Ryosei Sakai

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.

Activin increases bone mass and mechanical strength of lumbar vertebrae in aged ovariectomized rats

Activin is a member of the transforming growth factor-beta superfamily and is thought to be involved in the regulation of bone formation due to its presence in bone tissue and its osteogenic activity both in vitro and in vivo. We recently found that systemic administration of activin increased both tibial bone mass and mechanical strength in young growing rats. The present study investigated the effects of activin in aged ovariectomized (ovx) rats. Twelve-month-old Fischer rats were ovariectomized and maintained for 10 months. Recombinant human activin A (activin) or human parathyroid hormone 1-34 (PTH) was administered intramuscularly three times a week for 12 weeks. Activin (1 and 5 microg/kg) markedly increased lumbar vertebral bone mineral content and bone mineral density. Activin also increased the mechanical strength of the vertebral body, which was highly correlated to the bone mineral density of the vertebral body. The maximal response in bone mass and strength was observed at 1 microg/kg of activin, which was approximately equal to that induced by PTH at 40 microg/kg. Peripheral quantitative computed tomography revealed that activin enlarged the cross-sectional size of the vertebrae without changing the foramen area, indicating its effects on cortical shells. Histomorphometric analysis of cancellous bone of vertebral body in similar experiment showed that activin (3 microg/kg) increased bone volume and the mineralizing surface, although its effects were less than PTH. The present results indicate that low doses of activin are effective against vertebral bone loss in aged ovx rats.

The measurement of activin/EDF in mouse serum: Evidence for extragonadal production

Many studies have shown that activin/EDF mediates local physiological events at various sites. In this study, the authors confirmed the presence of activin in mouse serum by high performance liquid chromatography (HPLC) monitored by a specific bioassay. The retention time of the active fraction in HPLC was identical to that of authentic activin A, and the activity was neutralized by follistatin. That the serum activin levels in ovariectomized and aged mice were decreased suggests that the serum activin was generated partly by ovary (35%), but also by extragonadal organs. Activin and inhibin are structurally closely related, and both are involved in many physiological processes including control of follicle stimulating hormone secretion by the pituitary. The regulation of serum activin, however, appeared to differ from that of inhibin.

Activin release from bone coupled to bone resorption in organ culture of neonatal mouse calvaria.

Activin, a member of the transforming growth factor-beta (TGF-beta) superfamily, is present in the bone matrix and assumed to be involved in the regulation of bone formation. In the present study, we investigated whether the release of activin from bone is coupled with bone resorption. Neonatal mouse calvaria were cultured in the presence of various stimulators of bone resorption (parathyroid hormone [PTH], interleukin-1beta, prostaglandin E2) for up to 72 h, and the activin activity in the medium was measured using a specific bioassay for activin. Activin activity was accumulated in proportion to the time- and dose-dependent increase in calcium release from bone into the medium (bone resorption). An inhibition of PTH-dependent bone resorption by a bisphosphonate, disodium dichlormethane-1,1-bisphosphonic acid (Cl2MBP), completely blocked release of activin activity from bone into the medium. In primary culture of calvarial cells, however, neither PTH nor Cl2MBP affected activin production. These findings indicate that release of activin activity from bone tissue is strongly coupled to bone resorption. Because activin possesses osteogenic activities, activin released locally from bone might be involved in the regulation of bone formation in the physiological process of bone remodeling, as has been suggested for TGF-beta.

Metabolomics and its Potential for Assessment of Adequacy and Safety of Amino Acid Intake

In this short paper, we suggest some concepts and methods that may be useful in considering a metabolomic approach to the study of amino acid adequacy and safety. We suggest that the analysis of a metabolomic subset, such as amino acids, may yield useful information, and that correlation-based analyses could be useful in the analysis of metabolomic data to determine which metabolites may be responsible for the effects of excessive intakes of amino acids. The use of correlation-based analyses would allow the use of arbitrarily scaled pseudoquantitative data, making it possible to analyze unidentified peaks, which may be observed when the object sample has gone through chromatographic analysis, together with known ones. We suggest that these basic concepts and methods could serve as the basis for a metabolomic approach for the assessment of the range of adequacy of amino acid intakes.

Invivo treatment with erythroid differentiation factor (EDF / activin a) increases erythroid precursors (CFU-E and BFU-E) in mice

The in vivo effect of human EDF on erythroid precursors (CFU-E and BFU-E) was investigated in normal and bled mice. In anemic (bled) mice, EDF treatment led to significant dose-dependent rises in the CFU-E and BFU-E levels of bone marrow. An elevation in the level of CFU-E was also seen in the spleen. In normal mice, a significant elevation in the level of bone marrow BFU-E was observed. Thus, EDF has an effect on erythropoiesis in anemic and normal mice in vivo.

