Takahiko Muramatsu

Novel, Objective, Multivariate Biomarkers Composed of Plasma Amino Acid Profiles for the Diagnosis and Assessment of Inflammatory Bowel Disease

Background Inflammatory bowel disease (IBD) is a chronic intestinal disorder that is associated with a limited number of clinical biomarkers. In order to facilitate the diagnosis of IBD and assess its disease activity, we investigated the potential of novel multivariate indexes using statistical modeling of plasma amino acid concentrations (aminogram). Methodology and Principal Findings We measured fasting plasma aminograms in 387 IBD patients (Crohns disease (CD), n = 165; ulcerative colitis (UC), n = 222) and 210 healthy controls. Based on Fisher linear classifiers, multivariate indexes were developed from the aminogram in discovery samples (CD, n = 102; UC, n = 102; age and sex-matched healthy controls, n = 102) and internally validated. The indexes were used to discriminate between CD or UC patients and healthy controls, as well as between patients with active disease and those in remission. We assessed index performances using the area under the curve of the receiver operating characteristic (ROC AUC). We observed significant alterations to the plasma aminogram, including histidine and tryptophan. The multivariate indexes established from plasma aminograms were able to distinguish CD or UC patients from healthy controls with ROC AUCs of 0.940 (95% confidence interval (CI): 0.898–0.983) and 0.894 (95%CI: 0.853–0.935), respectively in validation samples (CD, n = 63; UC, n = 120; healthy controls, n = 108). In addition, other indexes appeared to be a measure of disease activity. These indexes distinguished active CD or UC patients from each remission patients with ROC AUCs of 0.894 (95%CI: 0.853–0.935) and 0.849 (95%CI: 0.770–0.928), and correlated with clinical disease activity indexes for CD (rs = 0.592, 95%CI: 0.385–0.742, p<0.001) or UC (rs = 0.598, 95%CI: 0.452–0.713, p<0.001), respectively. Conclusions and Significance In this study, we demonstrated that established multivariate indexes composed of plasma amino acid profiles can serve as novel, non-invasive, objective biomarkers for the diagnosis and monitoring of IBD, providing us with new insights into the pathophysiology of the disease.

Malnutrition impairs interferon signaling through mTOR and FoxO pathways in patients with chronic hepatitis C.

BACKGROUND & AIMS Patients with advanced chronic hepatitis C (CH-C) often are malnourished, but the effects of malnutrition on interferon (IFN) signaling and response to treatment have not been determined. We assessed the importance of the nutritional state of the liver on IFN signaling and treatment response. METHODS We studied data from 168 patients with CH-C who were treated with the combination of pegylated-IFN and ribavirin. Plasma concentrations of amino acids were measured by mass spectrometry. Liver gene expression profiles were obtained from 91 patients. Huh-7 cells were used to evaluate the IFN signaling pathway, mammalian target of rapamycin complex 1 (mTORC1), and forkhead box O (FoxO). Antiviral signaling induced by branched-chain amino acids (BCAAs) was determined using the in vitro hepatitis C virus replication system. RESULTS Multivariate logistic regression analysis showed that Fischers ratio was associated significantly with nonresponders, independent of interleukin-28B polymorphisms or the histologic stage of the liver. Fischers ratio was correlated inversely with the expression of BCAA transaminase 1, and was affected by hepatic mTORC1 signaling. IFN stimulation was impaired substantially in Huh-7 cells grown in medium that was low in amino acid concentration, through repressed mTORC1 signaling, and increased Socs3 expression, which was regulated by Foxo3a. BCAA could restore impaired IFN signaling and inhibit hepatitis C virus replication under conditions of malnutrition. CONCLUSIONS Malnutrition impaired IFN signaling by inhibiting mTORC1 and activating Socs3 signaling through Foxo3a. Increasing BCAAs to up-regulate IFN signaling might be used as a new therapeutic approach for patients with advanced CH-C.

Predictive Properties of Plasma Amino Acid Profile for Cardiovascular Disease in Patients with Type 2 Diabetes

