Metabolite Concentration Profiles Research Articles

Childhood Obesity Is Associated with Changes in the Serum Metabolite Profile

Objective: The human serum metabolite profile is reflective of metabolic processes, including pathophysiological changes characteristic of diseases. Therefore, investigation of serum metabolite concentrations in obese children might give new insights into biological mechanisms associated with childhood obesity. Methods: Serum samples of 80 obese and 40 normal-weight children between 6 and 15 years of age were analyzed using a mass spectrometry-based metabolomics approach targeting 163 metabolites. Metabolite concentrations and metabolite ratios were compared between obese and normal-weight children as well as between children of different pubertal stages. Results: Metabolite concentration profiles of obese children could be distinguished from those of normal-weight children. After correction for multiple testing, we observed 14 metabolites (glutamine, methionine, proline, nine phospholipids, and two acylcarnitines, p < 3.8 × 10^–4) and 69 metabolite ratios (p < 6.0 × 10^–6) to be significantly altered in obese children. The identified metabolite markers are indicative of oxidative stress and of changes in sphingomyelin metabolism, in β-oxidation, and in pathways associated with energy expenditure. In contrast, pubertal stage was not associated with metabolite concentration differences. Conclusion: Our study shows that childhood obesity influences the composition of the serum metabolome. If replicated in larger studies, the altered metabolites might be considered as potential biomarkers in the generation of new hypotheses on the biological mechanisms behind obesity.

Differences between Human Plasma and Serum Metabolite Profiles

BackgroundHuman plasma and serum are widely used matrices in clinical and biological studies. However, different collecting procedures and the coagulation cascade influence concentrations of both proteins and metabolites in these matrices. The effects on metabolite concentration profiles have not been fully characterized.Methodology/Principal FindingsWe analyzed the concentrations of 163 metabolites in plasma and serum samples collected simultaneously from 377 fasting individuals. To ensure data quality, 41 metabolites with low measurement stability were excluded from further analysis. In addition, plasma and corresponding serum samples from 83 individuals were re-measured in the same plates and mean correlation coefficients (r) of all metabolites between the duplicates were 0.83 and 0.80 in plasma and serum, respectively, indicating significantly better stability of plasma compared to serum (p = 0.01). Metabolite profiles from plasma and serum were clearly distinct with 104 metabolites showing significantly higher concentrations in serum. In particular, 9 metabolites showed relative concentration differences larger than 20%. Despite differences in absolute concentration between the two matrices, for most metabolites the overall correlation was high (mean r = 0.81±0.10), which reflects a proportional change in concentration. Furthermore, when two groups of individuals with different phenotypes were compared with each other using both matrices, more metabolites with significantly different concentrations could be identified in serum than in plasma. For example, when 51 type 2 diabetes (T2D) patients were compared with 326 non-T2D individuals, 15 more significantly different metabolites were found in serum, in addition to the 25 common to both matrices.Conclusions/SignificanceOur study shows that reproducibility was good in both plasma and serum, and better in plasma. Furthermore, as long as the same blood preparation procedure is used, either matrix should generate similar results in clinical and biological studies. The higher metabolite concentrations in serum, however, make it possible to provide more sensitive results in biomarker detection.

