瘦素水平与代谢综合症的关系

随着代谢综合症在世界范围内的广为流行,已经引起人们的高度重视.代谢综合征以肥胖和代谢异常为特征,胰岛素抵抗为主要的病理机制.瘦素主要来源于脂肪组织,是调节体内脂肪储量和维持能量平衡的一种内分泌激素.瘦素缺乏和瘦素抵抗不仅可以直接引起胰岛素抵抗,而且可以通过导致肥胖继而参与胰岛素抵抗的发生,最终引起代谢综合征.瘦素作为一种新的代谢综合征致病因子,参与代谢综合征的发生发展,故调节瘦素水平为临床治疗代谢综合症提供了新的思路和方法.本文综述了瘦素水平与代谢综合症的关系,以及调节瘦素水平治疗代谢综合征的方法.     

Effect of aeration intensity on the biochemical composition of baker's yeast. I. Factors affecting the type of metabolism

Efforts were made to eliminate the influence of other factors as far as possible in order to obtain reliable results on the effects of oxygen on the growth of baker's yeast. A cultivation method is presented which permits the study of the effects of aeration intensity under conditions where the influence of catabolite repression is eliminated. A completely synthetic medium with glucose as the only carbon and energy source is also described. The capacity of yeast to perform aerobic metabolism varies when cultivated under different intensities of aeration. A clear maximum is observed for growth with 10% oxygen in the aerating gas mixture. Under conditions where catabolite repression does not function yeast has the potential for oxidative metabolism even under oxygen-limited growth. The main agent controlling the ability of yeast to support growth using only the oxidative metabolism is the available oxygen. At high oxygen tensions the metabolism is disturbed.     

Novel multi-mode ultra performance liquid chromatography-tandem mass spectrometry assay for profiling enantiomeric hydroxywarfarins and warfarin in human plasma

Coumadin (R/S-warfarin) is a commonly prescribed anticoagulant for over ～20 million Americans. Although highly efficacious, positive clinical outcomes during warfarin therapy depend on maintaining a narrow therapeutic range for the drug. This goal is challenging due to large inter-individual variability in patient response, which has been attributed to diversity in drug metabolism. Warfarin is given as a racemic mixture and evidence suggest differences of R and S-warfarin in their therapeutic activities and metabolism. Previous investigation of warfarin metabolism has been hampered by the inability to quantify the individual enantiomers. To overcome this limitation a multi-mode LC-MS/MS method is reported. This strategy combines phenyl based reverse phase chromatography with chiral phase chromatography prior to quantitation by liquid chromatography tandem mass spectrometry. This approach was made possible through advances in UPLC technology producing narrow peaks suitable for transferring to a second column. The reported method separated individual R and S enantiomers of hydroxywarfarin and warfarin. All four possible isomers of 10-hydroxywarfarin were resolved to reveal unprecedented insights into the stereo-specific metabolism of warfarin. Characterization of the method demonstrated that it is robust and sensitive with inter-day coefficients of error between <7% and a detection limit of 2 nM in sample or 10 fmol on column for each analyte. Individual metabolites may be suitable surrogate biomarkers or predictive markers that predict warfarin dose, adverse interactions, or other important clinical outcomes during anticoagulant therapy. Consequently, the metabolite profiles obtained through this dual phase UPLC-MS/MS method are expected to increase our understanding of the role warfarin metabolism plays in patient response to therapy and yield new strategies to improve patient outcomes.     

Comparison of several methods to calculate reaeration in streams,and their effects on estimation of metabolism

Metabolism is an integrative measurement of stream and river ecosystem functioning, and thus, could be used to assess impairment. Stream metabolism is measured by different methods which often yield contrasting results. Furthermore, open-channel measurements of metabolism, which offer the best potential for continuous monitoring of stream functioning, rely on calculations of gas exchange with the atmosphere, for which a plethora of methods exists. Therefore, to incorporate metabolism in stream monitoring programs, it is necessary to determine which methods yield comparable results under a given set of environmental conditions. We studied 21 streams in the Basque Country (northern Spain), ranging widely in physical characteristics and water quality. We calculated reaeration during summer baseflows using three different approaches: the night-time drop in oxygen, the lag between noon and peak oxygen concentration, and ten empirical equations relating depth and velocity with reaeration coefficients obtained from the literature. Differences among methods were very large, especially at the shallower sites. The results obtained with most empirical equations were highly correlated, but showed little agreement with the night-time and peak lag methods. We then analyzed the response of reaeration rate to river stage: reaeration calculated by the night-time method during 1 year of continuous monitoring was regressed against discharge at each site, and the resulting model was compared to the results of empirical equations, using software HecRas 2.2 to model hydraulic conditions at different river stages. The shape of reaeration-discharge plots differed greatly and in a site-dependent manner, and there was little agreement between methods. Finally, we investigated the effects of reaeration rate on estimates of metabolism. The choice of method greatly affected the estimates of both primary production and respiration. The empirical equations, except E₇ and E_10, yielded the most unrealistic estimations of stream metabolism. Overall, the night-time method, especially when regressed against discharge, seems to be the most robust and reliable among those tested, with the energy dissipation method (E₁₀) appearing to be a viable alternative when the night-time method does not work.     

