Capillary Electrophoresis Time-of-flight Mass Spectrometry Research Articles

Subnormothermic ex vivo lung perfusion possibly protects against ischemia-reperfusion injury via the mTORC-HIF-1α pathway.

Ex vivo lung perfusion (EVLP) is a useful technique for evaluating and repairing donor lungs for transplantation. However, studies examining the effects of perfusate temperature on graft function are limited. Thus, this study aimed to examine these effects during EVLP on ischemic-reperfusion injury in the donor lung. Twenty-four male Sprague-Dawley rats were randomly divided into three groups, as follows: no treatment (sham group, n=5), normothermic EVLP (37 °C, n=5), and subnormothermic EVLP (30 °C, n=5). Lung function analyses, including oxygen capacity (OC), compliance, and pulmonary vascular resistance (PVR), were performed hourly during EVLP. Further, after 4 h of EVLP, histological evaluation of the right lobe was performed using the lung injury severity (LIS) scale. The expression levels of inflammatory cytokines such as tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, and IL-18 were evaluated. Metabolomic analysis of left lung tissues was conducted using capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS) after 4 h of EVLP in the EVLP groups and after 1 h of cold preservation in the sham group. Compared with those in the normothermic group, in the subnormothermic group, functional parameters during EVLP and subsequent histologic results were significantly superior, expression levels of inflammatory cytokines such as TNF-α, IL-1β, IL-6, and IL-18 were significantly lower, and glycolytic activity was significantly decreased. Furthermore, expression levels of mammalian target of rapamycin complex (mTORC), hypoxia-inducible factor (HIF) 1α, and nucleotide-binding domain, leucine-rich-containing family pyrin domain containing 3 (NLRP3) and its effector caspase-1 were significantly lower in the subnormothermic group than in the normothermic group. EVLP with subnormothermic perfusion improves lung graft function by reducing the expression of pro-inflammatory cytokines and glycolytic activity during EVLP. Additionally, EVLP can be a useful target for the improvement of graft function after transplantation.

Dietary supplement of mushrooms promotes SCFA production and moderately associates with IgA production: A pilot clinical study.

Mushrooms are rich in dietary fiber, and fiber intake has been reported to increase the levels of short-chain fatty acids (SCFAs). It has also been reported that SCFAs promote immunoglobulin A (IgA) production, indicating involvement in systemic immunity. The objective of this study was to evaluate the effects of mushroom consumption on the amount of intestinal IgA. We also aimed to comprehensively evaluate the gut microbiota and intestinal metabolome and to conduct an exploratory analysis of their relationship with IgA. Healthy adults (n = 80) were enrolled in a parallel group trial. Participants consumed a diet with mushrooms or a placebo diet once daily for 4 weeks. Gut microbiota profiles were assessed by sequencing the bacterial 16S ribosomal RNA-encoding gene. Intestinal metabolome profiles were analyzed using capillary electrophoresis-time of flight mass spectrometry (CE-TOFMS). Mushroom consumption tended to increase IgA levels at 4 weeks of consumption compared to those in the control group (p = 0.0807; Hedges' g = 0.480). The mushroom group had significantly higher levels of intestinal SCFAs, such as butyrate and propionate, than the control group (p = 0.001 and 0.020; Hedges' g = 0.824 and 0.474, respectively). Correlation analysis between the changes in the amount of intestinal IgA and the baseline features of the intestinal environment showed that the increasing amount of intestinal IgA was positively correlated with the baseline levels of SCFAs (Spearman's R = 0.559 and 0.419 for butyrate and propionate, respectively). Consumption of mushrooms significantly increased the intestinal SCFAs and IgA in some subjects. The increase in intestinal IgA levels was more prominent in subjects with higher SCFA levels at baseline. This finding provides evidence that mushroom alters the intestinal environment, but the intensity of the effect still depends on the baseline intestinal environment. This trial was registered at www.umin.ac.jp as UMIN000043979.

Analysis of metabolites of red seabream (Pagrus major) from different geographical origins by capillary electrophoresis time-of-flight mass spectrometry.

