c5e7","added_by":"auto","created_at":"2025-09-04 10:43:37","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":259486,"visible":true,"origin":"","legend":"\u003cp\u003eLife span assay of DSW using \u003cem\u003eC.elegans\u003c/em\u003e model.\u003c/p\u003e\n\u003cp\u003e(A) Optical images of \u003cem\u003eC.elegans\u003c/em\u003e at observed day 7. 1; control (MiliQ), 2; positive control (Metfolmin), 3; 2.5% DSW containing medium and 4; 10% DSW containing medium\u003c/p\u003e\n\u003cp\u003e(B) Kaplan meier curve of each condition. Black; control (MiliQ), Pink; positive control (Metfolmin), Sky blue; 2.5% DSW containing medium and Blue; 10% DSW containing medium. The arrow indicated the collection time points of \u003cem\u003eC.elegans\u003c/em\u003ebodies for DL-AA kinetics.\u003c/p\u003e\n\u003cp\u003e(C) DL-Asp kinetix extracted from \u003cem\u003eC.elegans\u003c/em\u003e bodies. The statistical analysis was conducted by Dunnet’s t-test compared with control group (*：p\u0026lt;0.05, **：p\u0026lt;0.01, ***：p\u0026lt;0.005).\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7397638/v1/e7c159ee59457e1028bbe15e.png"},{"id":94475315,"identity":"7dc103b3-7165-418e-90e8-e04480b37a60","added_by":"auto","created_at":"2025-10-27 15:52:09","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2556194,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7397638/v1/6eef2d35-24bb-494a-aa32-3b83a782fc68.pdf"},{"id":90583471,"identity":"2b08fe35-6f75-482c-8cc2-34ddf2c296e3","added_by":"auto","created_at":"2025-09-04 10:51:37","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":389772,"visible":true,"origin":"","legend":"","description":"","filename":"SupportingInformationSFR2025.docx","url":"https://assets-eu.researchsquare.com/files/rs-7397638/v1/ef0447fe9b18002591c4c43b.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"High-throughput DL-amino acid analysis of deep sea water from Toyama Bay and anti-aging activity assessment using C. elegans","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIn Japan, deep sea water (DSW) is defined as water deeper than 200 m below the sea surface. DSW is characterized by high purity, stability at low temperatures, high mineral concentrations, and the presence of bioactive nutritional species\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Consequently, its applications have expanded into the fields of energy, agriculture, food, cosmetics, and public health\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. In human healthcare, several clinical trials have reported beneficial effects on hypercholesterolemia, allergy, and \u003cem\u003eHelicobacter pylori\u003c/em\u003e infection\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Because the efficacy of DSW has only been partially established, mechanistic studies have attempted to identify which components in the \u0026ldquo;solution\u0026rdquo; are effective. Several studies have reported the relative efficacy of DSW in maintaining mineral balance\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. For example, Lee et al. demonstrated that the hardness levels of DSW, particularly the Mg and Ca content ratios (Mg/Ca\u0026thinsp;=\u0026thinsp;3/1), were critical for reducing hepatic cholesterol production\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Fukui et al. investigated the hardness of an adjusted solution containing DSW in relation to obesity in mice fed a high-fat diet\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. They also analyzed the mineral composition of DSW using liquid chromatography\u0026ndash;inductively coupled plasma mass spectrometry (LC\u0026ndash;ICP\u0026ndash;MS) and found that high levels of Mg and Ca may play a key role in its efficacy. Although reasonable explanations have been proposed regarding the role of minerals in these studies, almost no research has focused on amino acids (AAs) in DSW.\u003c/p\u003e\u003cp\u003eA few studies have explored the contribution of AAs in DSW. For example, a previous study detected 17 types of AAs in DSW from the Gulf of Mexico, suggesting that organic compounds may contribute to its efficacy\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Ikari et al. analyzed the relationship between the kynurenine signal in DSW from Japan and the stress response in \u003cem\u003eParalichthys olivaceus\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. As expected, these studies have detected extremely high amounts of minerals such as Mg in DSW, with AAs partially contributing as effective factors. However, to date, analytical results for AAs in DSW remain limited.\u003c/p\u003e\u003cp\u003eAAs are generally proteinogenic L-AAs (20 types). Both D- and L-isomers exist, with high amounts of L-forms detected in biological specimens. Several studies have detected D-AAs in biological samples and reported various effects on living organisms\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. For example, \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eD\u003c/span\u003e-Ser acts as a neurotransmitter for the NMDA receptor\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e, while D-Ala is a precursor for neurotransmitters and osmotic compounds for marine life such as shrimp and crabs\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. In addition, the utility of \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eD\u003c/span\u003e-AAs, including \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eD\u003c/span\u003e-Ala and \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eD\u003c/span\u003e-Ser, has been recognized in recent biological research. \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eD\u003c/span\u003e-AAs are produced and used by microorganisms such as bacteria and plankton\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Notably, one study suggested that \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eD\u003c/span\u003e-AAs are selectively used by deep-sea microorganisms\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e, which may support the presence and role of \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eD\u003c/span\u003e-AAs in living organisms in DSW. However, analytical research on AAs in DSW is limited, and even less research on D-AAs is available. Furthermore, because DSW is highly diluted, a sensitive and selective method is necessary to analyze \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eDL\u003c/span\u003e-AAs in DSW.