Significant Changes In Metabolite Levels Research Articles

Association of levels of metabolites with the safe margin of rectal cancer surgery: a metabolomics study

BackgroundRectal cancer is one of the most lethal of gastrointestinal malignancies. Metabonomics has gradually developed as a convenient, inexpensive and non-destructive technique for the study of cancers.MethodsA total of 150 tissue samples from 25 rectal cancer patients were analyzed by liquid chromatography–mass spectrometry (LC–MS), and 6 tissue samples were collected from each patient (group 1: tumor; group 2: 0.5 cm from tumor; group 3:1 cm from tumor; group 4:2 cm from tumor; group 5:3 cm from tumor and group 6:5 cm from tumor). The differential metabolites of tumor tissues and 5 cm from the tumor (normal tissues) were first selected. The differential metabolites between tumor tissues and normal tissues were regrouped by hierarchical clustering analysis, and further selected by discriminant analysis according to the regrouping of clustering results. The potential safe margin of clinical T(cT)1,cT2 stage rectal cancer and cT3,cT4 stage rectal cancer at the metabolomic level was further identified by observing the changes in the level of differential metabolites within the samples from group 1 to group 6.ResultsWe found 22 specific metabolites to distinguish tumor tissue and normal tissue. The most significant changes in metabolite levels were observed at 0.5 cm (cT1, cT2) and 2.0 cm (cT3, cT4) from the tumor, while the changes in the tissues afterwards showed a stable trend.ConclusionsThere are differential metabolites between tumor tissues and normal tissues in rectal cancer. Based on our limited sample size, the safe distal incision margin for rectal cancer surgery in metabolites may be 0.5 cm in patients with cT1 and cT2 stage rectal cancer and 2.0 cm in patients with cT3 and cT4 stage rectal cancer.

Porphyromonas somerae Invasion of Endometrial Cancer Cells

Recent evidence suggests an association between endometrial cancer and the understudied bacterial species Porphyromonas somerae. This association was demonstrated in previous work that indicated a significantly enriched abundance of P. somerae in the uterine microbiome of endometrial cancer patients. Given the known associations of the Porphyromonas genus and oral cancer, we hypothesized that P. somerae may play a similar pathogenic role in endometrial cancer via intracellular activity. Before testing our hypothesis, we first characterized P. somerae biology, as current background data is limited. These novel characterizations include growth curves in liquid medium and susceptibility tests to antibiotics. We tested our hypothesis by examining growth changes in response to 17β-estradiol, a known risk factor for endometrial cancer, followed by metabolomic profiling in the presence and absence of 17β-estradiol. We found that P. somerae exhibits increased growth in the presence of 17β-estradiol of various concentrations. However, we did not find significant changes in metabolite levels in response to 17β-estradiol. To study direct host-microbe interactions, we used in vitro invasion assays under hypoxic conditions and found evidence for intracellular invasion of P. somerae in endometrial adenocarcinoma cells. We also examined these interactions in the presence of 17β-estradiol but did not observe changes in invasion frequency. Invasion was shown using three lines of evidence including visualization via differential staining and brightfield microscopy, increased frequency of bacterial recovery after co-culturing, and in silico methods to detail relevant genomic and transcriptomic components. These results underscore potential intracellular phenotypes of P. somerae within the uterine microbiome. Furthermore, these results raise new questions pertaining to the role of P. somerae in the progression of endometrial cancer.

The Effect of Soybean Oil on Lipid Metabolism in Mucor circinelloides WJ11 by Metabolomic Analysis

Oleaginous fungus Mucor circinelloides utilizes different carbon sources associated with sustainable production of lipids, using soybean oil as carbon source, the fungus accumulated more intracellular lipids. Nonetheless, the metabolic changes in M. circinelloides upon vegetable oil as main carbon source have not been yet reported. Therefore, this study was conducted to investigate the metabolomics of M. circinelloides WJ11 cultivated in mixed glucose and soybean oil as carbon source and to reveal its effects on lipid metabolism. M. circinelloides strain WJ11 was cultured under optimized conditions in a fermenter and the biomass samples were collected after 24 h for lipid metabolite investigation. The frozen biomass samples were subjected to various chromatographic analyses like liquid chromatography mass spectrometry analysis and high resolution-mass spectrometry for metabolite profiling. A total of 438 differential metabolites were identified, of which 48 were up-regulated and 33 were down-regulated. Among them, lyso-phosphatidic acid and monoglyceride were up-regulated whereas phosphatidylglycerol and lyso-phosphatidylglycerol were down-regulated in the experimental group (soybean oil and glucose as mixed carbon sources) compared with the control group (glucose as single carbon source). Significant changes in metabolite levels correlated to lipid synthesis were identified. This study showed that the addition of soybean oil to the medium favors the triacylglycerol synthesis in M. circinelloides. Our results can also be applied to the investigation of other microorganisms and would contribute to further genetic engineering for higher lipid accumulation in fungi.