Occurrence of Phthalate Metabolites in Human Urine from Several Asian Countries

The occurrence of 14 phthalate metabolites was found in human urine samples collected from seven Asian countries: China, India, Japan, Korea, Kuwait, Malaysia, and Vietnam. Phthalate metabolites were found in all samples, indicating widespread exposure of humans to phthalates in these Asian countries. The highest total (the sum of 14 phthalates) phthalate metabolite concentrations were found in samples collected from Kuwait (median: 1050 ng/mL), followed in decreasing order by samples from India (389 ng/mL), China (234 ng/mL), Vietnam (133 ng/mL), Japan (120 ng/mL), Korea (117 ng/mL), and Malaysia (94.9 ng/mL). The creatinine-adjusted median concentrations of total phthalates for urine samples from Kuwait, India, China, Vietnam, Japan, Korea, and Malaysia were 692, 506, 289, 119, 103, 104, and 169 μg/g creatinine, respectively. Monomethyl phthalate (mMP), monoethyl phthalate (mEP), mono (2-isobutyl phthalate) (miBP), mono-n-butyl phthalate (mBP), and metabolites of di-(2-ethylhexyl) phthalate (DEHP) were the dominant compounds, collectively accounting for >95% of the total concentrations in the samples from the seven countries. The profiles of urinary phthalate metabolite concentrations varied among the samples collected from the seven countries. Urine samples from Kuwait contained the highest concentrations of mEP (median: 391 ng/mL), mBP (94.1 ng/mL), and the metabolites of DEHP (202 ng/mL), whereas samples from China and Japan contained the highest concentrations of miBP (50.8 ng/mL) and mMP (17.5 ng/mL), respectively. mEP was the predominant metabolite in urine samples from India and Kuwait (accounting for 49% of the total), mBP and miBP were the predominant compounds in samples from China (52%), and DEHP metabolites were the predominant compounds in samples from Korea (46%) and Vietnam (52%). Based on the urinary concentrations of mEP, mBP, miBP, and DEHP metabolites of the samples from the seven Asian countries, we estimated daily intake rates of diethyl phthalate (DEP), dibutyl phthalate (DBP), and DEHP. The results indicated that people in the seven Asian countries are exposed to DEP, DBP, and DEHP at levels well below the reference doses (RfD) suggested as unsafe by the U.S. Environmental Protection Agency (EPA). The estimated exposure doses to DEHP in Kuwait, however, were above the RfD recommended by the EPA.

Determination of the Intracellular Concentrations of Metabolites in Escherichia coli Collected during the Exponential and Stationary Growth Phases using Liquid Chromatography-Mass Spectrometry

In the present study, we demonstrate that SRM LC-MS/MS method developed by Luo et al. (ref. 10) can be successfully applied to the quantitative analysis of intracellular metabolites in E. coli that are collected at the exponential and statio- nary growth phases. A focus is given on measuring the changes in the concentrations of intracellular metabolites in batch cultures, which were induced during both the dynamically changing exponential and stationary growth phases. The following intracellular metabolites are quantified in the exponential and stationary phases of E. coli growth, using the SRM mode of a triple quadrupole mass spectrometer: glucose-1-phosphate, fructose-1,6-bisphosphate, phos- phoenolpyruvate, pyruvate, acetyl-coenzyme A, 6-phosphogluconate, ribulose-5-phosphate, xylulose-5-phosphate, erythrose-4-phosphate. The determined intracellular metabolite concentration profiles are shown to be in a good agree- ment with the growth profiles of E. coli, which clearly indicates that SRM LC-MS/MS can be successfully used for following the metabolite changes induced at different growth stages.

Climate change and tropical marine agriculture

The coral reef ecosystem forms part of a 'seascape' that includes land-based ecosystems such as mangroves and forests, and ideally should form a complete system for conservation and management. Aquaculture, including artisanal fishing for fish and invertebrates, shrimp farming, and seaweed farming, is a major part of the farming and gleaning practices of many tropical communities, particularly on small islands, and depends upon the integrity of the reefs. Climate change is making major impacts on these communities, not least through global warming and high CO(2) concentrations. Corals grow within very narrow limits of temperature, provide livelihoods for millions of people in tropical areas, and are under serious threat from a variety of environmental and climate extremes. Corals survive and grow through a symbiotic relationship with photosynthetic algae: zooxanthellae. Such systems apply highly co-operative regulation to minimize the fluctuation of metabolite concentration profiles in the face of transient perturbations. This review will discuss research on how climate influences reef ecosystems, and how science can lead to conservation actions, with benefits for the human populations reliant on the reefs for their survival.