Red seabream (Pagrus major), a migratory fish, is characterized by high protein levels in the muscle. South Korean and Japanese red seabreams have a general distribution pattern; however, distinguishing them based on their geographical origin is difficult. In this study, we used capillary electrophoresis time-of-flight mass spectrometry (CE-TOF/MS) to analyze the red seabream muscle metabolome to investigate how can distinguish the origin of the fish. The metabolites were extracted using 50% acetonitrile in water. Chromatographic separation was successfully used to classify the metabolite profiles of Japanese and South Korean red seabream. Principal component analysis and hierarchical cluster analysis showed good ability to categorize the samples according to their origin. Amino acids showed the greatest quantitative difference in South Korean and Japanese muscle samples. Specifically, the L-alanine, L-glutamic acid, L-isoleucine, dimethylglycine, and L-valine levels in Japanese red seabream samples were significantly higher than those in South Korean samples. In contrast, the levels of trimethylamine N-oxide and inosine monophosphate in South Korean muscle samples were significantly higher than those in Japanese red muscle samples. The monitored metabolite profiles suggest that South Korean and Japanese red seabreams can be identified on the basis of amino acid levels.

Untargeted Metabolomics reveals Doenjang metabolites affected by manufacturing process and microorganisms

Doenjang is a traditional Korean fermented soybean-based food that is now produced industrially as traditional Doenjang and modernized Doenjang, depending on the conditions of the manufacturing process. In this study, the effect of the production process on Doenjang metabolites was analyzed using untargeted capillary electrophoresis time-of-flight mass spectrometry (CE-TOF/MS). Of the 247 metabolites detected in 19 traditional and three modernized Doenjang samples, the level of 55 metabolites were significantly different between the two production groups. The S-plot of the orthogonal projection to latent structures-discriminant analysis (OPLS-DA) revealed that nine compounds (tryptamine, 2-phenylethylamine, citrulline, gamma-aminobutyric acid, putrescine, tyramine, 2-aminoisobutyric acid, stachydrine, and N5-ethylglutamine) were highly distributed in the traditional group, and six compounds (arginine, citric acid, choline, cytidine, hypoxanthine, and glucaric acid) were considered distinguishable metabolites of the modernized group. Microbial community analysis indicated that the levels of these metabolites were significantly altered by the presence of Bacillus spp., Enterococcus faecium, Tetragenococcus halophilus, Aspergillus oryzae, Penicillium spp., and Saccharomyces cerevisiae. These findings will give better understanding of the influence of the manufacturing process on Doenjang production in terms of both microbial activity and metabolite profiles.

Metabolomic Signatures of Ocean Acidification Stress in the Coral Acropora millepora

The rapid acidification of seawater poses a significant threat to the persistence of coral reefs. However, taxa‐specific, mechanistic understandings of holobiont responses to ocean acidification (OA) stress remain largely unknown. Understanding these mechanisms is crucial to uncovering predictive markers of OA stress. This could subsequently be used to assist field‐based conservation efforts. We hypothesized that exposure to elevated pCO2 levels would decrease the abundance of primary metabolites such as amino acids and carbohydrates and increase the production of secondary metabolites, specifically those involved in cnidarian‐symbiodinium cell signaling. In this study, we applied untargeted capillary electrophoresis time‐of‐flight mass spectrometry (CE‐TOFMS) to profile changes in the intracellular polar and semi‐polar metabolite composition of the coral holobiont after exposure to elevated pCO2 concentrations. Nubbins of Acropora millepora were exposed to ambient (410 µatm) or elevated (805 µatm) pCO2 levels under controlled conditions over a period of 10 days. Measurement of treatment‐induced bleaching was performed by quantification of symbiodinium cell density and chlorophyll a concentration. We hope to identify molecules in the metabolite profiles associated with the individual cellular responses of holobiont members to osmotic stress. Additionally, we hope to provide further insight into unknown roles of secondary and tertiary metabolite pools in cellular homeostasis and acclimation to thermal stress in the coral holobiont, and specifically the cnidarian‐symbiodinium symbiosis. Our goal is for these findings to assist conservation efforts, specifically in the development of rapid tests for field‐based conservation efforts.