Identification of Pre-Diagnostic Metabolic Patterns for Glioma Using Subset Analysis of Matched Repeated Time Points.

Simple SummaryReprogramming of cellular metabolism is a major hallmark of cancer cells, and play an important role in tumor initiation and progression. The aim of our study is to discover circulating early metabolic markers of brain tumors, as discovery and development of reliable predictive molecular markers are needed for precision oncology applications. We use a study design tailored to minimize confounding factors and a novel machine learning and visualization approach (SMART) to identify a panel of 15 interlinked metabolites related to glioma development. The presented SMART strategy facilitates early molecular marker discovery and can be used for many types of molecular data.Here, we present a strategy for early molecular marker pattern detection—Subset analysis of Matched Repeated Time points (SMART)—used in a mass-spectrometry-based metabolomics study of repeated blood samples from future glioma patients and their matched controls. The outcome from SMART is a predictive time span when disease-related changes are detectable, defined by time to diagnosis and time between longitudinal sampling, and visualization of molecular marker patterns related to future disease. For glioma, we detect significant changes in metabolite levels as early as eight years before diagnosis, with longitudinal follow up within seven years. Elevated blood plasma levels of myo-inositol, cysteine, N-acetylglucosamine, creatinine, glycine, proline, erythronic-, 4-hydroxyphenylacetic-, uric-, and aceturic acid were particularly evident in glioma cases. We use data simulation to ensure non-random events and a separate data set for biomarker validation. The latent biomarker, consisting of 15 interlinked and significantly altered metabolites, shows a strong correlation to oxidative metabolism, glutathione biosynthesis and monosaccharide metabolism, linked to known early events in tumor development. This study highlights the benefits of progression pattern analysis and provide a tool for the discovery of early markers of disease.

L-Aspartate, L-Ornithine and L-Ornithine-L-Aspartate (LOLA) and Their Impact on Brain Energy Metabolism.

L-Ornithine-L-aspartate (LOLA), a crystalline salt, is used primarily in the management of hepatic encephalopathy. The degree to which it might penetrate the brain, and the effects it might have on metabolism in brain are poorly understood. Here, to investigate the effects of LOLA on brain energy metabolism we incubated brain cortical tissue slices from guinea pig (Cavea porcellus) with the constituent amino acids of LOLA, L-ornithine or L-aspartate, as well as LOLA, in the presence of [1-13C]D-glucose and [1,2-13C]acetate; these labelled substrates are useful indicators of brain metabolic activity. L-Ornithine produced significant "sedative" effects on brain slice metabolism, most likely via conversion of ornithine to GABA via the ornithine aminotransferase pathway, while L-aspartate showed concentration-dependent excitatory effects. The metabolic effects of LOLA reflected a mix of these two different processes and were concentration-dependent. We also investigated the effect of an intraperitoneal bolus injection of L-ornithine, L-aspartate or LOLA on levels of metabolites in kidney, liver and brain cortex and brain stem in mice (C57Bl6J) 1 h later. No significant changes in metabolite levels were seen following the bolus injection of L-aspartate, most likely due to rapid metabolism of aspartate before reaching the target tissue. Brain cortex glutamate was decreased by L-ornithine but no other brain effects were observed with any other compound. Kidney levels of aspartate were increased after injection of L-ornithine and LOLA which may be due to interference by ornithine with the kidney urea cycle. It is likely that without optimising chronic intravenous infusion, LOLA has minimal impact on healthy brain energy metabolism due to systemic clearance and the blood - brain barrier.

1H MR spectroscopy of the motor cortex immediately following transcranial direct current stimulation at 7 Tesla.

Transcranial direct current stimulation (tDCS) is a form of non-invasive brain stimulation that may modulate cortical excitability, metabolite concentration, and human behaviour. The supplementary motor area (SMA) has been largely ignored as a potential target for tDCS neurorehabilitation but is an important region in motor compensation after brain injury with strong efferent connections to the primary motor cortex (M1). The objective of this work was to measure tissue metabolite changes in the human motor cortex immediately following tDCS. We hypothesized that bihemispheric tDCS would change levels of metabolites involved in neuromodulation including N-acetylaspartate (NAA), glutamate (Glu), and creatine (tCr). In this single-blind, randomized, cross-over study, fifteen healthy adults aged 21–60 participated in two 7T MRI sessions, to identify changes in metabolite concentrations by magnetic resonance spectroscopy. Immediately after 20 minutes of tDCS, there were no significant changes in metabolite levels or metabolite ratios comparing tDCS to sham. However there was a trend toward increased NAA/tCr concentration (p = 0.08) in M1 under the stimulating cathode. There was a strong, positive correlation between the change in the absolute concentration of NAA and the change in the absolute concentration of tCr (p<0.001) suggesting an effect of tDCS. Both NAA and creatine are important markers of neurometabolism. Our findings provide novel insight into the modulation of neural metabolites in the motor cortex immediately following application of bihemispheric tDCS.