Photosynthetic metabolism of C 3 plants shows highly cooperative regulation under changing environments: A systems biological analysis

We studied the robustness of photosynthetic metabolism in the chloroplasts of C(3) plants under drought stress and at high CO(2) concentration conditions by using a method called Minimization of Metabolic Adjustment Dynamic Flux Balance Analysis (M_DFBA). Photosynthetic metabolism in the chloroplasts of C(3) plants applies highly cooperative regulation to minimize the fluctuation of metabolite concentration profiles in the face of transient perturbations. Our work suggests that highly cooperative regulation assures the robustness of the biological system and that there is closer cooperation under perturbation conditions than under normal conditions. This results in minimizing fluctuations in the profiles of metabolite concentrations, which is the key to maintaining a system's function. Our methods help in understanding such phenomena and the mechanisms of robustness for complex metabolic networks in dynamic processes.

Metabolite-based clustering and visualization of mass spectrometry data using one-dimensional self-organizing maps

BackgroundOne of the goals of global metabolomic analysis is to identify metabolic markers that are hidden within a large background of data originating from high-throughput analytical measurements. Metabolite-based clustering is an unsupervised approach for marker identification based on grouping similar concentration profiles of putative metabolites. A major problem of this approach is that in general there is no prior information about an adequate number of clusters.ResultsWe present an approach for data mining on metabolite intensity profiles as obtained from mass spectrometry measurements. We propose one-dimensional self-organizing maps for metabolite-based clustering and visualization of marker candidates. In a case study on the wound response of Arabidopsis thaliana, based on metabolite profile intensities from eight different experimental conditions, we show how the clustering and visualization capabilities can be used to identify relevant groups of markers.ConclusionOur specialized realization of self-organizing maps is well-suitable to gain insight into complex pattern variation in a large set of metabolite profiles. In comparison to other methods our visualization approach facilitates the identification of interesting groups of metabolites by means of a convenient overview on relevant intensity patterns. In particular, the visualization effectively supports researchers in analyzing many putative clusters when the true number of biologically meaningful groups is unknown.

Investigations of structure and metabolism within Shewanella oneidensis MR-1 biofilms

Biofilms possess spatially and temporally varying metabolite concentration profiles at the macroscopic and microscopic scales. This results in varying growth environments that may ultimately drive species diversity, determine biofilm structure and the spatial distribution of the community members. Using non-invasive nuclear magnetic resonance (NMR) microscopic imaging/spectroscopy and confocal imaging, we investigated the kinetics and stratification of anaerobic metabolism within live biofilms of the dissimilatory metal-reducing bacterium Shewanella oneidensis strain MR-1. Biofilms were pre-grown using a defined minimal medium in a constant-depth film bioreactor and subsequently transferred to an in-magnet sample chamber under laminar flow for NMR measurements. Biofilms generated in this manner were subjected to changing substrate/electron acceptor combinations (fumarate, dimethyl sulfoxide, and nitrate) and the metabolic responses measured. Localized NMR spectroscopy was used to non-invasively measure hydrogen-containing metabolites at high temporal resolution (4.5 min) under O 2-limited conditions. Reduction of electron acceptor under anaerobic conditions was immediately observed upon switching feed solutions indicating that no gene induction (transcriptional response) was needed for MR-1 to switch metabolism from O 2 to fumarate, dimethyl sulfoxide or nitrate. In parallel experiments, confocal microscopy was used with constitutively expressed fluorescent reporters to independently investigate changes in population response to the availability of electron acceptor and to probe metabolic competition under O 2-limited conditions. A clearer understanding of the metabolic diversity and plasticity of the biofilm mode of growth as well as how these factors relate to environmental fitness is made possible through the use of non-invasive and non-destructive techniques such as described herein.

