Proteomics of appetite-regulating system influenced by menstrual cycle and intensive exercise in female athletes: a pilot study.

Female athletes who endure intense training are at risk of developing the 'female athlete triad,' making energy intake management crucial. However, the fluctuations in estradiol and progesterone levels throughout the menstrual cycle present a challenge in maintaining consistent energy intake. This study aimed to uncover the underlying factors associated with appetite regulation linked to menstrual phases and exercise using proteomic approach. Five female athletes engaged in 60 min of bicycle exercise, followed by 90 min of rest, during both the follicular and luteal phases. Serum samples were collected before, during, and after exercise, and the serum proteome was analyzed using 2D-gel electrophoresis. A total of 511 spots were detected in the subjects' serum profiles, with significant decreases observed in haptoglobin during the luteal phase and complement component 3 during bicycle training. Unsupervised learning with a generalized estimating equation analysis showed that serum peptide YY (PYY), an appetite suppressor, significantly influenced the fluctuations of serum proteins induced by exercise (p < 0.05). Regression analysis demonstrated a positive correlation between PYY and serum IgM (R = 0.87), implying that the intestinal environment and the immune response in female athletes may contribute to appetite regulation.

Mental stress objective screening for workers using urinary neurotransmitters.

BACKGROUND: Almost 10% of the population develop depression or anxiety disorder during their lifetime. Considering that people who are exposed to high stress are more likely to develop mental disorders, it is important to detect and remove mental stress before depression or anxiety disorder develops. We aimed to develop an objective screening test that quantifies mental stress in workers so that they can recognize and remove it before the disorder develops. METHODS: We obtained urine specimens from 100 healthy volunteers (49 men and 51 women; age = 48.2 ± 10.8 years) after they received medical checks and answered the Brief Job Stress Questionnaire (BJSQ). Participants were divided into high- and low- stress groups according to their total BJSQ scores. We further analyzed six urinary neurotransmitters (dopamine, serotonin, 5-hydoroxyindoleacetic acid, gamma-aminobutyric acid, homovanillic acid, and vanillylmandelic acid) using liquid chromatography-mass spectrometry to compare their levels between the two groups. RESULTS: We obtained the concentrations of the six analytes from 100 examinees and revealed that the levels of urinary dopamine (p = 0.0042) and homovanillic acid (p = 0.020) were significantly lower in the high-stress group than those in the low-stress group. No biases were observed between the two groups in 36 laboratory items. The stress index generated from the six neurotransmitter concentrations recognized high-stress group significantly. Moreover, we discovered that the level of each urinary neurotransmitter changed depending on various stress factors, such as dissatisfaction, physical fatigue, stomach and intestine problems, poor appetite, poor working environments, sleep disturbance, isolation, worry, or insecurity. CONCLUSION: We revealed that urinary neurotransmitters could be a promising indicator to determine underlying mental stress. This study provides clues for scientists to develop a screening test not only for workers but also for patients with depression.

Acidic Activated Charcoal Prevents Obesity and Insulin Resistance in High-Fat Diet-Fed Mice.

Obesity is becoming a major public health problem worldwide. Making charcoal from wood ("Sumi-yaki") has been a traditional activity in the southern part of Nagano Prefecture for centuries, with activated charcoal having reported detoxifying effects. However, it is unclear whether activated charcoal also possesses anti-obesity properties. Additionally, since activated charcoal is usually alkaline and might be affected by gastric juice, we evaluated the effect of acidic activated charcoal on high-fat diet (HFD)-induced obesity. This study demonstrated that co-treatment of acidic activated charcoal with a HFD significantly improved obesity and insulin resistance in mice in a dose-dependent manner. Metabolomic analysis of cecal contents revealed that neutral lipids, cholesterol, and bile acids were excreted at markedly higher levels in feces with charcoal treatment. Moreover, the hepatic expressions of genes encoding cholesterol 7 alpha-hydroxylase and hydroxymethylglutaryl-CoA reductase/synthase 1 were up-regulated by activated charcoal, likely reflecting the enhanced excretions from the intestine and the enterohepatic circulation of cholesterol and bile acids. No damage or abnormalities were detected in the gastrointestinal tract, liver, pancreas, and lung. In conclusion, acidic activated charcoal may be able to attenuate HFD-induced weight gain and insulin resistance without serious adverse effects. These findings indicate a novel function of charcoal to prevent obesity, metabolic syndrome, and related diseases.