Prevention of cardiovascular disease (CVD) is an important therapeutic object of diabetes care. This study assessed whether an index based on plasma free amino acid (PFAA) profiles could predict the onset of CVD in diabetic patients. The baseline concentrations of 31 PFAAs were measured with high-performance liquid chromatography-electrospray ionization-mass spectrometry in 385 Japanese patients with type 2 diabetes registered in 2001 for our prospective observational follow-up study. During 10 years of follow-up, 63 patients developed cardiovascular composite endpoints (myocardial infarction, angina pectoris, worsening of heart failure and stroke). Using the PFAA profiles and clinical information, an index (CVD-AI) consisting of six amino acids to predict the onset of any endpoints was retrospectively constructed. CVD-AI levels were significantly higher in patients who did than did not develop CVD. The area under the receiver-operator characteristic curve of CVD-AI (0.72 [95% confidence interval (CI): 0.64–0.79]) showed equal or slightly better discriminatory capacity than urinary albumin excretion rate (0.69 [95% CI: 0.62–0.77]) on predicting endpoints. A multivariate Cox proportional hazards regression analysis showed that the high level of CVD-AI was identified as an independent risk factor for CVD (adjusted hazard ratio: 2.86 [95% CI: 1.57–5.19]). This predictive effect of CVD-AI was observed even in patients with normoalbuminuria, as well as those with albuminuria. In conclusion, these results suggest that CVD-AI based on PFAA profiles is useful for identifying diabetic patients at risk for CVD regardless of the degree of albuminuria, or for improving the discriminative capability by combining it with albuminuria.

Validation of an analytical method for human plasma free amino acids by high-performance liquid chromatography ionization mass spectrometry using automated precolumn derivatization.

The analysis of human plasma free amino acids is important for diagnosing the health of individuals, because their concentrations are known to vary with various diseases. The development of valid, reliable, and high-throughput analytical methods for amino acids analysis is an essential requirement in clinical applications. In the present study, we have developed an automated precolumn derivatization amino acid analytical method based on high-performance liquid chromatography/electrospray ionization mass spectrometry (so-called UF-Amino Station). This method enabled the separation of at least 38 types of physiological amino acids within 8min, and the interval time between injections was 12min. We also validated this method for 21 major types of free amino acids in human plasma samples. The results of the specificity, linearity, accuracy, repeatability, intermediate precision, reproducibility, limits of detections, lower limits of quantification, carry over, and sample solution stability were sufficient to allow for the measurement of amino acids in human plasma samples. Our developed method should be suitable for use in clinical fields.

Reference intervals for plasma-free amino acid in a Japanese population

Background Plasma amino acid concentrations vary with various diseases. Although reference intervals are useful in daily clinical practice, no reference intervals have been reported for plasma amino acids in a large Japanese population. Methods Reference individuals were selected from 7685 subjects examined with the Japanese Ningen Dock in 2008. A total of 1890 individuals were selected based on exclusion criteria, and the reference samples were selected after the outlier samples for each amino acid concentration were excluded. The lower limit of the reference intervals for the plasma amino acid concentrations was set at the 2.5th percentile and the upper limit at the 97.5th percentile. Results By use of the nested analysis of variance, we analysed a large dataset of plasma samples and the effects of background factors (sex, age and body mass index [BMI]) on the plasma amino acid concentrations. Most amino acid concentrations were related to sex, especially those of branched-chained amino acid. The citrulline, glutamine, ornithine and lysine concentrations were related to age. The glutamate concentration was related to body mass index. Conclusions The concentrations of most amino acids are more strongly related to sex than to age or body mass index. Our results indicate that the reference intervals for plasma amino acid concentrations should be stratified by sex when the background factors of age and body mass index are considered.

Plasma Amino Acid Profiles in Collagen Disease Patients with Interstitial Lung Disease

Interstitial lung disease (ILD) is frequently associated with collagen diseases, and is designated collagen vascular disease-associated ILD (CVD-ILD) that influences the prognosis of the disease. Acute-onset diffuse ILD (AoDILD) occurs in patients with collagen disease with or without underlying CVD-ILD. The prognosis of AoDILD is quite poor. It has been reported that plasma amino acid profiles are altered in rheumatoid arthritis (RA) patients. Here, we investigated the plasma amino acid profiles to determine whether they may be useful for diagnosing CVDILD or AoDILD in collagen disease. Plasma amino acid levels were analyzed using liquid chromatography/electrospray ionization tandem mass spectrometry in 64 RA patients with or without CVD-ILD, and 15 collagen disease patients with AoDILD. By using support vector machine (svm) analysis, the svm index (AA) was generated from amino acid profiles and the svm index (AA, KL6) was from amino acid profiles together with Krebs von den lungen-6 (KL-6). Plasma lysine levels were higher in RA patients with ILD than in those without. The optimized cut-off level of svm index (AA) was determined for CVD-ILD in RA, and the specificity and the sensitivity were 86.8% and 65.4%, respectively. These values for the svm index (AA, KL6) were 81.6% and 88.5%. The plasma methionine and phenylalanine levels were significantly increased in the AoDILD state, whereas Fischer’s ratio was decreased. This is the first report of plasma amino acid profiles in CVD-ILD and AoDILD in collagen disease. The svm index (AA, KL6) will be a better marker for diagnosing CVD-ILD in RA than KL-6 alone, though svm index (AA) is not. The plasma amino acid profiles could be a better marker for AoDILD in collagen disease patients.