Prediction Methods for Nicotine Clearance Using Cotinine and 3-Hydroxy-Cotinine Spot Saliva Samples II. Model Application

To develop and compare methods that predict individual nicotine (NIC) clearance, which reflects CYP2A6 activity, using random saliva cotinine (COT) and trans 3'-hydroxycotinine (3HC) measurements. COT and 3HC saliva concentrations in smokers were simulated utilizing a mechanistic population pharmacokinetic model of NIC metabolism that was adapted from the one described in a companion paper. Four methods to predict NIC clearance using the metabolites concentrations were compared. The precision bias, and the fraction of predictions that are made with an absolute error below 25% were the performance measures evaluated. Four prediction methods were compared: (M1) reference method, an intercept slope model of the metabolite concentration ratios ([3HC]/[COT]) (M2) an intercept slope model of the natural logarithm of the metabolite ratios (M3) a spline of the logarithm of the metabolite ratios (M4) Maximal Posteriori Bayesian estimate of NIC clearance conditioned on the model, COT and 3HC concentrations. In addition, the effect of smoking patterns on the concentrations of COT and 3HC was evaluated. The precision, accuracy, and the fraction of predictions with an absolute error below 25%, were higher for methods M2-M4 compared to method M1. However, the differences between M2 and M4 were small. Additionally, smoking pattern did not affect the metabolite concentration profiles. Predicting NIC clearance using an intercept slope model of the natural logarithm of the ratio of 3HC to COT appears to be a relatively simple method that is better than using the metabolite ratio directly. This method has a bias of approximately -10%, precision of approximately 60%. The fraction of estimates below an absolute error of 25% is 43%. These results support use of M2 to estimate CYP2A6 activity in smokers in the clinical setting.

In vivo kinetics with rapid perturbation experiments in Saccharomyces cerevisiae using a second-generation BioScope

We present a robust second-generation BioScope: a system for continuous perturbation experiments. Firstly, the BioScope design parameters (i.e., pressure drop, overall oxygen (O 2) and carbon dioxide (CO 2) mass transfer, mean residence time distribution and plug flow characteristics) were evaluated. The average overall mass transfer coefficients were estimated to be 1.8 E−5 m s −1 for O 2 and 0.34 E−5 m s −1 for CO 2. It was determined that the O 2/CO 2 permeable membrane accounted for 75% and 95% of the overall resistance for O 2 and CO 2, respectively. The Peclet number ( Pe) of the system was found to be >500 for liquid flow rates between 1 and 4 ml min −1, ensuring plug flow characteristics. Secondly, steady-state intracellular metabolite concentrations obtained using direct rapid sampling from the fermentor were compared with those obtained by rapid sampling via the pre-perturbation sample port of the BioScope. With both methods the same metabolite levels were obtained. Thirdly, glucose perturbation experiments were carried out directly in the fermentor as well as in the BioScope, whereby steady-state Saccharomyces cerevisiae cells from a glucose/ethanol limited chemostat were perturbed by increasing the extracellular glucose concentration from 0.11 to 2.8 mM. Intracellular and extracellular metabolite levels were measured within a time window of 180 s. It was observed that the dynamic metabolite concentration profiles obtained from both perturbations were nearly the same, with the exception of the C4 metabolites of the TCA cycle, which might be due to differences in culture age.

Modelling of calcium dynamics in brain energy metabolism and Alzheimer's disease

Functional imaging techniques play a major role in the study of brain activation by monitoring the changes in blood flow and energy metabolism. In order to interpret functional neuroimaging data better, the existing mathematical models describing the links that may exist between electrical activity, energy metabolism and hemodynamics in literature are thoroughly analyzed for their advantages and disadvantages in terms of their prediction of available experimental data. Then, these models are combined within a single model that includes membrane ionic currents, glycolysis, mitochondrial activity, exchanges through the blood–brain barrier, as well as brain hemodynamics. Particular attention is paid to the transport and storage of calcium ions in neurons since calcium is not only an important molecule for signalling in neurons, but it is also essential for memory storage. Multiple efforts have underlined the importance of calcium dependent cellular processes in the biochemical characterization of Alzheimer's disease (AD), suggesting that abnormalities in calcium homeostasis might be involved in the pathophysiology of the disease. The ultimate goal of this study is to investigate the hypotheses about the physiological or biochemical changes in health and disease and to correlate them to measurable physiological parameters obtained from functional neuroimaging data as in the time course of blood oxygenation level dependent (BOLD) signal. When calcium dynamics are included in the model, both BOLD signal and metabolite concentration profiles are shown to exhibit temporal behaviour consistent with the experimental data found in literature. In the case of Alzheimer's disease, the effect of halved cerebral blood flow increase results in a negative BOLD signal implying suppressed neural activity.