\u003c/p\u003e\u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eDL\u003c/span\u003e-AAs cannot be analyzed using conventional liquid chromatography\u0026ndash;tandem mass spectrometry (LC-MS/MS) methods because enantiomers exhibit identical physical properties. Several methods employing chiral column technology or pre-column chiral derivatization have been reported. Chiral derivatization enables enantioseparation in conventional LC mode and highly sensitive detection by improving ionization efficiency for MS detection\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Several methods employing innovative derivatization reagents have been successfully used to analyze \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eDL\u003c/span\u003e-AAs in various biological samples, including our methodologies\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Although these methods achieve rapid analysis (~\u0026thinsp;20 min/measurement) of proteinogenic AAs with enantioseparation, higher-throughput analyses are required to meet the demands of big data generation. In this study, we screened novel reagents and developed methods that are more rapid than those used in previous studies. In addition, DSW from Toyama Bay, Japan was analyzed to profile \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eDL\u003c/span\u003e-AA concentrations.\u003c/p\u003e\u003cp\u003eIn addition to this analysis, the effectiveness of DSW was evaluated using \u003cem\u003eCaenorhabditis elegans\u003c/em\u003e (\u003cem\u003eC. elegans\u003c/em\u003e) in a lifespan assay. The nematode \u003cem\u003eC. elegans\u003c/em\u003e is approximately 1 mm in length (~\u0026thinsp;1000 cells in the body) with a transparent body that facilitates the observation of organ tissues. It possesses muscles, a digestive tract, a nervous system, epithelium, and reproductive organs. Moreover, its short lifespan of approximately 20 d makes it suitable for lifespan assays to reduce generation time\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. The life extension performance of DSW was assessed using \u003cem\u003eC. elegans\u003c/em\u003e, and \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eDL\u003c/span\u003e-AA kinetics were analyzed using the developed method.\u003c/p\u003e"},{"header":"Experimental","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eReagents and chemicals\u003c/h2\u003e\u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eDL\u003c/span\u003e-AA standards, including glycine (Gly, not optically active), alanine (Ala), serine (Ser), threonine (Thr), proline (Pro), valine (Val), leucine (Leu), isoleucine (Ile), aspartic acid (Asp), asparagine (Asn), glutamic acid (Glu), glutamine (Gln), methionine (Met), phenylalanine (Phe), tyrosine (Tyr), tryptophan (Trp), histidine (His), arginine (Arg), and lysine (Lys), were purchased from Tokyo Chemical Industry (Tokyo, Japan). Working solutions of each metabolite were prepared in acetonitrile/water (1:1) at a concentration of 10 mmol/L. For reagent synthesis, 2,4-dichloro-6-methoxy-1,3,5-triazine (DCMT) was also purchased from Tokyo Chemical Industry. Acetonitrile (CH\u003csub\u003e3\u003c/sub\u003eCN), methanol (CH\u003csub\u003e3\u003c/sub\u003eOH), ethyl acetate (EAAC), \u003cem\u003en\u003c/em\u003e-hexane (Hex), pyridine, triethylamine (TEA), hydrochloric acid (HCl), sodium bicarbonate (NaHCO\u003csub\u003e3\u003c/sub\u003e), ammonium formate, sodium chloride (NaCl), and LC-MS grade formic acid (FA) were obtained from Fujifilm Wako Pure Chemical Co. (Osaka, Japan). Purified water was sourced from a Milli-Q EQ7000 system (Merck, Darmstadt, Germany).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eScreening of the ideal enantiochemical tagging reagent for DL-AA analysis\u003c/h3\u003e\n\u003cp\u003eDCMT powder (360 mg, 2.0 mmol) was dissolved in 20 mL CH\u003csub\u003e3\u003c/sub\u003eCN. In a separate flask, L-AAs (L-Phe, L-Pro, L-Val, or L-Leu; each 2.0 mmol) were dissolved in 20 mL of a mixture of H\u003csub\u003e2\u003c/sub\u003eO and MeOH (1:4 v/v). These solutions were mixed for 3 h at room temperature, with 35 \u0026micro;L of TEA added every 30 min. After the reaction was complete, the mixture was extracted into the aqueous layer of a water/Hex phase, and a small amount of HCl was added. It was then extracted with EAAC and Hex (1:1 v/v), washed with H\u003csub\u003e2\u003c/sub\u003eO- and NaCl-saturated solutions, and evaporated under reduced pressure. The resulting residues were purified by preparative TLC (silica gel 70 F\u003csub\u003e254\u003c/sub\u003e PLC, 1.0 mm, Fujifilm Wako Pure Chemical Co.) using an eluent of EAAC and Hex (1:1 v/v).\u003c/p\u003e\n\u003ch3\u003eSynthesize of reagents i.e., 2,4-Dichloro-6-methoxy-1,3,5-triazine-D-Leu\u003c/h3\u003e\n\u003cp\u003eDCMT powder (360 mg, 2.0 mmol) was dissolved in 20 mL of CH\u003csub\u003e3\u003c/sub\u003eCN. In another flask, D-Leu (262 mg, 2.0 mmol) was dissolved in 20 mL of an H\u003csub\u003e2\u003c/sub\u003eO\u0026ndash;MeOH mixture (1:4 v/v). These solutions were mixed for 3 h at room temperature, with 35 \u0026micro;L of TEA added every 30 min. After the reaction was complete, the mixture was extracted into the aqueous layer of a water/Hex phase, and a small amount of HCl was added. Subsequently, it was extracted with EAAC and Hex (1:1 v/v), washed with H\u003csub\u003e2\u003c/sub\u003eO- and NaCl-saturated solutions, and evaporated under reduced pressure. The resulting residue was redissolved in 4 mL of H\u003csub\u003e2\u003c/sub\u003eO/MeOH (1:1 v/v) and purified using a PU 714M pump, SC 762 system controller, and PLC 761 fraction collector (GL Sciences, Tokyo, Japan). The preparative column used was a TSKgel ODS-80Ts column (5 \u0026micro;m, 25 cm \u0026times; 