Drugs with antidepressant properties affect tryptophan metabolites differently in rodent models with depression-like behavior.

The metabolism of tryptophan through kynurenine and serotonin pathways is linked to depression. Here, effects of different drugs with antidepressant properties (vortioxetine, fluoxetine, and ketamine) on various tryptophan metabolites in different brain regions and plasma were examined using tandem mass spectrometry (LC-MS/MS), in Flinders Sensitive Line rats, a genetic rat model of depression, and its controls: Flinders Sensitive Line and Sprague-Dawley rats. Protein levels of kynurenine pathway enzymes were measured in the brains and livers of these rat strains. Furthermore, effects of vortioxetine on tryptophan metabolites were assessed in the cortical regions of lupus mice (MRL/MpJ-FasIpr ), a murine model of increased depression-like behavior associated with inflammation. Sustained vortioxetine or fluoxetine (at doses aimed to fully occupy serotonin transporter via food or drinking water for at least 14days) reduced levels of the excitotoxin quinolinic acid (QUIN) in various brain regions in all rats. Furthermore, chronic vortioxetine reduced levels of QUIN in MRL/MpJ-FasIpr mice. Acute i.p. administration of fluoxetine (10mg/kg) or vortioxetine (10mg/kg) led to reduced brain 5-hydroxyindoleacetic acid in Sprague-Dawley rats (2, 4, 6, and 8h) and a similar trend was evident in Flinders Sensitive Line and Flinders Sensitive Line rats after 4h. In contrast, single or repeated administration of ketamine (15mg/kg i.p.) did not induce significant changes in metabolite levels. In conclusion, sustained vortioxetine and fluoxetine administration decreased QUIN independent of species, while ketamine was ineffective. These results support the hypothesis that modulating tryptophan metabolism may be part of the mechanism of action for some antidepressants.

Metabolic Profiles and Free Radical Scavenging Activity of Cordyceps bassiana Fruiting Bodies According to Developmental Stage

The metabolic profiles of Cordyceps bassiana according to fruiting body developmental stage were investigated using gas chromatography-mass spectrometry. We were able to detect 62 metabolites, including 48 metabolites from 70% methanol extracts and 14 metabolites from 100% n-hexane extracts. These metabolites were classified as alcohols, amino acids, organic acids, phosphoric acids, purine nucleosides and bases, sugars, saturated fatty acids, unsaturated fatty acids, or fatty amides. Significant changes in metabolite levels were found according to developmental stage. Relative levels of amino acids, purine nucleosides, and sugars were higher in development stage 3 than in the other stages. Among the amino acids, valine, isoleucine, lysine, histidine, glutamine, and aspartic acid, which are associated with ABC transporters and aminoacyl-tRNA biosynthesis, also showed higher levels in stage 3 samples. The free radical scavenging activities, which were significantly higher in stage 3 than in the other stages, showed a positive correlation with purine nucleoside metabolites such as adenosine, guanosine, and inosine. These results not only show metabolic profiles, but also suggest the metabolic pathways associated with fruiting body development stages in cultivated C. bassiana.

High-throughput NMR based metabolic profiling of Braeburn apple in relation to internal browning

NMR is a valuable tool for metabolomics due to its short analysis time and reproducibility. However, this technique remains little used due to its high cost. Recently, cheaper NMR machines for high-throughput screening have been developed. In this study, NMR was used to study the effect of several pre- and postharvest factors on apple metabolite levels during air and controlled atmosphere storage, including metabolic changes related to the incidence of internal browning. The results show that the selected fertilizer treatments and fruit side (green versus red side) did not affect the metabolite levels. However, the different postharvest storage conditions (optimal CA and brown inducing CA) did result in significant changes in metabolite levels. In addition, differences (e.g., pyruvate, citrate, fumarate, alanine, chlorogenate, methanol, ethanol, acetaldehyde and acetoine) between brown and unaffected apples stored under the applied CA conditions could be demonstrated.