Oxidized Albumin and Cartilage Acidic Protein-1 as Blood Biomarkers to Predict Ischemic Stroke Outcomes.

Background: There is high demand for blood biomarkers that reflect the therapeutic response or predict the outcomes of patients with acute ischemic stroke (AIS); however, few biomarkers have been evidentially verified to date. This study evaluated two proteins, oxidized albumin (OxHSA) and cartilage acidic protein-1 (CRTAC1), as potential prognostic markers of AIS. Methods: The ratio of OxHSA to normal albumin (%OxHSA) and the level of CRTAC1 in the sera of 74 AIS patients were analyzed on admission (day 0), and at 1 and 7 days after admission. AIS patients were divided into two groups according to their modified Rankin Scale (mRS) at 3 months after discharge: the low-mRS (mRS < 2) group included 48 patients and the high-mRS (mRS ≥ 2) group included 26 patients. The differences in %OxHSA and CRTAC1 between the two groups on days 0, 1, and 7 were evaluated. Results: The mean %OxHSA values of the high-mRS group on days 0, 1, and 7 were significantly higher than those of the low-mRS group (p < 0.05). The CRTAC1 levels continuously increased from day 0 to day 7, and those of the high-mRS group were significantly higher than those of the low-mRS group on day 7 (p < 0.05). Conclusions: These results suggest that higher %OxHSA and CRTAC1 are associated with poor outcomes in AIS patients. An index that combines %OxHSA and CRTAC1 can accurately predict the outcomes of AIS patients.

Multiblock metabolomics: An approach to elucidate whole-body metabolism with multiblock principal component analysis.

Principal component analysis (PCA) is a useful tool for omics analysis to identify underlying factors and visualize relationships between biomarkers. However, this approach is limited in addressing life complexity and further improvement is required. This study aimed to develop a new approach that combines mass spectrometry-based metabolomics with multiblock PCA to elucidate the whole-body global metabolic network, thereby generating comparable metabolite maps to clarify the metabolic relationships among several organs. To evaluate the newly developed method, Zucker diabetic fatty (ZDF) rats (n = 6) were used as type 2 diabetic models and Sprague Dawley (SD) rats (n = 6) as controls. Metabolites in the heart, kidney, and liver were analyzed by capillary electrophoresis and liquid chromatography mass spectrometry, respectively, and the detected metabolites were analyzed by multiblock PCA. More than 300 metabolites were detected in the heart, kidney, and liver. When the metabolites obtained from the three organs were analyzed with multiblock PCA, the score and loading maps obtained were highly synchronized and their metabolism patterns were visually comparable. A significant finding in this study was the different expression patterns in lipid metabolism among the three organs; notably triacylglycerols with polyunsaturated fatty acids or less unsaturated fatty acids showed specific accumulation patterns depending on the organs.

Lipidomics links oxidized phosphatidylcholines and coronary arteritis in Kawasaki disease.