Metabolic Rewiring by Human Placenta-Derived Mesenchymal Stem Cell Therapy Promotes Rejuvenation in Aged Female Rats.

Aging is a degenerative process involving cell function deterioration, leading to altered metabolic pathways, increased metabolite diversity, and dysregulated metabolism. Previously, we reported that human placenta-derived mesenchymal stem cells (hPD-MSCs) have therapeutic effects on ovarian aging. This study aimed to identify hPD-MSC therapy-induced responses at the metabolite and protein levels and serum biomarker(s) of aging and/or rejuvenation. We observed weight loss after hPD-MSC therapy. Importantly, insulin-like growth factor-I (IGF-I), known prolongs healthy life spans, were markedly elevated in serum. Capillary electrophoresis-time-of-flight mass spectrometry (CE-TOF/MS) analysis identified 176 metabolites, among which the levels of 3-hydroxybutyric acid, glycocholic acid, and taurine, which are associated with health and longevity, were enhanced after hPD-MSC stimulation. Furthermore, after hPD-MSC therapy, the levels of vitamin B6 and its metabolite pyridoxal 5′-phosphate were markedly increased in the serum and liver, respectively. Interestingly, hPD-MSC therapy promoted serotonin production due to increased vitamin B6 metabolism rates. Increased liver serotonin levels after multiple-injection therapy altered the expression of mRNAs and proteins associated with hepatocyte proliferation and mitochondrial biogenesis. Changes in metabolites in circulation after hPD-MSC therapy can be used to identify biomarker(s) of aging and/or rejuvenation. In addition, serotonin is a valuable therapeutic target for reversing aging-associated liver degeneration.

Metabolomic analysis of plasma and intramuscular adipose tissue between Wagyu and Holstein cattle.

In this experiment, we studied the effects of breed differences in intramuscular adipogenic capacity on the metabolomic profiles of plasma and intramuscular adipose tissue between Wagyu (high intramuscular adipogenic capacity) and Holstein (low intramuscular adipogenic capacity) using capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS). We showed that the intramuscular fat content, intramuscular adipocyte size and the expression of adipogenic transcription factors (C/EBPβ and C/EBPα) of Wagyu were significantly higher than those of Holstein. Metabolites detected at significantly higher levels in Wagyu plasma were related to the tricarboxylic acid cycle, lipid synthesis, fatty acid metabolism, diabetes, and glucose homeostasis. In contrast, metabolites detected at significantly higher levels in Holstein plasma were related to choline metabolism, the ethanolamine pathway, glutathione homeostasis, nucleic acid metabolism, and amino acid metabolism. Metabolites detected at significantly higher levels in Holstein intramuscular adipose tissue were related to nucleic acid metabolism, amino acid metabolism, amino sugar metabolism, beta oxidation, and the ethanolamine pathway. There were no metabolites significantly higher levels in Wagyu intramuscular adipose tissue. These results indicate candidate biomarkers of breed differences in intramuscular adipogenic capacity between Wagyu and Holstein.

The study of skin hydration, anti-wrinkles function improvement of anti-aging cream with alpha-ketoglutarate.