Engineering a living cell to desired metabolite concentrations and fluxes: pathways with multifunctional enzymes

With molecular genetics enabling modulation of the concentrations of cellular enzymes, metabolic engineering becomes limited by the question of which modulations of the enzyme concentrations are required to bring about a desired pattern of cellular metabolism. In an earlier paper (Kholodenko et al. (1998). Biotechnol. Bioeng. 59, 239-247) we derived a method to determine the required modulations. This method, however, cannot be immediately applied to cellular pathways with enzymes catalyzing more than one step in metabolism (multifunctional enzymes). In the present paper we show to which extent the presence of multifunctional enzymes limits biotechological ambitions, which one might otherwise pursue in vain. In particular, it is impossible to change the concentration of a single intermediate and leave the rest of metabolism unperturbed if that intermediate interacts directly with a multifunctional enzyme. The analytical machinery of Metabolic Control Analysis is used to relate the desired and ensuing changes in the metabolic pattern. An explicit solution to this problem of engineering metabolism is then given in the form of a single matrix equation.     

降钙素与骨质疏松的治疗

骨质疏松症是常见的老年性疾病,日前尚无有效的预防和治疗手段。降钙素是一种多肽激素,能调节体内钙的代谢,对骨质疏松症有一定的疗效。本文简要阐述了降钙素用于治疗骨质疏松症的机理、方法、效果以及应用前景。     

Measurement of fast dynamic intracellular pH in Saccharomyces cerevisiae using benzoic acid pulse

pH affects many processes on cell metabolism, such as enzyme kinetics. To enhance the understanding of the living cells, it is therefore indispensable to have a method to monitor the pH in living cells. To accomplish this, a dynamic intracellular pH measurement method applying low concentration benzoic acid pulse was developed. The method was thoroughly validated and successfully implemented for measuring fast dynamic intracellular pH of Saccharomyces cerevisiae in response to a glucose pulse perturbation performed in the BioSCOPE set-up. Fast drop in intracellular pH followed by partial alkalinization was observed following the pulse. The low concentration benzoic acid pulse which was implemented in the method avoids the undesirable effects that may be introduced by benzoic acid to cell metabolism.     

Mathematical modeling of living cell metabolism using the method of steady-state stoichiometric flux balance

This approach uses a set of algebraic linear equations for reaction rates (the method of steady-state stoichiometric flux balance) to model the purposeful metabolism of the living self-reproducing biochemical system (i.e. cell), which persists in steady-state growth. Linear programming (SIMPLEX method) is used to derive the solution for the model equations set (determining reaction rates which provide flux balance at given conditions). Here, we demonstrate the approach through the mathematical modeling of steady-state metabolism in Saccharomyces cerevisiae mitochondria.     

物质代谢及其资源环境效应研究进展

从物质代谢的视角研究物质在社会经济系统中的流通过程,不仅能深入理解人类活动与自然环境之间的关系,更有助于实现资源的合理利用和可持续发展.本文在对国内外有关物质代谢的研究方法、在不同空间尺度的应用、资源和环境效应的研究进展进行综述的基础上,较全面的对上述研究中已取得的成果以及尚待完善之处进行总结.物质代谢研究应从过去的单一理论和方法向多学科和方法交叉融合的方向发展,以解决复杂的代谢问题.随着城市在全球生态环境中的重要性日益凸显,应加强传统的代谢研究与地理空间分析的结合,并将人类福祉等评价指标纳入物质代谢的研究范畴,进而从物质代谢的视角提出节约资源、降低环境负荷、提高环境-经济-社会协调发展的可持续管理对策.     