20.0 mm i.d., Tosoh Corporation). The elution conditions were as follows: mobile phase, H\u003csub\u003e2\u003c/sub\u003eO/MeOH mixture containing 0.1% (v/v) FA; gradient elution (MeOH% (min)): 20 (0\u0026ndash;1), 70 (1\u0026ndash;200), 95 (200\u0026ndash;205), and 5 (205\u0026ndash;210). The flow rate of the mobile phase was 5.0 mL/min. Purity (HPLC%) was determined as \u0026gt;\u0026thinsp;99% by photo diode array (190 nm\u0026ndash;600 nm, ACQUITY PDA, Waters). The \u003cem\u003em/z\u003c/em\u003e of the peak was 275.0904 (theoretical value; 275.0910 [M\u0026thinsp;+\u0026thinsp;H\u003csup\u003e+\u003c/sup\u003e]) by high-resolution mass spectrometry (Xevo G2-XS QTOF, Waters). Analytical conditions and results of these purity tests were shown in Supporting Information (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eInstrument operating conditions\u003c/h3\u003e\n\u003cp\u003eUltraperformance liquid chromatography\u0026ndash;electrospray ionization\u0026ndash;tandem mass spectrometry (UPLC-ESI-MS/MS) was performed using an ACQUITY UPLC H-class instrument (Waters, Milford, MA, USA) connected to a Xevo TQ-XS mass spectrometer (Waters, Milford, MA, USA). For MS detection of the derivatives, positive ESI and multiple reaction monitoring (MRM) modes were used. The analytical columns used were an ACQUITY\u0026trade; Premier BEH C18 column (1.7 \u0026micro;m, 100 \u0026times; 2.1 mm i.d., Waters) or an ADME-HR column (2.0 \u0026micro;m, 100 \u0026times; 2.1 mm i.d., Osaka Soda). A shorter ADME-HR column (2.0 \u0026micro;m, 50 \u0026times; 2.1 mm i.d., Osaka Soda) was employed under optimal conditions. The elution conditions for the ACQUITY\u0026trade; Premier BEH C18 column were as follows: mobile phase, a CH\u003csub\u003e3\u003c/sub\u003eCN/H\u003csub\u003e2\u003c/sub\u003eO mixture containing 0.1% (v/v) FA; gradient elution (CH\u003csub\u003e3\u003c/sub\u003eCN% (min)): 5 (0\u0026ndash;0.5), 42 (0.5\u0026ndash;16), 98 (16\u0026ndash;18), and 5 (18\u0026ndash;20). The flow rate of the mobile phase was 0.4 mL/min. The elution conditions for the ADME-HR column were as follows: mobile phase, a CH\u003csub\u003e3\u003c/sub\u003eCN/H\u003csub\u003e2\u003c/sub\u003eO mixture containing 10 mmol/L ammonium formate; gradient elution (CH\u003csub\u003e3\u003c/sub\u003eCN% (min)): 2 (0\u0026ndash;0.3), 25 (0.3\u0026ndash;7.5), 98 (7.5\u0026ndash;8.5), and 2 (8.5\u0026ndash;10). The flow rate of the mobile phase was 0.5 mL/min. The elution conditions for the shorter ADME-HR column were as follows: mobile phase, a CH\u003csub\u003e3\u003c/sub\u003eCN/H\u003csub\u003e2\u003c/sub\u003eO mixture containing 10 mmol/L ammonium formate; gradient elution (CH\u003csub\u003e3\u003c/sub\u003eCN% (min)): 2 (0\u0026ndash;0.2), 39 (0.2\u0026ndash;4.5), 98 (4.5\u0026ndash;5.5), and 2 (5.5\u0026ndash;7). Another set of conditions was also evaluated as follows: mobile phase, a CH\u003csub\u003e3\u003c/sub\u003eCN/H\u003csub\u003e2\u003c/sub\u003eO mixture containing 0.1% (v/v) FA; gradient elution (CH\u003csub\u003e3\u003c/sub\u003eCN% (min)): 2 (0\u0026ndash;0.2), 25 (0.2\u0026ndash;9), 98 (9\u0026ndash;10), and 2 (10\u0026ndash;11). The flow rate of the mobile phase was 0.5 mL/min. The MS/MS conditions are described in detail in the Supporting Information (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eDerivatization conditions of AAs\u003c/h3\u003e\n\u003cp\u003eAn aliquot of 10 \u0026micro;L of a 1 \u0026micro;mol/L \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eDL\u003c/span\u003e-AA mixture was added to 10 \u0026micro;L of 20 mmol/L CMT-D-Leu in CH\u003csub\u003e3\u003c/sub\u003eCN, and 10 \u0026micro;L of 100 mmol/L NaHCO\u003csub\u003e3\u003c/sub\u003e in H\u003csub\u003e2\u003c/sub\u003eO. These mixtures were reacted for 90 min at 60\u0026deg;C. After the reaction was complete, the mixture was evaporated under reduced pressure and redissolved in 100 \u0026micro;L of 1% FA in CH\u003csub\u003e3\u003c/sub\u003eCN/H\u003csub\u003e2\u003c/sub\u003eO (2:98, v/v).\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eValidation of \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eDL\u003c/span\u003e-AA quantification analysis of DSW\u003c/h2\u003e\u003cp\u003eA mixture of 1900 \u0026micro;L DSW and 100 \u0026micro;L of \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eDL\u003c/span\u003e-AA solutions at various concentrations (0\u0026ndash;50 \u0026micro;M) was desalinated by adding it to 9 mL of MeOH. These mixtures were centrifuged at 4000 \u0026times; \u003cem\u003eg\u003c/em\u003e for 5 min at 4\u0026deg;C. The supernatant (1 mL) was then added to 10 \u0026micro;L of CMT-D-Leu in CH\u003csub\u003e3\u003c/sub\u003eCN and 50 \u0026micro;L of NaHCO\u003csub\u003e3\u003c/sub\u003e in H\u003csub\u003e2\u003c/sub\u003eO. The resulting mixtures were subsequently used for derivatization. Calibration curves were constructed by plotting the peak area against the sample concentration. These mixtures were reacted for 120 min at 60\u0026deg;C. After the reaction was complete, the mixture was evaporated under reduced pressure and redissolved in 100 \u0026micro;L of 2% FA in CH\u003csub\u003e3\u003c/sub\u003eCN/H\u003csub\u003e2\u003c/sub\u003eO (2:98, v/v). A recovery test from DSW was performed at three concentrations. Intra- and inter-day precisions were assessed by quantifying \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eDL\u003c/span\u003e-AAs in DSW over 3 d (n\u0026thinsp;=\u0026thinsp;3 each day).