IntroductionThe tricarboxylic acid (TCA) cycle is a key metabolic pathway for driving the generation of mitochondrial energy in all oxidative organisms. Alpha‐ketoglutarate (Alpha‐KG), a precursor of glutamine, is known as a crucial intermediate of the TCA cycle and plays a pivotal role in multiple metabolic processes. As a precursor of glutamate and glutamine, AKG acts as an antioxidant agent as it directly reacts with hydrogen peroxide with formation of succinate, water, and carbon dioxide; meanwhile, it discharges plenty of ATP by oxidative decarboxylation. Several studies reported that Alpha‐KG is a key participant in the detoxification of reactive oxygen species and acts as an integral part of the oxidative defense machinery. However, few studies have been reported on the efficacy of Alpha‐KG in the maintenance of skin functions. This study demonstrated that Alpha‐KG has beneficial effects on skin hydration and barrier function and that fermentation is an effective way to enhance the synthesis of Alpha‐KG in yeast, which possesses mitochondria.Methods Evaluation of promoting effects on epidermal keratinocyte proliferation: Keratinocytes were incubated with a test sample, and the degree of proliferation was determined by MTT [3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide] assay. Evaluation of promoting effects on mRNA expression of genes related to skin hydration and barrier function: Keratinocytes were incubated with a test sample, and gene expression levels of filaggrin (FLG), serine palmitoyltransferase (SPT), and involucrin (IVL) were analyzed by real‐time RT‐PCR. Analysis of Alpha‐KG in rice fermented liquid: Alpha‐KG in rice fermented liquid was quantitatively analyzed by capillary electrophoresis time‐of‐flight mass spectrometry (CE‐TOF‐MS). Clinical study testing methods and VISIA testing: After 28 days of treatment use the cream with Alpha‐KG and control sample without Alpha‐KG, instrumentation measurements were adopted to assess skin wrinkles, texture, elasticity, and firmness, tested by the VISIA‐CR.ResultsImmediately after using the sample for D0, the skin wrinkles were significantly reduced by 23.64%. After using the sample for D7, the average clinical score of outer corner wrinkles was significantly reduced by 15.23%, and nasolabial groove wrinkles were significantly reduced by 25.68%. After using the sample for D56, the mean clinical evaluation score of crow's feet decreased significantly by 25.42%; the average score of clinical evaluation of skin firmness increased significantly by 41.40%; the skin gloss increased significantly by 28.67%.ConclusionIt was demonstrated that Alpha‐KG is expected to promote skin hydration and barrier function by the activation of cell proliferation and the up‐regulation of mRNA expression of genes related to the maintenance of epidermal function in in vitro tests. In order to develop a cosmetic ingredient including Alpha‐KG, we focused on fermentation with yeast as an efficient production method of Alpha‐KG and found that fermentation of rice with yeast efficiently produced Alpha‐KG. Taken together, it is considered that rice fermented liquid containing Alpha‐KG could be a promising ingredient for skin care products. Through the clinical testing, rice fermented liquid containing Alpha‐KG could be a promising cosmetic ingredient for skin care products.

Analyzing the Metabolomic Profile of Yellowtail (Seriola quinquerdiata) by Capillary Electrophoresis-Time of Flight Mass Spectrometry to Determine Geographical Origin.

Country-of-origin violations have occurred in which some merchants have fraudulently sold cheap Japanese yellowtail (Seriola quinqueradiata) by presenting them as domestic Korean products. There are many methods for determining the origins of marine organisms, such as molecular genetic methods and isotope analysis. However, this study aimed to develop a method for determining the origins of aquatic products using metabolite analysis technology. Ten yellowtail each from Korea and Japan were analyzed by capillary electrophoresis–time of flight/mass spectrometry (CETOF/MS). Hierarchical cluster analysis (HCA) and principal component analysis (PCA) results showed highly differing aspects between the Korean and Japanese samples. In the tricarboxylic acid (TCA) cycle, citric, malic, oxaloglutaric, and fumaric acids exhibited significant differences between Korean and Japanese yellowtail. Sixteen of the twenty essential amino acids analyzed as metabolites also differed significantly. All amino acids were involved in protein digestion, absorption, and metabolism. All 16 amino acid contents were higher in Japanese yellowtail than in Korean yellowtail, except for glutamine. The fasting period was found to be the biggest factor contributing to the difference in amino acid contents, in addition to environmental factors (including feeding habits). These significant differences indicated that metabolomics could be used to determine geographical origin.