AIMS: Coronary arteritis is a life-threatening complication that may arise in the acute stage of Kawasaki disease (KD), the leading cause of systemic vasculitis in childhood. Various microorganisms and molecular pathogens have been reported to cause KD. However, little is known about the key molecules that contribute to the development of coronary arteritis in KD. METHODS AND RESULTS: To identify causative molecules for coronary arteritis in KD, we prospectively recruited 105 patients with KD and 65 disease controls in four different parts of Japan from 2015 to 2018. During this period, we conducted lipidomics analyses of their sera using liquid chromatography-mass spectrometry (LC-MS). The comprehensive LC-MS system detected a total of 27 776 molecules harbouring the unique retention time and m/z values. In the first cohort of 57 KD patients, we found that a fraction of these molecules showed enrichment patterns that varied with the sampling region and season. Among them, 28 molecules were recurrently identified in KD patients but not in controls. The second and third cohorts of 48 more patients with KD revealed that these molecules were correlated with inflammatory markers (leucocyte counts and C-reactive proteins) in the acute stage. Notably, two of these molecules (m/z values: 822.55 and 834.59) were significantly associated with the development of coronary arteritis in the acute stage of KD. Their fragmentation patterns in the tandem MS/MS analysis were consistent with those of oxidized phosphatidylcholines (PCs). Further LC-MS/MS analysis supported the concept that reactive oxygen species caused the non-selective oxidization of PCs in KD patients. In addition, the concentrations of LOX-1 ligand containing apolipoprotein B in the plasma of KD patients were significantly higher than in controls. CONCLUSION: These data suggest that inflammatory signals activated by oxidized phospholipids are involved in the pathogenesis of coronary arteritis in KD. Because the present study recruited only Japanese patients, further examinations are required to determine whether oxidized PCs might be useful biomarkers for the development of coronary arteritis in broad populations of KD.

A Potential Serum N-glycan Biomarker for Hepatitis C Virus-Related Early-Stage Hepatocellular Carcinoma with Liver Cirrhosis.

Detection of early-stage hepatocellular carcinoma (HCC) is beneficial for prolonging patient survival. However, the serum markers currently used show limited ability to identify early-stage HCC. In this study, we explored human serum N-glycans as sensitive markers to diagnose HCC in patients with cirrhosis. Using a simplified fluorescence-labeled N-glycan preparation method, we examined non-sialylated and sialylated N-glycan profiles from 71 healthy controls and 111 patients with hepatitis and/or liver cirrhosis (LC) with or without HCC. We found that the level of serum N-glycan A2G1(6)FB, a biantennary N-glycan containing core fucose and bisecting GlcNAc residues, was significantly higher in hepatitis C virus (HCV)-infected cirrhotic patients with HCC than in those without HCC. In addition, A2G1(6)FB was detectable in HCV-infected patients with early-stage HCC and could be a more accurate marker than alpha-fetoprotein (AFP) or protein induced by vitamin K absence or antagonists-II (PIVKA-II). Moreover, there was no apparent correlation between the levels of A2G1(6)FB and those of AFP or PIVKA-II. Thus, simultaneous use of A2G1(6)FB and traditional biomarkers could improve the accuracy of HCC diagnosis in HCV-infected patients with LC, suggesting that A2G1(6)FB may be a reliable biomarker for early-stage HCC patients.

Utility of Comprehensive Serum Glycopeptide Spectra Analysis (CSGSA) for the Detection of Early Stage Epithelial Ovarian Cancer.

Comprehensive serum glycopeptide spectra analysis (CSGSA) evaluates >10,000 serum glycopeptides and identifies unique glycopeptide peaks and patterns via supervised orthogonal partial least-squares discriminant modeling. CSGSA was more accurate than cancer antigen 125 (CA125) or human epididymis protein 4 (HE4) for detecting early stage epithelial ovarian cancer. Combined CSGSA, CA125, and HE4 had improved diagnostic performance. Thus, CSGSA may be a useful screening tool for detecting early stage epithelial ovarian cancer.

Comprehensive Serum Glycopeptide Spectra Analysis Combined with Artificial Intelligence (CSGSA-AI) to Diagnose Early-Stage Ovarian Cancer.

Ovarian cancer is a leading cause of deaths among gynecological cancers, and a method to detect early-stage epithelial ovarian cancer (EOC) is urgently needed. We aimed to develop an artificial intelligence (AI)-based comprehensive serum glycopeptide spectra analysis (CSGSA-AI) method in combination with convolutional neural network (CNN) to detect aberrant glycans in serum samples of patients with EOC. We converted serum glycopeptide expression patterns into two-dimensional (2D) barcodes to let CNN learn and distinguish between EOC and non-EOC. CNN was trained using 60% samples and validated using 40% samples. We observed that principal component analysis-based alignment of glycopeptides to generate 2D barcodes significantly increased the diagnostic accuracy (88%) of the method. When CNN was trained with 2D barcodes colored on the basis of serum levels of CA125 and HE4, a diagnostic accuracy of 95% was achieved. We believe that this simple and low-cost method will increase the detection of EOC.

