Measurement of Tryptophan Metabolites in Healthy Old Men and Patients of Type 2 Diabetes Mellitus (T2DM)

Background: Plasma levels of tryptophan (TRP) metabolites have not been measured extensively in patients of type 2 diabetes mellitus (T2DM). Methods: Metabolites analysis was performed by a liquid chromatograph tandem mass spectrometer, the LCMS-8060 quadrupole mass spectrometer combined with Nexera X2 liquid chromatograph system (Shimadzu Corporation, Kyoto, Japan). Body mass index (BMI) and TRP metabolites have been measured in healthy old men (n = 20) and patients of T2DM (n = 20). TRP metabolites were measured by using the ultrahigh speed liquid chromatography-mass spectros-copy (Shimadzu Corporation). Results: The plasma levels of 5-hydroxytryptophan (5-HTRP), 5-hydroxyindoleacetic acid (5-HIAA), kynurenic acid (KNA), 3-hydroxykynurenine (3-HKN), and 3-hydroxyanthranilic acid (3-HAA) were higher in patients of T2DM than healthy old men. Since 5HTRP and 5-HIAA belong to the serotonin pathway, and KNA, 3-HKN, and 3-HAA belong to the KN pathway of TRP metabolism, these pathways were activated more in the patients of T2DM. Since plasma levels of Indole-3-acetic acid were not elevated in T2DM, that pathway was not activated more in T2DM. Serotonin levels were not increased but 5-HIAA levels were increased in the plasma of T2DM patients, which may mean that serotonin was quickly metabolized to 5-HIAA in the patients of T2DM. Conclusion: Plasma levels of tryptophan metabolites in serotonin and kynurenine pathways increased in T2DM patients. Obesity expressed by BMI may not influence tryptophan metabolism in healthy old men and T2DM patients. These results indicate that our new method of the simultaneous measurements of all the tryptophan metabolites is the powerful measure to identify factor related to endogenous stresses seen in DM.

Improvement of the Assay Method for Steviol Glycosides in the JECFA Specifications

Steviol glycosides are natural sweetener constituents found in the leaves of Stevia rebaudiana Bertoni (Asteraceae). The specifications for steviol glycosides were established by the Joint FAO/WHO Expert Committee on Food Additives (JECFA) in 2008, although there was a call in the following year for the modification of this assay method to enable the determination of nine steviol glycosides rather than just seven. In response, based on a proposed method by the Japan Stevia Association, we developed an improved method by changing the HPLC conditions and including the use of an octadecylsilyl column instead of an amino-bonded column to enable the rapid and reliable determination of the nine steviol glycosides by an isocratic HPLC-UV method. With the developed method, the nine steviol glycosides can be separately determined, and identified using individual reference chemicals as standards, unlike the previous identification method, which was based on the relative retention times. In addition, the single stevioside quantification standard was replaced with both stevioside and rebaudioside A quantification standards. Importantly, the validation of the developed method was successful. The limits of quantification for the nine steviol glycosides were between 0.2% and 0.6%. The developed assay method for the nine steviol glycosides was proposed to JECFA and adopted as the revised assay method for the steviol glycosides specifications at its 73rd meeting in 2010.

Correlation between LIFG and Autonomic Activation during Stressful Tasks:A Functional Near-infrared Spectroscopy (fNIRS) Study

It remains unclear whether language tasks in one＇s first （L1） or second （L2） language can cause stress responses and whether frontal, autonomic and behavioral responses to stressful tasks are correlated. In this study, we studied 22 Chinese subjects whose L2 was English and measured the cerebral blood oxygenation in their frontal lobe by using functional near-infrared spectroscopy （fNIRS） as par- ticipants engaged in a mental arithmetic task （MAT） and verbal fluency tasks （VFTs） in L1 （Chinese） and L2 （English）. To examine the activated cortical areas, we estimated the channel location based on Montreal Neurological Institute （MNI） standard brain space by using a-probabilistic estimation method. We evaluated heart rate （HR） changes to analyze autonomic nervous system （ANS） functioning. We found that the MAT and VFTs induced greater increases in HR than did the control （Ctrl） task. Further- more, subjects developed greater increases in HR in the MAT and VFTt~ than they did in the VFTL1. Compared with the Ctrl task, the MAT and both VFTLland VFTL2 produced robust and widespread bi- lateral activation of the frontal cortex. Interestingly, partial correlation analysis indicated that the activity in the left inferior frontal gyrus （LIFG） [Brodmarm＇s area （BA） 47] was consistently correlated with the increases in HR across the three tasks （MAT, VFTL2, and VFTL1）, after controlling for the performance data. The present results suggested that a VFT in L2 may be more stressful than in L1. The LIFG may affect the activation of the sympathetic system induced by stressful tasks, includin~ MATs and VFTs.