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eLifespan assay using C. elegans model\u003c/h3\u003e\n\u003cp\u003eThe lifespan assay was conducted following procedures described in a previous study. The biomaterials used in this study are described in the Supplementary Data. Briefly, the wild-type nematode \u003cem\u003eC. elegans\u003c/em\u003e (Bristol strain N2) was used as a model organism. \u003cem\u003eC. elegans\u003c/em\u003e stocks were maintained on nematode growth medium (NGM) plates seeded with 50 \u0026micro;L \u003cem\u003eE. coli\u003c/em\u003e (DH5αFT) at 20\u0026deg;C as a food source. Eggs were collected from \u003cem\u003eC. elegans\u003c/em\u003e by lysing them using KOH and NaClO. These eggs were left in Milli-Q water overnight to synchronize \u003cem\u003eC. elegans\u003c/em\u003e at the L1 stage. After the worms reached L1, they were transferred onto NGM plates with \u003cem\u003eE. coli\u003c/em\u003e until the L4 stage. On the third day, 50 \u0026micro;M 5-Fluoro-2\u0026rsquo;-deoxyuridine (FUDR; FUJIFILM, Miyazaki, Japan) was added to suppress reproduction. On the fourth day, \u003cem\u003eC. elegans\u003c/em\u003e were exposed to liquid culture medium composed of 75 \u0026micro;L FUDR, 550 \u0026micro;L S-basal, 1.5 \u0026micro;L 2% cholesterol, 75 \u0026micro;L solvent (DSW, 25% DSW, 500 mM metformin, or Milli-Q water as control), and 50 \u0026micro;L Milli-Q water containing \u003cem\u003eC. elegans\u003c/em\u003e. Next, 750 \u0026micro;L of this liquid culture was added to each well of a 24-well plate containing a cell culture insert with a 0.4 \u0026micro;m translucent polyethylene terephthalate (PET) membrane (ThinCerts; Greiner Bio-One, Kremsm\u0026uuml;nster, Austria). The membrane served as a net to contain the worms while allowing diffusion of liquid culture components across it. Each well contained approximately 30 worms. In addition, 500 \u0026micro;L of liquid culture outside the insert was replaced three times per week. The number of live worms was counted three times per week. The worms were considered dead if they showed no movement. Samples of \u003cem\u003eC. elegans\u003c/em\u003e were collected on days 0, 7, and 14 after exposure. \u003cem\u003eC. elegans\u003c/em\u003e were collected from the well and diluted to 400 \u0026micro;L. The samples were crushed with beads (3 g of 3 mm beads and 0.7 g of 1 mm beads) using a Multi-beads Shocker (YASUI KIKAI, Osaka, Japan). Subsequently, 200 \u0026micro;L of supernatant was added to 800 \u0026micro;L of CH\u003csub\u003e3\u003c/sub\u003eCN for deproteinization by centrifugation at 10000 \u0026times; g for 5 min at 4\u0026deg;C. Next, 800 \u0026micro;L of the resulting supernatant was evaporated under reduced pressure and redissolved in 100 \u0026micro;L of CH\u003csub\u003e3\u003c/sub\u003eCN/H\u003csub\u003e2\u003c/sub\u003eO (1:1, v/v). Then, 50 \u0026micro;L of the redissolved solution was reacted with 10 \u0026micro;L of CMT-D-Leu in CH\u003csub\u003e3\u003c/sub\u003eCN and 10 \u0026micro;L of NaHCO\u003csub\u003e3\u003c/sub\u003e for 1 h at 60\u0026deg;C. After the reaction was complete, the mixture was evaporated under reduced pressure and redissolved in 2% FA in CH\u003csub\u003e3\u003c/sub\u003eCN/H\u003csub\u003e2\u003c/sub\u003eO (2:98, v/v).\u003c/p\u003e"},{"header":"Results and discussion","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eScreening of reagent structure for DL-AA separation\u003c/h2\u003e\u003cp\u003eChiral derivatization reagents were synthesized and evaluated to develop a rapid method for screening \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eDL\u003c/span\u003e-AAs. DMT-(\u003cem\u003eS\u003c/em\u003e)-Pro-OSu is a reagent that enables selective detection of DL-AAs with high sensitivity, owing to ESI efficiency enhancement by the triazine moiety\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. However, this method requires five different LC modes and a total of 40 min to complete detection. In this study, we designed and developed a novel reagent. Bhushan et al. previously developed several chlorotriazine-type chiral derivatization reagents for DL-AA separation\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e, enabling a moderate separation speed using the standard LC mode. Therefore, we aimed to enhance separation efficiency by modifying the optically active sites in the reagent. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e1\u003c/span\u003e shows the screened structures of the candidate reagents. All the reagents possess an asymmetric carbon at the α-AA moiety. Semi-purified reagents obtained using pTLC were employed to evaluate the separation efficacy for all the \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eDL\u003c/span\u003e-AAs. Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e compares the chromatographic resolutions between enantiomers for all the target compounds. The best separation was achieved using derivatives of the CMT-L-Leu reagent. The L-AAs eluted earlier than the D-AAs, which is undesirable because the detection sensitivity of D-AAs is thought to be lower than that of L-AAs. In principle, the retention order can be reversed by inverting the asymmetric carbon in the reagent moiety; therefore, CMT-D-Leu was selected as the optimal reagent.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eResolutions of all target DL-AAs.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eDL-AA\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"4\" nameend=\"c5\" namest=\"c2\"\u003e\u003cp\u003eStructure of enantio-chemical-tag site\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eL-Leu\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eL-Phe\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eL-Pro\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eL-Val\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAla\u003c/p\u003e\u003cp\u003eSer\u003c/p\u003e\u003cp\u003ePro\u003c/p\u003e\u003cp\u003eVal\u003c/p\u003e\u003cp\u003eThr\u003c/p\u003e\u003cp\u003eIle\u003c/p\u003e\u003cp\u003eLeu\u003c/p\u003e\u003cp\u003eAsn\u003c/p\u003e\u003cp\u003eAsp\u003c/p\u003e\u003cp\u003eGln\u003c/p\u003e\u003cp\u003eLys\u003c/p\u003e\u003cp\u003eGlu\u003c/p\u003e\u003cp\u003eMet\u003c/p\u003e\u003cp\u003eHis\u003c/p\u003e\u003cp\u003ePhe\u003c/p\u003e\u003cp\u003eArg\u003c/p\u003e\u003cp\u003eTyr\u003c/p\u003e\u003cp\u003eTrp\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5.98\u003c/p\u003e\u003cp\u003e2.45\u003c/p\u003e\u003cp\u003e1.44\u003c/p\u003e\u003cp\u003e6.87\u003c/p\u003e\u003cp\u003e6.91\u003c/p\u003e\u003cp\u003e6.36\u003c/p\u003e\u003cp\u003e7.62\u003c/p\u003e\u003cp\u003e0.64\u003c/p\u003e\u003cp\u003e1.54\u003c/p\u003e\u003cp\u003e1.10\