Inter-Laboratory Comparison of Metabolite Measurements for Metabolomics Data Integration.

BACKGROUND: One of the current problems in the field of metabolomics is the difficulty in integrating data collected using different equipment at different facilities, because many metabolomic methods have been developed independently and are unique to each laboratory. METHODS: In this study, we examined whether different analytical methods among 12 different laboratories provided comparable relative quantification data for certain metabolites. Identical samples extracted from two cell lines (HT-29 and AsPc-1) were distributed to each facility, and hydrophilic and hydrophobic metabolite analyses were performed using the daily routine protocols of each laboratory. RESULTS: The results indicate that there was no difference in the relative quantitative data (HT-29/AsPc-1) for about half of the measured metabolites among the laboratories and assay methods. Data review also revealed that errors in relative quantification were derived from issues such as erroneous peak identification, insufficient peak separation, a difference in detection sensitivity, derivatization reactions, and extraction solvent interference. CONCLUSION: The results indicated that relative quantification data obtained at different facilities and at different times would be integrated and compared by using a reference materials shared for data normalization.

Comprehensive Serum Glycopeptide Spectra Analysis (CSGSA): A Potential New Tool for Early Detection of Ovarian Cancer.

OBJECTIVES: To conduct a comprehensive glycopeptide spectra analysis of serum between cancer and non-cancer patients to identify early biomarkers of epithelial ovarian cancer (EOC). METHODS: Approximately 30,000 glycopeptide peaks were detected from the digested serum glycoproteins of 39 EOC patients (23 early-stage, 16 advanced-stage) and 45 non-cancer patients (27 leiomyoma and ovarian cyst cases, 18 endometrioma cases) by liquid chromatography mass spectrometry (LC-MS). The differential glycopeptide peak spectra were analyzed to distinguish between cancer and non-cancer groups by employing multivariate analysis including principal component analysis (PCA), orthogonal partial least squares discriminant analysis (OPLS-DA) and heat maps. RESULTS: Examined spectral peaks were filtered down to 2281 serum quantitative glycopeptide signatures for differentiation between ovarian cancer and controls using multivariate analysis. The OPLS-DA model using cross-validation parameters R2 and Q2 and score plots of the serum samples significantly differentiated the EOC group from the non-cancer control group. In addition, women with early-stage clear cell carcinoma and endometriomas were clearly distinguished from each other by OPLS-DA as well as by PCA and heat maps. CONCLUSIONS: Our study demonstrates the potential of comprehensive serum glycoprotein analysis as a useful tool for ovarian cancer detection.

Serum leucine-rich alpha-2-glycoprotein-1 with fucosylated triantennary N-glycan: a novel colorectal cancer marker.

BACKGROUND: Carcinoembryonic antigen (CEA) and carbohydrate antigen (CA)19-9 are used in clinical practice as tumor markers to diagnose or monitor colorectal cancer (CRC) patients, However, their specificities and sensitivities are not ideal, and novel alternatives are needed. In this study, mass spectrometry was used to search for screening markers, focusing on glycan alterations of glycoproteins in the sera of CRC patients. METHODS: Glycopeptides were prepared from serum glycoproteins separated from blood samples of 80 CRC patients and 50 healthy volunteers, and their levels were measured by liquid chromatography time-of flight mass spectrometry (LC-TOF-MS). RESULTS: Leucine-rich alpha-2-glycoprotein-1 with fucosylated triantennary N-glycan (LRG-FTG) was identified as CRC marker after evaluating 30,000 candidate glycopeptide peaks. The average LRG-FTG level in CRC patients (1.25 ± 0.973 U/mL) was much higher than that in healthy volunteers (0.496 ± 0.433 U/mL, P < 10- 10), and its sensitivity and specificity exceeded those of CA19-9. The combination of CEA and LRG-FTG showed a complementary effect and had better sensitivity (84%), specificity (90%), and AUC (0.91 by ROC analysis) than each marker alone or any other previously reported marker. LRG-FTG alone or combined with CEA also corresponded well with patient response to treatment. CONCLUSIONS: We identified LRG-FTG as a new CRC marker, with a sensitivity and specificity exceeding CA19-9. The combination of LRG-FTG and CEA showed much higher sensitivity and specificity than each marker alone. Further validation beyond this initial exploratory cohort is warranted.