钙、磷摄入对骨代谢及骨组织形态影响的研究进展

骨代谢始终贯穿于动物的生命过程之中,而机体摄入的钙、磷水平和钙、磷比例又是骨代谢的重要影响因素。钙、磷摄入水平和比例的改变会使甲状旁腺激素（PTH）、降钙素（CT）和1α,25-双羟维生素D3[1,25-（OH）2D3]的水平产生变化,影响RANK／RANKL／OPG系统对骨细胞功能的调控,进而对骨代谢及骨组织形态产生影响。骨唾液酸蛋白（Bone Sialoprotein,BSP）是目前评价成骨细胞分化和骨骼矿化情况的新指标,研究BSP表达水平的变化规律,可以从分子水平解释钙、磷摄入水平和比例对骨组织形态影响的机理。而骨组织形态计量学（Bone Histomorphometry）则是研究钙、磷摄入水平和比例对骨组织形态影响的重要方法。     

Tryptophan 2,3-dioxygenase in chick embryo hepatocytes: studies in ovo and in culture

Tryptophan dioxygenase is a hemoprotein in its active form, which has a relatively low affinity for heme. From previous studies in rats, the ratio of holoenzyme/total enzyme activity of tryptophan dioxygenase has been proposed to reflect the size of a "free" heme pool in hepatocytes. Chick embryo hepatocytes in ovo and in culture are other systems that have proven useful for study of hepatic heme metabolism and its control. Heretofore, there have been few studies of tryptophan dioxygenase activity in chick embryo hepatocytes. As part of studies on hepatic heme metabolism, using two different assays, we have measured tryptophan dioxygenase activity and percentage of heme saturation of the enzyme in chick embryo livers cells in ovo and in culture. One method of assay relies on endogenous formamidase to generate the final product, kynurenine, which is measured directly, whereas the other method uses a chemical hydrolysis step to form kynurenine which is further diazotized prior to measurement. The latter method is shown to be preferable for studies with chick embryo hepatocytes. In addition, we show that (i) tryptophan dioxygenase activity is present and can be increased by tryptophan and phenobarbital-like drugs in chick embryo hepatocytes in ovo; (ii) total enzyme activity falls markedly in cultured hepatocytes despite the presence of high concentrations of glucocorticoids in the culture medium; and (iii) under all conditions studied thus far in the cultures, the enzyme is nearly saturated with heme. Results are discussed in relation to regulation of heme metabolism in chick embryo hepatocytes.     

Bioenergetic and proteomic profiling to screen small molecule inhibitors that target cancer metabolisms

Cancer cells can reprogram their metabolic machinery to survive. This altered metabolism, which is distinct from the metabolism of normal cells, is thought to be a possible target for the development of new cancer therapies. In this study, we constructed a screening system that focuses on bioenergetic profiles (specifically oxygen consumption rate and extracellular acidification rate) and characteristic proteomic changes. Thus, small molecules that target cancer-specific metabolism were investigated. We screened the chemical library of RIKEN Natural Products Depository (NPDepo) and found that unantimycin A, which was recently isolated from the fraction library of microbial metabolites, and NPL40330, which is derived from a chemical library, inhibit mitochondrial respiration. Furthermore, we developed an in vitro reconstitution assay method for mitochondrial electron transport chain using semi-intact cells with specific substrates for each complex of the mitochondrial electron transport chain. Our findings revealed that NPL40330 and unantimycin A target mitochondrial complexes I and III, respectively.     

A thin-layer chromatographic method for separation of thymidine and deoxyuridine nucleotides

A thin-layer chromatographic method for the separation of thymidine and deoxyuridine nucleotides and nucleosides is described. This procedure involves the following sequence of steps: (i) Ion-exchange thin-layer chromatography to afford separation into fractions of increasing degree of phosphorylation, (ii) conversion of each fraction into an equivalent mixture of thymine and uracil through the combined actions of alkaline phosphatase and thymidine phosphorylase, and (iii) partition thin-layer chromatographic separation of thymine and uracil. A key feature of the method is the specificity afforded by the second step which converts only thymidine and deoxyuridine nucleotides and nucleosides to the corresponding pyrimidine bases. An application of the method to the study of [³H]deoxyuridine metabolism in L1210 cells, as well as the effect of methotrexate on this metabolism is also described.     

Circadian Variation of the Human Metabolome Captured by Real-Time Breath Analysis

Circadian clocks play a significant role in the correct timing of physiological metabolism, and clock disruption might lead to pathological changes of metabolism. One interesting method to assess the current state of metabolism is metabolomics. Metabolomics tries to capture the entirety of small molecules, i.e. the building blocks of metabolism, in a given matrix, such as blood, saliva or urine. Using mass spectrometric approaches we and others have shown that a significant portion of the human metabolome in saliva and blood exhibits circadian modulation; independent of food intake or sleep/wake rhythms. Recent advances in mass spectrometry techniques have introduced completely non-invasive breathprinting; a method to instantaneously assess small metabolites in human breath. In this proof-of-principle study, we extend these findings about the impact of circadian clocks on metabolomics to exhaled breath. As previously established, our method allows for real-time analysis of a rich matrix during frequent non-invasive sampling. We sampled the breath of three healthy, non-smoking human volunteers in hourly intervals for 24 hours during total sleep deprivation, and found 111 features in the breath of all individuals, 36–49% of which showed significant circadian variation in at least one individual. Our data suggest that real-time mass spectrometric "breathprinting" has high potential to become a useful tool to understand circadian metabolism, and develop new biomarkers to easily and in real-time assess circadian clock phase and function in experimental and clinical settings.     