u003c/p\u003e\u003cp\u003e0.66\u003c/p\u003e\u003cp\u003e2.44\u003c/p\u003e\u003cp\u003e6.17\u003c/p\u003e\u003cp\u003e0.64\u003c/p\u003e\u003cp\u003e8.26\u003c/p\u003e\u003cp\u003e1.69\u003c/p\u003e\u003cp\u003e3.79\u003c/p\u003e\u003cp\u003e4.54\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e6.99\u003c/p\u003e\u003cp\u003e2.72\u003c/p\u003e\u003cp\u003e3.12\u003c/p\u003e\u003cp\u003e0.00\u003c/p\u003e\u003cp\u003e7.89\u003c/p\u003e\u003cp\u003e0.00\u003c/p\u003e\u003cp\u003e0.00\u003c/p\u003e\u003cp\u003e0.17\u003c/p\u003e\u003cp\u003e0.78\u003c/p\u003e\u003cp\u003e1.94\u003c/p\u003e\u003cp\u003e0.44\u003c/p\u003e\u003cp\u003e4.07\u003c/p\u003e\u003cp\u003e6.21\u003c/p\u003e\u003cp\u003e0.29\u003c/p\u003e\u003cp\u003e3.12\u003c/p\u003e\u003cp\u003e1.69\u003c/p\u003e\u003cp\u003e4.99\u003c/p\u003e\u003cp\u003e5.68\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.83\u003c/p\u003e\u003cp\u003e1.05\u003c/p\u003e\u003cp\u003e1.39\u003c/p\u003e\u003cp\u003e2.73\u003c/p\u003e\u003cp\u003e2.49\u003c/p\u003e\u003cp\u003e2.61\u003c/p\u003e\u003cp\u003e0.35\u003c/p\u003e\u003cp\u003e1.77\u003c/p\u003e\u003cp\u003e0.33\u003c/p\u003e\u003cp\u003e2.99\u003c/p\u003e\u003cp\u003e2.26\u003c/p\u003e\u003cp\u003e0.91\u003c/p\u003e\u003cp\u003e1.99\u003c/p\u003e\u003cp\u003e0.45\u003c/p\u003e\u003cp\u003e2.08\u003c/p\u003e\u003cp\u003e2.40\u003c/p\u003e\u003cp\u003e1.40\u003c/p\u003e\u003cp\u003e0.16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e4.90\u003c/p\u003e\u003cp\u003e2.27\u003c/p\u003e\u003cp\u003e1.86\u003c/p\u003e\u003cp\u003e5.38\u003c/p\u003e\u003cp\u003e5.27\u003c/p\u003e\u003cp\u003e4.59\u003c/p\u003e\u003cp\u003e5.01\u003c/p\u003e\u003cp\u003e0.19\u003c/p\u003e\u003cp\u003e1.81\u003c/p\u003e\u003cp\u003e1.27\u003c/p\u003e\u003cp\u003e0.00\u003c/p\u003e\u003cp\u003e2.99\u003c/p\u003e\u003cp\u003e4.88\u003c/p\u003e\u003cp\u003e0.54\u003c/p\u003e\u003cp\u003e3.83\u003c/p\u003e\u003cp\u003e1.60\u003c/p\u003e\u003cp\u003e5.63\u003c/p\u003e\u003cp\u003e3.46\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"5\"\u003eResolutions were calculated by the following equation: Rs\u0026thinsp;=\u0026thinsp;1.18\u0026times;(t\u003csub\u003eRD\u003c/sub\u003e-t\u003csub\u003eRL\u003c/sub\u003e)/(W\u003csub\u003e0.5D\u003c/sub\u003e+W\u003csub\u003e0.5L\u003c/sub\u003e), where t\u003csub\u003eRD\u003c/sub\u003e and t\u003csub\u003eRL\u003c/sub\u003e indicated retention time of D- and L- form peak and W\u003csub\u003e0.5D\u003c/sub\u003e and W\u003csub\u003e0.5L\u003c/sub\u003e indicated half time of peak of D- and L- form.\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eFigure S2A shows the MRM chromatograms of DL-AA separation using CMT-D-Leu under conventional octadecylsilane-type columns and FA acidic conditions. All the targeted DL-AAs were separated, except for Asn and His. Increasing the basicity of the mobile phase partially resolved this issue; however, Asp and Glu remained unseparated (Figure S2B). Therefore, further separation was performed by varying the column type. In a previous study on DMT-(\u003cem\u003eS\u003c/em\u003e)-Pro-OSu, an ADME column was employed, showing an improved retention pattern compared with the conventional column. Similarly, in this study, an ADME column was evaluated for DL-AA separation. Figure S3A shows the MRM chromatograms obtained under acidic conditions, indicating insufficient separation of Asn and His. However, as shown in Figure S3B, good separation was achieved for all other targets in 10 mmol/L ammonium formate condition. Although all the targeted DL-AAs were completely separated within 10 min per run, the detection sensitivities of Asp and Glu were more than 50 times lower than those of the other compounds. These derivatives include tricarboxylic acids, thus suggesting that significantly stronger interactions affected peak performance and/or detection sensitivities. Therefore, a shorter ADME column (50 mm) was employed to improve throughput, and divided identical methods were developed within a single analytical system (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The limit of quantification (LOQ), defined as the concentration with \u003cem\u003eS/N\u003c/em\u003e\u0026thinsp;=\u0026thinsp;10, was determined. Values of 0.33\u0026ndash;54.9 pmol/L on column (in vial) were obtained under the final conditions (Table S2). These sensitivities were sufficient to detect rare AAs in DSW. Based on these results, a high-throughput and highly sensitive method using the optimal reagent, CMT-D-Leu, was developed.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eOptimization of the reaction conditions and validation\u003c/h2\u003e\u003cp\u003eThe reaction conditions for \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eDL\u003c/span\u003e-AAs were optimized in high concentrations of mineral water for DSW analysis. Initially, interference from the minerals in the DSW hindered the reaction (data not shown). Therefore, a method was developed to reduce mineral content in DSW. We considered the differences in solubility between the minerals and AAs. When nine times the sample volume of methanol was added, significant precipitation was observed in the sample tube. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003e shows the reaction time course between 10 and 120 min for five representative AAs. A 100 mmol/L NaHCO\u003csub\u003e3\u003c/sub\u003e solution was used as the basic catalyst. The maximum and plateau responses were reached after 60 min. Under these reaction conditions, the recovery rate of DSW was examined. Table S3 shows the results for the recovery rate and precision. The spiking concentrations were set at 1.0, 0.5, and 0.2 \u0026micro;mol/L for \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eL\u003c/span\u003e-AAs and 0.2, 0.1, and 0.04 \u0026micro;mol/L for \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eD\u003c/span\u003e-AAs. The results indicate that the spiked concentrations were fully recovered using the standard spiking calibration method.