Evaluating “Cosmetic Therapy” by Using Near-Infrared Spectroscopy

This study examined the effect of cosmetic therapy on frontal lobe activation as revealed by topographic near-infrared spectroscopy (NIRS). We evaluated emotional responses to a photograph of a face with/without makeup by 22 healthy female volunteers (mean age, 52 ± 10.5 years). The results of the first-round analysis showed a significant increase of oxy-Hb in the frontal lobe area when the subject looked at a photograph of herself made up as compared to not made up. In a later round of analysis, we divided the subjects into 2 groups having contrasting scores on the Profile of Mood States-Short Form Japanese version. One group was classed as “high vigor” (a common standard pattern) and the other as “low vigor” (depression-tendency pattern). The made-up/not made-up difference did not have any effect on the oxy-Hb level in the frontal lobe in the high vigor group. In contrast, makeup produced a significant increase in the oxy-Hb level over a wide frontal area in the low vigor group, which indicated widespread frontal lobe activation. This result indicates a beneficial effect of cosmetic therapy on the brain function of patients with depression and/ or dementia.

Differences of Plasma Levels of Tryptophan, Serotonin, 5-Hydroxyindole Acetic Acid, and Kynurenine between Healthy People and Patients of Major Monopolar Depression at Various Age and Gender

Background: It is not well analyzed whether there are differences in plasma levels of tryptophan (TRP) metabolites between healthy control people (HC) and patients of major monopolar depression (MMD). Methods: Ultra high-speed liquid chromatography/mass spectrometry has been used for the simultaneous determination of plasma levels of tryptophan metabolites in depressive patients. Results: There are no significant differences between plasma levels of TRP between HC and MMD. Plasma levels of TRP of HC are higher in young men, young women, old men, and old women in this order. Serotonin (5-HT) levels are higher in MMD than HC. Plasma levels of 5-HIAA of HC are also higher than those of patients of MMD. Plasma levels of kynurenine (KYN) of healthy old men and old women are higher than those of young men and old women. Plasma levels of KYN are higher in old women and young men of MMD than those of HC. Conclusion: Plasma levels of 5-HT are higher in patients of MMD than those of HC, which may suggest that use of drugs inhibiting the 5-HT transportation may increase plasma levels of 5-HT in MMD.

Rapid Determination of Residual Quinolones in Honey Samples by Fast HPLC with an On-Line Sample Pretreatment System

A method of on-line pretreatment coupled to HPLC with fluorescence detection was developed and validated for the determination of nine quinolones in honey samples. This method simplified the complicated process of sample pretreatment and reduced sample treatment time. Recovery of the quinolones was between 92% - 101% for spiked honey samples. The limit of detection was 0.22 - 3.78 ng/mL (signal/noise ratio = 3). There was good linear correlation between HPLC peak area and concentration of the quinolones, with a linear range of 1.0 - 100.0 ng/mL and correlation coefficients >0.9997. The method proposed was validated for detecting quinolones in honey samples.

LO-RAY-LIGH@ Diffraction Gratings in UV-VIS Spectroscopy

It has been more than half a century since the release of the first Shimadzu UV-VIS （UV-visible） spectrophotometer QB-50 in 1952, and during this time more than 160,000 UV-VIS spectrometers have been produced and installed in a wide variety of different applications. A lot of technical innovations have been implemented to improve the performance and significantly reduce the stray light levels. The latest innovation during development of sophisticated spectrophotometers is based on a new holographic exposure method and optimized etching process which has made it possible to produce both high-efficient and exceptionally low stray light gratings. These LO-RAY-LIGH~ gratings have guaranteed values of stray light at the intermediate position between zero-order and first-order lights. The values are measured by Shimadzu＇s laser stray-light-measuring system. The latest development in the series of UV-VIS spectrophotometers is the UV-2700 which is a true double beam double monochromator system in a compact design for high-precision spectral analysis of a wide range of samples including organic and inorganic compounds, biological samples, optical materials and photovoltaics. The high performance optical system is designed with ＂LO-RAY-LIGH＂ diffraction gratings, featuring highest efficiency and exceptionally low stray light. The spectrophotometer operates in the wavelength range from 185 nm to 900 nm and allows highly sophisticated applications such as direct measurement of high density samples up to 8 absorbance units without dilution.