Metabolism of MDCK cells during cell growth and influenza virus production in large-scale microcarrier culture

The production of equine influenza in Madin–Darby canine kidney (MDCK) cells in large-scale microcarrier culture is described with detailed on- and off-line analytical data during cell growth and virus replication. Metabolite concentration profiles for glucose, glutamine, lactate and ammonium are shown. Lactate and ammonium concentrations were always below inhibiting levels. Concentration profiles for essential and non-essential amino acids of the cell culture medium are discussed. During cell growth proline was released into the medium with a significant rate while two amino acids, serine and methionine were almost depleted. After infection, virus titer increased after a delay of 10–16 h whereas first changes in amino acid metabolism could be observed within 4 h post-infection. Here, glutamate and aspartate increase correlated to virus release kinetics, indicating cell disruption and apoptosis. Starting with a moi of 0.025 resulted in a maximum virus yield of 2.4log HA/100 μl at 44 h post-infection.

Expansion dynamics, metabolite composition and substance transfer of the primary root growth zone ofZea maysL. grown in different external nutrient availabilities

ABSTRACTA combined analysis of growth and metabolite composition was performed in primary roots ofZea maysL. (Var. Alexander). The seedlings were hydroponically cultivated either in pure water or in complete nutrient solution. The overall root growth performance was similar in both treatments. Yet, digital image sequence processing methods resolved, that growth distribution and oscillatory movements within the growth zone depended strongly on external nutrient availability. Metabolite concentration profiles were similar in both treatments for most investigated metabolites, indicating a thorough mobilization of nutrient resources from the seed, but concentrations of glutamine, glutamic acid, NO3–, NH4+, malate and citrate showed pronounced differences between treatments. No diurnal variations in metabolite concentrations were found. Deposition rate profiles were in general more similar to relative elemental growth rate profiles than concentration profiles and were not affected by the treatment. Major ions were deposited maximally in front of the centre of growth activity, while greatest hexose deposition was found behind that. Relative to their abundance in the root growth zone, net rates of transfer from mature tissue were highest for sucrose, glutamic acid and aspartic acid, whereas glucose, fructose and most amino acids inversely showed high net rates of transfer out of the root growth zone, indicating a high catabolic rate for those substances there. NO3–, but not other nutrients, was transferred to a great extent from the root growth zone to the mature tissue in nutrient solution. Overall, the results show, that a careful analysis of growth dynamics allows quantifying and interpreting a number of important flux parameters in the growing organ and that the performance of the primary root does not depend strongly on external nutrient availability.

Continuous stable production of von Willebrand Factor monoclonal antibody in spin filter bioreactor with bleeding technology

The characteristics of two different modes of perfusion culture, intermittent and continuous bleedings, were investigated by culturing the hybridoma cells producing von Willebrand Factor (vWF) monoclonal antibody (McAb) in a 15 L bioreactor without clogging the filter. Both culture methods exhibited similar profiles of cell density and metabolite concentrations during the culture period at the cell concentration of around 1×107 cells/mL. When the perfusion rate was increased, the intermittent bleeding culture showed problems of ammonia accumulation and decrease of cell viability. The continuous bleeding culture exhibited higher physiological activity than that of the intermittent bleeding culture in terms of nutrient consumption and metabolite production kinetics. But the analysis of specific oxygen consumption rate showed that the specific oxygen consumption rate of intermittent bleeding culture was similar to that of exponential growth phase. The continuous bleeding culture showed higher specific vWF McAb productivity and cumulative production than those of the intermittent bleeding culture. Finally we proved the possibility of long-term operation of continuous bleeding culture and produced approximately 40 g of vWF McAb in a 15 L bioreactor after one-month operation.