ALTERATION OF PLASMA-FREE AMINO ACIDS (PFAAS) AND AMINES COULD BE NOVEL BLOOD-BASED BIOMARKERS FOR DETECTION OF ALZHEIMER’S DISEASE AND FUTURE PROGRESSION

(i) P-value<0.05, (ii) fold change 1.5 and (iii) consistency variation tendency in three groups. After assessed by Ingenuity Pathway Analysis (IPA), we then performed parallel reaction monitoring (PRM) mass spectrometry to accurately quantify the remaining samples. Receiver Operating Characteristic was used to differentiate samples in the different groups. Results:A total of 35 candidate proteins could distinguish AD from MCI, and a total of 34 candidates could distinguish AD from NC, with an area under the ROC curve (AUC) above 0.7, but failed to distinguish MCI from NC according to the same criteria. Stratification by gender revealed improved differentiation between AD vs. NC, AD vs. MCI, and MCI vs. NC with satisfactory results of AUC 0.9, sensitivity 80% and specificity 80%. Conclusions: Gender difference may play an important role in the detection of plasma biomarkers for AD. For females, we recommend Complement C1r subcomponent, Inter-alpha-trypsin inhibitor heavy chain H4, Alpha-2-HS-glycoprotein, Insulin-like growth factor-binding protein complex acid labile subunit, Kininogen-1, Ficolin-2, Complement C3, Transthyretin, Syntaxin-binding protein 5 and Antithrombin-III to distinguish MCI from NC. For males, we recommend Kininogen-1, Glutathione peroxidase 3, Alpha-1-antitrypsin and Clusterin. Furthermore, Mesothelin, Phosphatidylcholine-sterol acyltransferase and Phosphatidylinositol-glycan-specific phospholipase D for females and Syntaxin-binding protein 5, Clusterin, and Alpha-1-antitrypsin et al. for males may predict the conversion from MCI to AD.

Abstract 4632: Probability of plasma amino acid concentration and its profile as a novel diagnostic marker for prostate cancer

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Introduction. Although there is no doubt on a plausible significance of prostate-specific antigen (PSA) on prostate cancer (PCa) diagnosis and managements, PSA screening for cancer has several problems especially on (1) specificity to discriminate cancers from benign lesions such as hypertrophy or inflammation, and (2) sensitivity to some cancers which PSA production is low or missing. Under the present circumstances, combination of other biomarkers with PSA is a way to overcome these problems. On the other hand, plasma amino acid-concentration reflects metabolic status of the body. Although maintained in healthy humans, it is known to change in various pathologic conditions including liver dysfunction and cancers, and therefore one could speculate that the profile of the change could be a promising biomarker (“AminoIndex”) of various cancers. Additionally, recent advances in technologies enable us to easily evaluate plasma amino acid profiles of a great number of samples. In this study, the potential of plasma amino acid profiling for prostate cancer detection was investigated. Subjects and Methods. Plasma samples were collected from Japanese prostate cancer patients those were consulted to Kanagawa Cancer Center Hospital and Yokohama Municipal Citizens Hospital and finally diagnosed by histopathological examination of biopsy specimens. Those of Japanese healthy controls were collected from subjects who were undergone comprehensive medical examination at Mitsui Memorial Hospital. Plasma amino acid-concentrations were measured by LC-MS, and compared between PCa patients and healthy controls. A multivariate logistic regression function was applied to obtain the index to discriminate PCa patients from controls using changes of plasma amino acid-concentrations. Results. Plasma concentrations of several amino acids changed significantly in PCa patients compared to control subjects. In particular, plasma levels of proline, alanine, and asparagine were increased while those of valine, and tryptophan were decreased in PCa patients significantly. The predicted amino acid profile index (“AminoIndex”) composed with these significantly changed amino acids showed high discrimination performance between PCa patients and healthy controls with more than 70% of area under ROC curve. The obtained index discriminated both patients of poorly differentiated cancer and the early stage cancer from control, and was independent of both age and serum PSA level. Conclusion and Perspectives. In this study, we demonstrated that plasma amino acid profile was altered in prostate cancer patients and the calculated “AminoIndex” could be a novel promising tool for detection of prostate cancer. PCa screening by the “AminoIndex” in conjunction with PSA could be a promising way to decrease both false positive and false negative cases. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4632.