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eQuantification results of DSW\u003c/h2\u003e\u003cp\u003eBased on the above results, a quantitative analysis of DSW was performed using the established method. Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e shows the quantification results for the DSW samples from Toyama Bay. The samples were analyzed three times over the course of a year to determine the stability of the DSW content. Owing to the low rate of ocean currents in DSW, stable concentrations were observed throughout the year. Relatively high concentrations of \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eD\u003c/span\u003e-Leu, \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eD\u003c/span\u003e-Val, \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eD\u003c/span\u003e-Ala, \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eD\u003c/span\u003e-Thr, and \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eD\u003c/span\u003e-Ser were quantified. Lower concentrations of \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eD\u003c/span\u003e-Glu, \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eD\u003c/span\u003e-Asn, and \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eD\u003c/span\u003e-Pro were also detected in DSW. These results suggest that the AA content profile varies with ocean water depth. To the best of our knowledge, this is the first study to quantify DL-AAs in DSW from Japan. Different effects of D-AAs have been reported; therefore, these results provide an interesting perspective to explain the efficacy of DSW and/or its concentration. The higher concentration of D-AAs in the DSW is suggested to result from marine snow\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Marine snow forms during the death cycle of plankton, marine bacteria, and living organisms such as fish. Most marine snow is thought to contain the remains of bacteria and plankton, suggesting that its composition includes various types of prokaryotes\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Prokaryotes generally use D-AAs for initiation and production. One study suggested that D-AAs are selectively used by deep-sea microorganisms. In addition, D-Asp, D-Glu, and D-Ala have been detected in seabed hydrothermal sediments from the Izena and Yoron Cauldrons, Okinawa Trough\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. These observations and our results suggest that DL-AA profiles may be suitable for analyzing marine areas, and their measurement is valuable not only for mechanistic research on DSW but also for discussing the specificity of the ecosystem.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eQuantification results of DSW from Toyama Bay.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"9\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003eSample Lot\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e240415\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e250109\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003e250519\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003eAverage\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c9\"\u003e\u003cp\u003eSE\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGly\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNull\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e153\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e372\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e115\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e213\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e80\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eAla\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e14.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e36.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e6.85\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e19.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e8.8\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eL\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e213\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e491\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e155\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e286\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e104\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eSer\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e10.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e30.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e19.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e20.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e5.8\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eL\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e464\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e630\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e329\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e474\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e87\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eThr\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e3.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e74.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e7.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" 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colname=\"c2\"\u003e\u003cp\u003eL\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e121\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e69.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eND\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e95.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e20.8\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eArg\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e3.19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eND\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e3.26\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e3.21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.04\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eL\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e179\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e182\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e208\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e189\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e9\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eHis\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2.55\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e3.69\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e8.05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e4.76\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e1.68\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eL\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e79.