Leucine and Protein Metabolism in Obese Zucker Rats

Branched-chain amino acids (BCAAs) are circulating nutrient signals for protein accretion, however, they increase in obesity and elevations appear to be prognostic of diabetes. To understand the mechanisms whereby obesity affects BCAAs and protein metabolism, we employed metabolomics and measured rates of [1-14C]-leucine metabolism, tissue-specific protein synthesis and branched-chain keto-acid (BCKA) dehydrogenase complex (BCKDC) activities. Male obese Zucker rats (11-weeks old) had increased body weight (BW, 53%), liver (107%) and fat (∼300%), but lower plantaris and gastrocnemius masses (−21–24%). Plasma BCAAs and BCKAs were elevated 45–69% and ∼100%, respectively, in obese rats. Processes facilitating these rises appeared to include increased dietary intake (23%), leucine (Leu) turnover and proteolysis [35% per g fat free mass (FFM), urinary markers of proteolysis: 3-methylhistidine (183%) and 4-hydroxyproline (766%)] and decreased BCKDC per g kidney, heart, gastrocnemius and liver (−47–66%). A process disposing of circulating BCAAs, protein synthesis, was increased 23–29% by obesity in whole-body (FFM corrected), gastrocnemius and liver. Despite the observed decreases in BCKDC activities per gm tissue, rates of whole-body Leu oxidation in obese rats were 22% and 59% higher normalized to BW and FFM, respectively. Consistently, urinary concentrations of eight BCAA catabolism-derived acylcarnitines were also elevated. The unexpected increase in BCAA oxidation may be due to a substrate effect in liver. Supporting this idea, BCKAs were elevated more in liver (193–418%) than plasma or muscle, and per g losses of hepatic BCKDC activities were completely offset by increased liver mass, in contrast to other tissues. In summary, our results indicate that plasma BCKAs may represent a more sensitive metabolic signature for obesity than BCAAs. Processes supporting elevated BCAA]BCKAs in the obese Zucker rat include increased dietary intake, Leu and protein turnover along with impaired BCKDC activity. Elevated BCAAs/BCKAs may contribute to observed elevations in protein synthesis and BCAA oxidation.

Leucine‐nitrogen metabolism in the brain of conscious rats: its role as a nitrogen carrier in glutamate synthesis in glial and neuronal metabolic compartments

The source of nitrogen (N) for the de novo synthesis of brain glutamate, glutamine and GABA remains controversial. Because leucine is readily transported into the brain and the brain contains high activities of branched‐chain aminotransferase (BCAT), we hypothesized that leucine is the predominant N‐precursor for brain glutamate synthesis. Conscious and unstressed rats administered with [U‐13C] and/or [15N]leucine as additions to the diet were killed at 0–9 h of continuous feeding. Plasma and brain leucine equilibrated rapidly and the brain leucine‐N turnover was more than 100%/min. The isotopic dilution of [U‐13C]leucine (brain/plasma ratio 0.61 ± 0.06) and [15N]leucine (0.23 ± 0.06) differed markedly, suggesting that 15% of cerebral leucine‐N turnover derived from proteolysis and 62% from leucine synthesis via reverse transamination. The rate of glutamate synthesis from leucine was 5 µmol/g/h and at least 50% of glutamate‐N originally derived from leucine. The enrichment of [5‐15N]glutamine was higher than [15N]ammonia in the brain, indicating glial ammonia generation from leucine via glutamate. The enrichment of [15N]GABA, [15N]aspartate, [15N]glutamate greater than [2‐15N]glutamine suggests direct incorporation of leucine‐N into both glial and neuronal glutamate. These findings provide a new insight for the role of leucine as N‐carrier from the plasma pool and within the cerebral compartments.

Involvement of activin in the regulation of bone metabolism

Osteogenic activities of activin, a member of TGF-beta superfamily, have been shown in both in vivo and in vitro studies. Local injection of activin promoted fracture healing in rat fibula fracture models. Since both activin and its receptor are expressed during fracture healing, activin would be involved in the healing process via autocrine and/or paracrine mode of action. Activin was abundantly stored also in normal bone matrix, presumably produced by osteoblasts in the process of normal bone formation. It was observed that activin was released in the culture of neonatal mouse calvaria, and the release was strongly coupled with bone resorption. Thus, activin could be involved in the regulation of bone remodeling as one of coupling factors, as was suggested for TGF-ss. Systemic administration of activin in aged ovariectomized rats, in which bone mass decreases due to uncoupling between bone resorption and formation, increased both bone mass and mechanical strength of vertebral bodies. These findings suggest physiological roles of activin in the regulation of bone formation, and further, its possible usefulness for the therapy of fracture and osteoporosis.

Screening of Toxicity Biomarkers for Methionine Excess in Rats

Although many animal studies have reported that dietary excess of methionine causes toxic changes including growth suppression and hemolytic anemia, the biochemical mechanism and biomarkers for methionine toxicity have not been well elucidated. The present study aimed to identify toxicity biomarkers from plasma metabolites in rats fed excessive methionine. Young growing rats were fed graded doses of additional methionine for 2 wk. Cluster analysis of multivariate correlations was performed on the physiological and toxicity variables with plasma metabolites detected by GC/MS, amino acid analyzer, and thiol-specific analysis. Indicative variables for hemolysis such as splenic nonheme iron content and plasma bilirubin were grouped in the same cluster as many methionine metabolites. Homocysteine and some undefined metabolites in this cluster were found to be strong discriminators between nontoxic and toxic levels of methionine intake. Product-to-precursor ratios of each methionine metabolite demonstrated that excessive methionine intake caused a marked decrease only in the ratio of cystathionine to homocysteine, suggesting that metabolism from homocysteine to cystathionine would be rate limiting in the disposal of excessive methionine. Collectively from these results, homocysteine appeared to be the most plausible biomarker to assess methionine excess as a surrogate marker both for toxicity and for setting a metabolic upper limit.