Effects of Gender, Age, and Clot Formation on the Measurements of Tryptophan Metabolites in Blood

Background: Tryptophan metabolism plays important roles in health and diseases. Although simultaneous measurements of tryptophan metabolites are successfully measured, influences of age, gender, and clot formation on the measurements have not been reported. Methods: We took blood from young and old Japanese men and women and compared plasma levels of tryptophan metabolites. We also took plasma and serum from the blood of middle-aged men (n = 10). Metabolites analysis was performed by a liquid chromatograph tandem mass spectrometer, the LCMS-8060 quadrupole mass spectrometer combined with Nexera X2 liquid chromatograph system (Shimadzu Corporation, Kyoto, Japan). Body mass index (BMI) and TRP metabolites have been measured in healthy young men (n = 48), young women (n = 47), old men (n = 44), and old women (n = 39). TRP metabolites were measured by using the ultrahigh speed liquid chromatography-mass spectroscopy (Shimadzu Corporation). Results: Tryptophan and its metabolites such as serotonin, 5-hydroxyindole acetic acid, indole-3-acetic acid, kynurenine, anthranilic acid, were higher in young women and old men than young men and old women. Plasma levels of 3-hydroxykynurenine and xanthurenic acid were lower in young women and old men. Comparison of plasma and serum indicates that most of metabolites were higher in serum than plasma except for 3-hydroxy-kynurenine and quinolinic acid. Conclusion: Metabolites of the upper stream of degradation of tryptophan were higher in young women and old men than young men and old women, which suggests that the degradation of tryptophan was accelerated in young men and old women than young women and old men. Serum preparation may activate tryptophan degradation resulting in higher levels of metabolites in serum than in plasma.

Comparison of Plasma Levels of Tryptophan Metabolites between Healthy People and Patients of Bipolar Depression at Various Age and Gender

Background: It is not well analyzed whether there are differences in plasma levels of tryptophan (TRP) metabolites between healthy control people (HC) and patients of type II bipolar depression (BDII). Methods: Ultra high-speed liquid chromatography/mass spectrometry has been used for the simultaneous determination of plasma levels of tryptophan metabolites in depressive patients. Results: Plasma levels of TRP are not different between HC and patients of BDII. Serotonin (5-HT) levels are higher in BDII than HC. Plasma levels of 5-HIAA of HC are higher than those of old women of BDII, but lower in young women of BDII. Plasma levels of kynurenine (KYN) of HC are not different from those of patients of BDII. Conclusion: Plasma levels of 5-HT are higher in patients of BDII than those of HC, which may suggest that use of drugs inhibiting the 5-HT transportation and lower transporter biding may increase plasma levels of 5-HT in patients of BD.

A multiplex method for detection of SARS-CoV-2 variants based on MALDI-TOF mass spectrometry

The recent outbreak of the coronavirus disease 2019(COVID-19)pandemic and the continuous evolution of the severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)have highlighted the significance of new detection methods for global monitoring and prevention.Although quantitative reverse transcription PCR(RT-qPCR),the current gold standard for diagnosis,performs excellently in genetic testing,its multiplexing capability is limited because of the signal crosstalk of various fluorophores.Herein,we present a highly effi-cient platform which combines 17-plex assays with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry(MALDI-TOF MS),enabling the targeting of 14 different mutation sites of the spike gene.Diagnosis using a set of 324 nasopharyngeal swabs or sputum clinical samples with SARS-CoV-2 MS method was identical to that with the RT-qPCR.The detection consistency of mutation sites was 97.9%(47/48)com-pared to Sanger sequencing without cross-reaction with other respiratory-related pathogens.Therefore,the MS method is highly potent to track and assess SARS-CoV-2 changes in a timely manner,thereby aiding the continuous response to viral variation and prevention of further transmission.

Analysis of keto-enol tautomers of curcumin by liquid chromatography/mass spectrometry

Keto-enol tautomers of curcumin were confirmed by reversed-phase liquid chromatography（RPLC）/ hybrid quadrupole ion trap/time-of-flight mass spectrometry（QIT/TOFMS）.Tautomers gave different MS/MS spectra in negative mode.Different mass spectra were also obtained by hydrogen/deuterium exchange LC/MS/MS in positive mode.Our results suggest that enol form is the major form in the solution（water/acetonitrile）.