TAMI-56. TARGETING AMINO ACID METABOLIC VULNERABILITIES IN IDH-MUTANT AND IDH-WILDTYPE GLIOMAS

Abstract IDH-wildtype glioma and IDH-mutant glioma have different genetical and metabolic background although their histological appearances are similar. The aim of the study is to reveal the difference in metabolites between IDH-wildtype glioma and IDH-mutant glioma, and to find the effective treatment targeting cancer metabolism according to the status of IDH in gliomas. Two artificial cell lines made from normal human astrocyte were used: NHAE6E7hTERTRas (IDH-wildtype) and NHAE6E7hTERTIDHmut (IDH-mutant). Capillary electrophoresis time-of-flight-mass spectrometry (CE-TOFMS) revealed that the amount of asparagine was lower in NHAE6E7hTERTRas cells compared with NHAE6E7hTERTIDHmut cells. L-asparaginase, which converts asparagine into aspartate, was more effective in the former cells than the latter cells. L-asparaginase induced autophagy and inhibition of autophagy by 3-MA suppressed L-asparaginase-induced antitumor effect. Adding asparagine into the culture medium rescued the antitumor effect of L-asparaginase. L-asparaginase increased the expression of asparagine synthetase (ASNS) and genetical or pharmacological inhibition of ASNS enhanced the antitumor effect of L-asparaginase. CE-TOFMS showed the lower amount of glutamine, glutamate and 2-oxoglutarate in NHAE6E7hTERTIDHmut cells than NHAE6E7hTERTRas cells. GLUD1 inhibitor inhibited proliferation by inducing higher ROS level and apoptosis in NHAE6E7hTERTIDHmut cells than NHAE6E7hTERTRas cells. ROS inhibitor, NAC suppressed GLUD1 inhibitor-induced ROS, apoptosis, and cytotoxicity in NHAE6E7hTERTIDHmut cells. Exogeneous dimethyl 2-oxoglutarate rescued the cytotoxicity induced by GLUD1 inhibitor. L-asparaginase and GLUD1 inhibitor will be new therapeutic option for IDH-wildtype glioma and IDH-mutant glioma, respectively.

Urinary Metabolome Analyses of Patients with Acute Kidney Injury Using Capillary Electrophoresis-Mass Spectrometry.

Acute kidney injury (AKI) is defined as a rapid decline in kidney function. The associated syndromes may lead to increased morbidity and mortality, but its early detection remains difficult. Using capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS), we analyzed the urinary metabolomic profile of patients admitted to the intensive care unit (ICU) after invasive surgery. Urine samples were collected at six time points: before surgery, at ICU admission and 6, 12, 24 and 48 h after. First, urine samples from 61 initial patients (non-AKI: 23, mild AKI: 24, severe AKI: 14) were measured, followed by the measurement of urine samples from 60 additional patients (non-AKI: 40, mild AKI: 20). Glycine and ethanolamine were decreased in patients with AKI compared with non-AKI patients at 6–24 h in the two groups. The linear statistical model constructed at each time point by machine learning achieved the best performance at 24 h (median AUC, area under the curve: 89%, cross-validated) for the 1st group. When cross-validated between the two groups, the AUC showed the best value of 70% at 12 h. These results identified metabolites and time points that show patterns specific to subjects who develop AKI, paving the way for the development of better biomarkers.

Quality Assessment of Untargeted Analytical Data in a Large-Scale Metabolomic Study.

Large-scale metabolomic studies have become common, and the reliability of the peak data produced by the various instruments is an important issue. However, less attention has been paid to the large number of uncharacterized peaks in untargeted metabolomics data. In this study, we tested various criteria to assess the reliability of 276 and 202 uncharacterized peaks that were detected in a gathered set of 30 plasma and urine quality control samples, respectively, using capillary electrophoresis-time-of-flight mass spectrometry (CE-TOFMS). The linear relationship between the amounts of pooled samples and the corresponding peak areas was one of the criteria used to select reliable peaks. We used samples from approximately 3000 participants in the Tsuruoka Metabolome Cohort Study to investigate patterns of the areas of these uncharacterized peaks among the samples and clustered the peaks by combining the patterns and differences in the migration times. Our assessment pipeline removed substantial numbers of unreliable or redundant peaks and detected 35 and 74 reliable uncharacterized peaks in plasma and urine, respectively, some of which may correspond to metabolites involved in important physiological processes such as disease progression. We propose that our assessment pipeline can be used to help establish large-scale untargeted clinical metabolomic studies.

Quantitative and Molecular Similarity Analyses of the Metabolites of Cold- and Hot-Natured Chinese Herbs.