Fully sialylated alpha-chain of complement 4-binding protein (A2160): a novel prognostic marker for epithelial ovarian carcinoma.

PURPOSE: Fully sialylated alpha-chain of complement 4-binding protein (A2160) is a member of the glycoprotein family and has recently been identified as a diagnostic biomarker for epithelial ovarian cancer. This study examined the utility of A2160 as a prognostic biomarker for this disease. METHODS: This is a retrospective analysis of prospectively collected plasma samples from 93 women with stage I-IV epithelial ovarian cancer who underwent primary cytoreductive surgery between 2009 and 2014. Pretreatment A2160 levels were correlated to clinico-pathological factors and survival outcome. RESULTS: Women with advanced-stage disease had significantly higher 2160 levels compared to those with early stage disease (stage I-II versus III-IV, median 2.17-2.70 versus 5.31-8.70 U/mL, P < 0.01). Women with high-grade serous ovarian carcinoma had higher A2160 levels compared to other histologies (6.60 versus 3.01 U/mL, P = 0.05). Women who had suboptimal cytoreduction had significantly higher A2160 levels than those who achieved optimal/complete cytoreduction (7.02 versus 2.30-3.17 U/mL, P < 0.01). On univariable analysis, higher A2160 levels were significantly associated with decreased progression-free survival (64-100 versus 1-33%ile, 5-year rates 53.4 versus 78.9%, P = 0.029). After controlling for age, CA-125 level, cytoreductive status, histology, and stage, higher A2160 levels remained an independent prognostic factor for decreased progression-free survival (adjusted-hazard ratio (HR) 2.48, 95% confidence interval (CI) 1.01-6.11, P = 0.049). Similarly, higher A2160 levels were independently associated with decreased cause-specific survival on multivariable analysis (adjusted-HR 3.07, 95% CI 1.19-7.93, P = 0.021). CONCLUSION: Our study suggests that A2160 may be a useful prognostic biomarker for epithelial ovarian cancer, and higher pretreatment levels of A2160 predicts poor survival outcome.

UPLC-MS/MS based diagnostics for epithelial ovarian cancer using fully sialylated C4-binding protein.

Serum levels of fully sialylated C4-binding protein (FS-C4BP) are remarkably elevated in patients with epithelial ovarian cancer (EOC) and can be used as a marker to distinguish ovarian clear cell carcinoma from endometrioma. This study aimed to develop a stable, robust and reliable liquid chromatography-hybrid mass spectrometry (UPLC-MS/MS) based diagnostic method that would generalize FS-C4BP as a clinical EOC biomarker. Glycopeptides derived from 20 µL of trypsin-digested serum glycoprotein were analyzed via UPLC equipped with an electrospray ionization time-of-flight mass spectrometer. This UPLC-MS/MS-based diagnostic method was optimized for FS-C4BP and validated using sera from 119 patients with EOC and 127 women without cancer. A1958 (C4BP peptide with two fully sialylated biantennary glycans) was selected as a marker of FS-C4BP because its level in serum was highest among FS-C4BP family members. Preparation and UPLC-MS/MS were optimized for A1958, and performance and robustness were significantly improved relative to our previous method. An area under the curve analysis of the FS-C4BP index receiver operating characteristic curve revealed that the ratio between A1958 and A1813 (C4BP peptide with two partially sialylated biantennary glycans) reached 85%. A combination of the FS-C4BP index and carbohydrate antigen-125 levels further enhanced the sensitivity and specificity.

Multifucosylated Alpha-1-acid Glycoprotein as a Novel Marker for Hepatocellular Carcinoma.