Metabolic analysis of four phenolic acids in rat by liquid chromatography-tandem mass spectrometry

A liquid chromatography-diode array detection-electrospray ionization ion trap mass spectrometry (LC-DAD-ESI-MS(n)) method was established for the analysis of danshensu, caffeic acid, ferulic acid and isoferulic acid in rat plasma, bile, urine and feces after oral administration or intravenous injection. Liquid-liquid extraction was employed for the preparation of biosamples, and the chromatographic separation was carried out using an Agilent Zorbax Extend C(18) reversed phase column and acetonitrile-0.1% formic acid as the mobile phase. Totally nineteen metabolites were detected and identified as prototype, methylated, hydroxylated, sulfated and glucuronized conjugates. The metabolism of the individual phenolic acids in biosamples was investigated, and the metabolic pathway was proposed. By comparing the metabolism of different compounds which shared similar structures, we were able to find that methylation was the main pathway of danshensu metabolism, and the double bond on the side chain was critical for the drug excretion via bile and the formation of glucuronized conjugates. The results proved that the established method was simple, sensitive and reliable, which could be used to detect and identify the structures of metabolites and to better understand their in vivo metabolism.     

Activation of calcium transport in synaptosomes by phosphatidylinositol-specific phospholipase C

The isotope labeling method was used to study the influence of phospholipases C of different origin and specificity on Ca2+ accumulation in rat brain synaptosomes. It was found that phospholipases C specific to phosphatidylinositides (PI) stimulate Ca2+ transport into synaptosomes, while non-specific phospholipase C, which hydrolyzes different membrane lipid fractions, decreases the Ca2+ content in synaptosomes. It is supposed that the stimulating effect of PI-specific phospholipases C is determined by the activation of PI metabolism, which results in an increase in the content of some PI metabolism products serving as Ca2+ ionophores in synaptosomal membranes. The inhibition of Ca2+ uptake by synaptosomes treated with non-specific phospholipase C is thought to result from partial disruption of synaptosomal membranes.     

On-line stoichiometry and identification of metabolic state under dynamic process conditions

A method for the on-line calculation of conversion rates and yield coefficients under dynamic process conditions was developed. The method is based on cumulated mass balances using a moving average method. Elemental balances were used to test the measured cumulated quantities for gross errors and inappropriate stoichiometry definition followed by data reconciliation and estimation of non-measured conversion rates, using a bioprocess set-up including multiple on-line analysis techniques. The quantitative potential of the proposed method is demonstrated by executing transient experiments in aerobic cultures of Saccharomyces cerevisiae on glucose. Rates and yield coefficients could be consistently quantified in shift-up, shift-down, and accelerostat experiments. The method shows the capability to describe quantitatively transient changes in metabolism including uncoupling of catabolism and anabolism, also for the case when multiple components of metabolism are not measured. The validity of the experiment can be evaluated on-line. Additionally, the method detects with high sensitivity inappropriate stoichiometry definition, such as a change in state of metabolism. It was shown that concentration values can be misleading for the identification of the metabolic state. In contrast, the proposed method provides a clear picture of the metabolic state and new physiological regulations could be revealed. Hence, the novelty of the proposed method is the on-line availability of consistent stoichiometric coefficients allowing a significant speed up in strain characterization and bioprocess development using minimal knowledge of the metabolism. Additionally, it opens up the use of transient experiments for physiological studies.     

ACoM: A classification method for elementary flux modes based on motif finding

Elementary flux mode analysis is a powerful tool for the theoretical study of metabolic networks. However, when the networks are complex, the determination of elementary flux modes leads to combinatorial explosion of their number which prevents from drawing simple conclusions from their analysis. To deal with this problem we have developed a method based on the Agglomeration of Common Motifs (ACoM) for classifying elementary flux modes. We applied this algorithm to describe the decomposition into elementary flux modes of the central carbon metabolism in Bacillus subtilis and of the yeast mitochondrial energy metabolism. ACoM helps to give biological meaning to the different elementary flux modes and to the relatedness between reactions. ACoM, which can be viewed as a bi-clustering method, can be of general use for sets of vectors with values 0, +1 or −1.