Background Based on the theory of traditional Chinese medicine, Chinese herbs possess four different medicinal properties: hot, warm, cold, and cool. These serve as a reference guide for these herbal medicines. However, the molecular mechanisms supporting their relevance remain unclear. Methods We performed metabolomics based on capillary electrophoresis-time-of-flight mass spectrometry (CE-TOF/MS) and multivariate data analysis for the structural identification of compounds of cold- and hot-natured Chinese herbs. Results To this end, 30 selected herbs were analyzed and a total of 416 metabolites were identified via CE-TOF/MS, of which 193 compounds were detected in most herbs. The observed profiles offered the potential to understand the mechanism of association between the compounds and nature of the Chinese herbs. Comparison of the similarity in terms of chemical and molecular structures and content revealed that hot-natured herbs contained more nucleotides. In contrast, principal component analysis revealed the presence of more amino acid compounds in cold-natured herbs. Conclusion Comparing the structural similarities between the samples using the Tanimoto coefficient revealed that a general non-specific structure was observed between cold- and hot-natured herbs; however, the distribution of the molecular groups seemed to contribute more toward the energy properties.

Reprogramming of glutamine metabolism via glutamine synthetase silencing induces cisplatin resistance in A2780 ovarian cancer cells

BackgroundCisplatin (CDDP) significantly prolongs survival in various cancers, but many patients also develop resistance that results in treatment failure. Thus, this study aimed to elucidate the underlying mechanisms by which ovarian cancer cells acquire CDDP resistance.MethodsWe evaluated the metabolic profiles in CDDP-sensitive ovarian cancer A2780 cells and CDDP-resistant A2780cis cells using capillary electrophoresis-time-of-flight mass spectrometry (CE-TOFMS). We further examined the expression of glutamine metabolism enzymes using real-time PCR and Western blot analyses. Cell viability was accessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.ResultsThe results showed that levels of glutamine, glutamate, and glutathione (GSH), a key drug resistance mediator synthesized from glutamate, were significantly elevated in A2780cis cells than those in A2780 cells. Furthermore, glutamine starvation decreased the GSH levels and CDDP resistance in A2780cis cells. Interestingly, the expression of glutamine synthetase (GS/GLUL), which synthesizes glutamine from glutamate and thereby negatively regulates GSH production, was almost completely suppressed in resistant A2780cis cells. In addition, treatment of A2780cis cells with 5-aza-2′-deoxycytidine, a DNA-demethylating agent, restored GS expression and reduced CDDP resistance. In contrast, GS knockdown in CDDP-sensitive A2780 cells induced CDDP resistance.ConclusionsThe results indicate that upregulation of GSH synthesis from glutamine via DNA methylation-mediated silencing of GS causes CDDP resistance in A2780cis cells. Therefore, glutamine metabolism could be a novel therapeutic target against CDDP resistance.

Liver metabolomic analysis in broiler chicks: Profiling the metabolites after oral administration of l-citrulline.

Hypothermia is directly linked to metabolism; however, it is still unknown how the overall metabolism is altered by oral administration of hypothermic agent, l-citrulline (l-Cit). The present study aimed to determine the characteristics of liver metabolites of chicks orally administered l-Cit to provide a greater understanding of its metabolism. Capillary electrophoresis-time-of-flight mass spectrometry (CE-TOFMS) and liquid chromatography-time-of-flight mass spectrometry (LC-TOFMS) were conducted on liver samples after oral administration of l-Cit. A total of 361 liver metabolites were identified. Although a small number of samples were used for each group, a principal component analysis and heatmap patterns confirmed that the composition of metabolites could be segregated from each other. Of the 361 compounds detected in the liver, 41 compounds, including amino acids related to the Cit-arginine (Arg) cycle, argininosuccinic acid, Arg, ornithine, and Cit, as well as gamma aminobutyric acid, glycine, histidine, and nicotinamide adenine dinucleotide were abundant in l-Cit-treated livers. In contrast, 24 compounds containing fatty acids, amino acids, and cyclic adenosine monophosphate were lower in the l-Cit group. These data imply that the active Cit-Arg cycle, TCA cycle metabolism, and a low activity in fatty acid metabolism occur in l-Cit-treated broiler chicks.