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e108\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e85.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e90.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e8.5\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eTyr\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e6.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e4.57\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eNC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e3.91\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e1.66\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eL\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e39.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e103\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e43.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e61.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e20.7\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003ePhe\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e3.34\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eND\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e2.38\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.78\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eL\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e39.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e62.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e23.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e41.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e11.3\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eTrp\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.55\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e7.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e4.19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e4.51\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e1.81\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eL\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eND\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e16.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1.62\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e9.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e6.04\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"9\"\u003eThese concentrations were indicated as nmol/L of DSW. NC: Not calculated because the value showed under LLOQ, ND: Not detected.\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eLifespan assay of C. elegans with DSW addition and DL-AA kinetics\u003c/h2\u003e\u003cp\u003eFinally, we investigated the mechanistic contribution of DSW using the \u003cem\u003eC. elegans\u003c/em\u003e model. Several studies have indicated that the benefits of DSW utility require continuous intake\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. An animal model, \u003cem\u003eC. elegans\u003c/em\u003e, was used in this study to evaluate lifespan during screening. The nematode \u003cem\u003eC. elegans\u003c/em\u003e is a small organism with a shorter lifespan than higher-order animals such as mice. In addition, it has few organs and a genome similar to that of humans. Therefore, this microorganism is considered suitable for use in lifespan assays employing a simple experimental system, such as a well chamber. In this study, previous reports using the Transwell port system to facilitate daytime treatments during the experimental schedule were referenced\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Figure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eA shows a microscopic image, and Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eB shows the resulting lifespan analysis curve. The movements of living organisms were clearly observed under microscopy. Observations throughout the lifespan revealed a clear prolongation of lifespan in the positive control and DSW-added samples (see also Table S4 as significant test). These results suggest that DSW demonstrates anti-aging efficacy in the \u003cem\u003eC. elegans\u003c/em\u003e model. Given that the AA concentrations were lower than those of the mineral components, their contribution to the prolonged effect may be limited. However, AAs are suspected to serve as synergistic factors alongside mineral effects. In addition, several D-AAs are present at low concentrations (~\u0026thinsp;10 nmol/L); therefore, they may contribute to the proliferation mechanism. Further studies, such as additional experiments with DL-AAs in DSW and/or desalinated DSW samples, are needed to elucidate the contribution of DL-AAs to the anti-aging effect. Furthermore, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eC shows the kinetics results for DL-Asp (days 7 and 14), which was the only AA exhibiting significant changes. The D% significantly decreased in the treated groups on day 14. A decrease in D% was also observed on day 7. D-Asp is attracting attention as an aging marker derived from the aging reaction of the proteogenic L-Asp moiety\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. These results suggest that D-Asp may be a suitable rapid biomarker for aging in \u003cem\u003eC. elegans\u003c/em\u003e. In addition, fluctuations in D-Asp could potentially serve as a faster marker for lifespan assays. Further validation studies may establish \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eD\u003c/span\u003e-Asp as a rapid biomarker for lifespan assays.