High-sensitivity and -specificity diagnostic techniques to detect early-stage hepatocellular carcinoma (HCC) are in high demand. Screening with serum HCC markers, such as alpha-fetoprotein, is not practical because they possess poor sensitivity and specificity. As such, we focused on glycan alterations of glycoproteins found in patient sera in an attempt to discover novel HCC markers that are more specific and sensitive than current HCC markers. Sera from 42 HCC patients and 80 controls, composed of 27 chronic hepatitis B patients, 26 chronic hepatitis C patients, and 27 healthy volunteers, were analyzed in this study. Glycopeptides obtained from serum proteins by trypsin digestion were enriched by ultrafiltration and Aleuria aurantia lectin-based affinity chromatography, followed by analysis using liquid chromatography time-of-flight mass spectrometry. The data were analyzed by our newly developed software, which calculates peak intensities and positions (m/z and elution time), aligns all sample peaks, and integrates all data into a single table. HCC markers were extracted from more than 30 000 detected glycopeptide peaks by t test, mean-fold change, and ROC analyses. As a result, we revealed that alpha-1-acid glycoprotein with multifucosylated tetraantennary N-glycans was significantly elevated in HCC patients, whereas the single fucosylated derivative was not.

Fully-sialylated alpha-chain of complement 4-binding protein: Diagnostic utility for ovarian clear cell carcinoma.

OBJECTIVE: While a certain fraction of endometriomas can develop de novo epithelial ovarian cancer (EOC) such as clear cell carcinoma (OCCC), there is currently no useful biomarker available for early detection of OCCC from endometriomas. The aim of this study was to describe the diagnostic utility of a novel biomarker for EOC especially for OCCC to distinguish from endometrioma. METHODS: More than 100,000 glycan structures of serum glycoproteins obtained from 134 pretreatment all stage EOC patients (including 45 OCCCs) and 159 non-cancer control women (including 36 endometriomas) were explored for a mass spectrum approach. Diagnostic accuracy of identified biomarker was compared to the one of CA-125 by comparing area under curve (AUC) and positive/negative predictive values (PPV and NPV). RESULTS: A2160, a fully-sialylated alpha-chain of complement 4-binding protein, was identified as a candidate target marker. A2160 was significantly elevated in all stages of OCCC compared to with endometriomas. Diagnostic accuracy of A2160 (cutoff 1.6U/mL) to distinguish early stage OCCC from endometrioma is significantly higher than that of CA-125 (cutoff 35IU/L): AUC for A2160 versus CA-125, 0.92 versus 0.67; PPV 95% versus 64%; and NPV 85% versus 58%. In addition, fully-sialylated glycans had a higher accuracy for diagnosing EOC as compared to partially-sialylated glycans of alpha-chain of complement 4-binding protein. CONCLUSION: Our study suggested that A2160 may be a useful biomarker to distinguish early-stage OCCC from endometrioma. This new biomarker can be potentially applied for the monitoring of endometrioma patients, making possible the early diagnosis of OCCC.

Outer arm fucosylation of N-glycans increases in sera of hepatocellular carcinoma patients.

Serum glycans are promising markers for early-stage cancer detection, but the research remains challenging because low concentrations of serum glycoproteins are secreted from early-stage tumors. We have established an N-glycan profiling method using liquid chromatography electrospray ionization-mass spectrometry with high sensitive derivative, trimethyl(4-aminophenyl)ammonium chloride (TMAPA). The mass sensitivity of TMAPA-labeled oligosaccharides was enhanced more than 50 times compared with 2-aminopyridine (PA) labeled oligosaccharides, and the analytical period was significantly shortened compared with traditional HPLC 2D-mapping. Using this method, we found about 28 major N-linked oligosaccharides in human sera, and we investigated their alterations in patients who developed hepatocellular carcinoma (HCC). We found that outer arm fucosylation (attached GlcNAc via an alpha 1-3/4 linkage) in highly branched oligosaccharides increased significantly in sera of HCC patients. Normalizing the level of outer arm fucosylation by taking into account platelet concentration allowed us to distinguish more clearly between HCC and LC patients.