Gut Environment Changes Due to Androgen Deprivation Therapy in Patients With Prostate Cancer

Background: It is estimated that up to 2040 there will be 1,017,712 new cases of prostate cancer worldwide. Androgen deprivation therapy (ADT) is widely used as a treatment option for all disease stages. ADT, and the resulting decline in androgen levels, may indirectly affect gut microbiota. Factors affecting gut microbiota are wide-ranging; however, literature is scare on the effects of ADT on gut microbiota and metabolome profiles in patients with prostate cancer. Methods: This 24-week observational study investigated the relationship between testosterone levels in Japanese patients with prostate cancer undergoing ADT, with changes in gut microbiota. Sequential faecal, and serum blood samples were collected 1 and 2 weeks before ADT, and 1, 4, 12, 24 weeks after ADT. Bacterial 16S rRNA gene-based microbiome analyses, and capillary electrophoresis-time-of-flight mass spectrometry (CE-TOFMS)-based metabolome analyses were performed. Findings: A total of 23 patients completed the study. The α- and s-diversity of gut microbiota decreased significantly at 24 weeks after ADT (p=0.017, p<0.001, respectively). Relative abundances of Proteobacteria, Gammaproteobacteria, Pseudomonadales, Pseudodmonas, and concentrations of urea, lactate, butyrate, 2-hydroxyisobutyrate and S-adenosylmethionine (SAM) changed significantly after ADT (p<0.05). There was a significant positive correlation between the abundance of Proteobacteria, a known dysbiosis indicator, and the concentration of lactate (R=0.49, p<0.01). Interpretation: The decline in testosterone levels resulted in detrimental changes in gut microbiota. This dysbiosis may contribute to an increase in frailty and an increased risk of adverse outcomes in patients with prostate cancer. Clinical Trial Registration Information: This study was also registered in the UMIN clinical registry (UMI21161). Funding Information: This work was supported in part by JSPS KAKENHI (18H04805 to S.F.), AMED-CREST (JP20gm1010009 to S.F.), JST ERATO (JPMJER1902 to S.F.), the Takeda Science Foundation (to S.F.), the Food Science Institute Foundation (to S.F.), the Programme for the Advancement of Research in Core Projects under Keio University’s Longevity Initiative (to S.F.). Declaration of Interests: The authors declare no conflicts of interest. Ethics Approval Statement: This study was conducted in accordance with the ethical principles of Declaration of Helsinki. The samples and clinical information used in this study were obtained by written informed consent, and with the approval of the institutional review boards of Juntendo University school of Medicine (Approval number: 14-117).

Low-Dose Ethanol Has Impacts on Plasma Levels of Metabolites Relating to Chronic Disease Risk in SAMP8 mice.

The effects of low-dose alcohol on experimental animals are unclear. This study examined plasma metabolites in senescence-accelerated mice 8 (SAMP8) given low-dose ethanol, and compared them with aging progress and skeletal muscle strength. Male SAMP8 mice (10-wk-old) were given drinking water containing 0% (control), 1%, 2%, or 5% (v/v) ethanol for 14 wk. Compared with the control group, only mice who consumed 1% ethanol experienced a lower senescence score at 18 and 23 wk, as well as an increased limb grip strength at 21 wk. Plasma metabolites of control, 1% and 2% ethanol groups were analyzed by capillary electrophoresis-time-of-flight mass spectrometry (CE-TOF/MS). Among the 7 metabolites affected by ethanol, notewhorthy is the positive association of the ethanol levels in drinking water with the levels of α-ketoglutarate (antioxidant and anti-inflammatory metabolite) and hippurate (antioxidant and microbial co-metabolite) (p<0.05). Intriguingly, the levels of 2-hydroxyisobutyrate (the biomarker of energy metabolism and microbial co-metabolite) were higher in the 1% ethanol group (p<0.05), but not in the 2% ethanol group as compared to the control. Furthermore, the levels of some of the metabolites affected were correlated with some variables in the grading score of senescence and muscle strength. This study provides a novel insight into how low-dose ethanol in SAMP8 mice modulates the levels of circulating metabolites relating to chronic disease risk.