\u003c/p\u003e\u003c/div\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn this study, a rapid \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eDL\u003c/span\u003e-AA screening method was developed using a novel enantiochemical tagging reagent, CMT-D-Leu. Screening of reagent columns enabled a 14 min gradient elution with good separation of 19 AAs for quantitative analysis. The detection sensitivity ranged from 10 to 100 pmol/L (LOD). Based on previous reports, this sensitivity level is sufficient to detect DL-AAs in sea water. Sea water samples were successfully analyzed, and several DL-AAs were quantified at 10\u0026ndash;100 nmol/L. Although these concentrations may be insufficient to directly account for the efficacy, the synergistic effects with minerals and higher activity of D-AAs compared to L-AAs suggest their potential contribution to the mechanism of DSW. The content profiles of DL-AAs in DSW can be used for mechanistic analysis and environmental research in marine studies. Furthermore, although further improvements to the method are needed, such as target expansion (DL-kynurenine, citrulline, allothreonine, and highly reactive thiol AAs such as cysteine), this novel tagging reagent shows promise for the rapid separation of additional targets. In addition, biological analysis using \u003cem\u003eC. elegans\u003c/em\u003e revealed the proliferative effects of DSW. Although concentrated D-AAs in DSW were not detected in the \u003cem\u003eC. elegans\u003c/em\u003e body, the aging marker D-Asp significantly decreased in the proliferated groups. This suggests that DSW reduced aging in \u003cem\u003eC. elegans\u003c/em\u003e, similar to the positive control. An anti-aging mechanistic study will be conducted in the future to determine the contribution of DL-AAs to DSW.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Airi Minami (Master’s student) for technical advice and experimental support. We also thank Yukinobu Saeki and Sunao Fujii (GOSHU, Toyama, Japan) for preparing the DSW used in this study. This study was supported by a Sasakawa Scientific Research Grant from the Japan Science Society.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eT. T. and H. I. contributed equally to this study. T. T. and K. I. conceived the study. H. I. and T. T. performed the experiments, data analysis, interpretation, and writing of the original draft. R. F., A. M., Y. S., and T. T. collected, adjusted, and analyzed \u003cem\u003eC. elegans\u003c/em\u003e samples and conducted observations.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional Information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe additional methods and data include the detailed LC-MS(/MS) conditions, purity test results, MRM chromatograms, LLOQ and validation results, and significant tests of lifespan assay were available in Supporting Information.\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFundings\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by a Sasakawa Scientific Research Grant from the Japan Science Society (No. 2024-6007).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eC. 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A\u003c/em\u003e, \u003cstrong\u003e2016\u003c/strong\u003e, \u003cem\u003e1467\u003c/em\u003e, 318-325.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Enantiochemical tagging, DL-amino acids, Deep sea water, C. elegans","lastPublishedDoi":"10.21203/rs.3.rs-7397638/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7397638/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eDeep sea water (DSW), defined as sea water deeper than 200 m, has notable applications in various fields such as energy, agriculture, food, cosmetics, and public health. Several studies have attributed its utility to mineral effects; however, its organic compounds have rarely been investigated. To emphasize the mechanistic evidence of DSW, a sensitive analytical method was developed for the individual analysis of \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eD\u003c/span\u003e- and \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eL\u003c/span\u003e-amino acids (AAs) using enantiochemical tagging\u0026ndash;liquid chromatography\u0026ndash;tandem mass spectrometry. A novel reagent, CMT-D-Leu, was developed to enable high-speed analysis of individual DL-AAs, achieving analysis of 19 \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eDL\u003c/span\u003e-AAs within 17 min. A limit of detection of 10\u0026ndash;100 pmol/L (in vial) was achieved, which was sufficient to reveal the DL-AA profiles in DSW. Three batches of DSW from Toyama Bay were subjected to quantitative analysis using the spiking standard method, detecting DL-AA concentrations of 10\u0026ndash;100 nmol/L. Notably, D-Leu, D-Val, D-Ala, D-Ser, D-Thr, and D-Glu were detected at higher concentrations than other D-AAs. Finally, a lifespan assay using the \u003cem\u003eC. elegans\u003c/em\u003e model showed that DSW exhibited a clear proliferative effect, similar to the positive control. Moreover, DL-AA kinetics analysis revealed a reduction in D-Asp, an aging marker, in the proliferated groups.\u003c/p\u003e","manuscriptTitle":"High-throughput DL-amino acid analysis of deep sea water from Toyama Bay and anti-aging activity assessment using C. elegans","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-04 10:43:32","doi":"10.21203/rs.3.rs-7397638/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9aec8762-0cb1-4ddb-9d8b-6a14c5eb20ce","owner":[],"postedDate":"September 4th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":54087272,"name":"Biological sciences/Biochemistry"},{"id":54087273,"name":"Biological sciences/Biological techniques"},{"id":54087274,"name":"Biological sciences/Biotechnology"},{"id":54087275,"name":"Physical sciences/Chemistry"},{"id":54087276,"name":"Earth and environmental sciences/Environmental sciences"}],"tags":[],"updatedAt":"2025-10-27T14:40:58+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-04 10:43:32","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7397638","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7397638","identity":"rs-7397638","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}