Effect of cold exposure on profiles of metabolic and endocrine responses and on responses to feeding and arginine injection in sheep1

The effect of cold exposure (0 degrees C) on profiles of total heat production (HP), energy metabolism, and blood metabolite and hormone concentrations were measured in nine shorn Suffolk rams. Blood metabolite and hormone responses to feeding and to i.v. arginine injection (.625 mmol/kg BW) and postprandial changes in HP were also measured. Heat production was greater (P < .05) during cold exposure than in the thermoneutral environment due to enhanced (P < .05) nonprotein oxidation. Protein oxidation and nitrogen balance remained unchanged during cold exposure. Plasma glucagon concentrations increased (P < .05) during the initial period of cold exposure. Heat production increased (P < .01) after the initiation of feeding in both environments. Heat production returned gradually to prefeeding values in the thermoneutral environment, but it remained close to the higher levels during cold exposure. Plasma insulin and glucagon concentrations increased (P < .05), and plasma growth hormone concentrations decreased (P < .10) after the initiation of feeding in both environments. Plasma glucagon responses to feeding (P < .05) and plasma insulin responses to arginine injection (P < .01) were reduced by cold exposure. We suggest that 1) enhanced HP in sheep exposed to cold is maintained by enhanced nonprotein oxidation and 2) endocrine responses to stimulants are influenced by cold exposure, even though profiles after cold exposure change little.

Tissue engineering of a bioartificial pancreas: modeling the cell environment and device function.

Cell-based implantable artificial tissues are most promising for the long-term treatment of endocrine diseases, such as diabetes. One type of a bioartificial pancreas device consists of calcium alginate microbeads containing insulin-secreting cells and is surrounded by a poly(L-lysine) (PLL) membrane. The membrane is semipermeable, allowing cellular nutrients and metabolites to diffuse through but excluding the antibodies and cytotoxic cells of the host, thus immunoprotecting the cells. The device can be modeled by writing the equations for diffusion of nutrients and metabolites through the polymer and for consumption of the former and production of the latter by the cells. In this paper, we describe the construction and analysis of such a model for alginate/PLL microbeads with insulin-secreting recombinant mouse pituitary AtT-20 and mouse insulinoma beta TC3 cells. Entrapped AtT-20 cells are a simplified model system, whereas microbeads with beta TC3 cells constitute a realistic artificial pancreatic device. Effective diffusivities of key compounds through the polymer with entrapped, inactivated AtT-20 spheroids were measured first. The kinetics of glucose and oxygen consumption and insulin secretion were modeled next, and the equations for diffusion and reaction were then combined to describe the entire system. The model was used to compute nutrient and metabolite concentration profiles in beads and the bead secretory response for different bead sizes and cell loadings. The size and loading necessary for the cells to be well nourished and for the beads to be rapidly responsive to step-ups and step-downs of secretion stimuli were evaluated. It was shown that if the cells are hypersensitive to glucose, i.e., they do not shut off secretion at the physiological glucose threshold but at a lower one, so are the microbeads. This work demonstrates the usefulness of mechanistic models with representative parameter values in optimizing the design of artificial tissues and in characterizing aspects of their behavior that are of importance for restoring in vivo function.

Variations in the nutrition of the canine intervertebral disc induced by motion.

In this experiment study, we have investigated some transport and metabolic parameters of the canine intervertebral disc induced by spinal motion. The movements of the spine were obtained during supervised exercise, where either moderate, violent, or specific movements were performed. The results show changes in the metabolic rates as well as in concentration profiles of metabolites of the exercised groups in comparison to the control group. An increasing aerobic metabolism was noted in the outer part of the annulus and in the central part of the nucleus pulposus, resulting in a reduction of the lactate concentration. These findings do indicate that spinal movements, over a longer period of time give rise to positive nutritional variations. Furthermore, the reported changes might be of significance also for the human lumbar disc problem, since previous studies have demonstrated nutritional similarities between the discs of dogs and men.