Nutritional and Functional Properties of Fermented Mixed Grains by Solid-State Fermentation with Bacillus amyloliquefaciens 245.

Fermented foods have several advantages, including increased nutritional value, improved bioavailability, and functional health properties. We examined that these outcomes were also observed in fermented mixed grains (FMG) containing wheat germ, wheat bran, oats, brown rice, barley, quinoa, and lentils following solid-state fermentation (SSF) by Bacillus amyloliquefaciens 245. The metabolic profile during fermentation was screened using capillary electrophoresis time-of-flight mass spectrometry (CE-TOF-MS). The amino acids were quantitatively measured for the validation of the changes in metabolites. The activity of enzymes (e.g., amylase, protease, and fibrinolysis) and antioxidant capacity was also assessed to elucidate the functionality of FMG. The essential amino acid contents gradually increased as fermentation progressed. As the metabolites involved in the urea cycle and polyamine pathway were changed by fermentation, arginine was used as a substance to produce citrulline, ornithine, and agmatine. FMG showed dramatic increases in enzyme activity. FMG incubated for 36 h also displayed higher total phenolic contents and free radical scavenging ability than MG. The data suggest that FMG produced by Bacillus amyloliquefaciens 245 possess improved nutritional and functional quality, leading to their potential use as dietary supplements.

Differential Metabolomics Profiles Identified by CE-TOFMS between High and Low Intramuscular Fat Amount in Fattening Pigs.

The amount of intramuscular fat (IMF) present in the loin eye area is one of the most important characteristics of high-quality pork. IMF measurements are currently impractical without a labor-intensive process. Metabolomic profiling could be used as an IMF indicator to avoid this process; however, no studies have investigated their use during the fattening period of pigs. This study examined the metabolite profiles in the plasma of two groups of pigs derived from the same Duroc genetic line and fed the same diet. Five plasma samples were collected from each individual the day before slaughter. Capillary electrophoresis-time of flight mass spectrometry (CE-TOFMS) was used to analyze the purified plasma from each sample. Principle component analysis (PCA) and partial least squares (PLS) were used to find the semi-quantitative values of the compounds. The results indicate that branched-chain amino acids are significantly associated with high IMF content, while amino acids are associated with low IMF content. These differences were validated using the quantification analyses by high-performance liquid chromatograph, which supported our results. These results suggest that the concentration of branched-chain amino acids in plasma could be an indicative biomarker for the IMF content in the loin eye area.

Metabolomic profiling of gastric cancer tissues identified potential biomarkers for predicting peritoneal recurrence

Metabolomics is useful for analyzing the nutrients necessary for cancer progression, as the proliferation is regulated by available nutrients. We studied the metabolomic profile of gastric cancer (GC) tissue to elucidate the associations between metabolism and recurrence. Cancer and adjacent non-cancerous tissues were obtained in a pair-wise manner from 140 patients with GC who underwent gastrectomy. Frozen tissues were homogenized and analyzed by capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS). Metabolites were further assessed based on the presence or absence of recurrence. Ninety-three metabolites were quantified. In cancer tissues, the lactate level was significantly higher and the adenylate energy charge was lower than in non-cancerous tissues. The Asp, β-Ala, GDP, and Gly levels were significantly lower in patients with recurrence than in those without. Based on ROC analyses to determine the cut-off values of the four metabolites, patients were categorized into groups at high risk and low risk of peritoneal recurrence. Logistic regression and Cox proportional hazard analyses identified β-Ala as an independent predictor of peritoneal recurrence (hazard ratio [HR] 5.21 [95% confidence interval 1.07-35.89], p = 0.029) and an independent prognostic factor for the overall survival (HR 3.44 [95% CI 1.65-7.14], p < 0.001). The metabolomic profiles of cancer tissues differed from those of non-cancerous tissues. In addition, four metabolites were significantly associated with recurrence in GC. β-Ala was both a significant predictor of peritoneal recurrence and a prognostic factor.