LC–MS Based Serum Metabolomics for Identification of Hepatocellular Carcinoma Biomarkers in Egyptian Cohort

Although hepatocellular carcinoma (HCC) has been subjected to continuous investigation and its symptoms are well-known, early stage diagnosis of this disease remains difficult and the survival rate after diagnosis is typically very low (3-5%). Early and accurate detection of metabolic changes in the sera of patients with liver cirrhosis can help improve the prognosis of HCC and lead to a better understanding of its mechanism at the molecular level, thus providing patients with in-time treatment of the disease. In this study, we compared metabolite levels in sera of 40 HCC patients and 49 cirrhosis patients from Egypt by using ultraperformance liquid chromatography coupled with quadrupole time-of-flight mass spectrometer (UPLC-QTOF MS). Following data preprocessing, the most relevant ions in distinguishing HCC cases from cirrhotic controls are selected by statistical methods. Putative metabolite identifications for these ions are obtained through mass-based database search. The identities of some of the putative identifications are verified by comparing their MS/MS fragmentation patterns and retention times with those from authentic compounds. Finally, the serum samples are reanalyzed for quantitation of selected metabolites as candidate biomarkers of HCC. This quantitation was performed using isotope dilution by selected reaction monitoring (SRM) on a triple quadrupole linear ion trap (QqQLIT) coupled to UPLC. Statistical analysis of the UPLC-QTOF data identified 274 monoisotopic ion masses with statistically significant differences in ion intensities between HCC cases and cirrhotic controls. Putative identifications were obtained for 158 ions by mass based search against databases. We verified the identities of selected putative identifications including glycholic acid (GCA), glycodeoxycholic acid (GDCA), 3β, 6β-dihydroxy-5β-cholan-24-oic acid, oleoyl carnitine, and Phe-Phe. SRM-based quantitation confirmed significant differences between HCC and cirrhotic controls in metabolite levels of bile acid metabolites, long chain carnitines and small peptide. Our study provides useful insight into appropriate experimental design and computational methods for serum biomarker discovery using LC-MS/MS based metabolomics. This study has led to the identification of candidate biomarkers with significant changes in metabolite levels between HCC cases and cirrhotic controls. This is the first MS-based metabolic biomarker discovery study on Egyptian subjects that led to the identification of candidate metabolites that discriminate early stage HCC from patients with liver cirrhosis.

Integrated pathway modules using time-course metabolic profiles and EST data from Milnesium tardigradum

BackgroundTardigrades are multicellular organisms, resistant to extreme environmental changes such as heat, drought, radiation and freezing. They outlast these conditions in an inactive form (tun) to escape damage to cellular structures and cell death. Tardigrades are apparently able to prevent or repair such damage and are therefore a crucial model organism for stress tolerance. Cultures of the tardigrade Milnesium tardigradum were dehydrated by removing the surrounding water to induce tun formation. During this process and the subsequent rehydration, metabolites were measured in a time series by GC-MS. Additionally expressed sequence tags are available, especially libraries generated from the active and inactive state. The aim of this integrated analysis is to trace changes in tardigrade metabolism and identify pathways responsible for their extreme resistance against physical stress.ResultsIn this study we propose a novel integrative approach for the analysis of metabolic networks to identify modules of joint shifts on the transcriptomic and metabolic levels. We derive a tardigrade-specific metabolic network represented as an undirected graph with 3,658 nodes (metabolites) and 4,378 edges (reactions). Time course metabolite profiles are used to score the network nodes showing a significant change over time. The edges are scored according to information on enzymes from the EST data. Using this combined information, we identify a key subnetwork (functional module) of concerted changes in metabolic pathways, specific for de- and rehydration. The module is enriched in reactions showing significant changes in metabolite levels and enzyme abundance during the transition. It resembles the cessation of a measurable metabolism (e.g. glycolysis and amino acid anabolism) during the tun formation, the production of storage metabolites and bioprotectants, such as DNA stabilizers, and the generation of amino acids and cellular components from monosaccharides as carbon and energy source during rehydration.ConclusionsThe functional module identifies relationships among changed metabolites (e.g. spermidine) and reactions and provides first insights into important altered metabolic pathways. With sparse and diverse data available, the presented integrated metabolite network approach is suitable to integrate all existing data and analyse it in a combined manner.

Metabolites in human breastmilk identified as markers for sample storage quality

Detailed understanding of how storage temperature affects specific breastmilk metabolites is scarce. Current storage recommendations of expressed breastmilk for either scientific or domestic use is based exclusively on studies of bacterial growth and immuno capacity. Metabolomic profiling could reveal significant changes in metabolite levels in breast milk due to different exposure conditions that may affect the overall transfer of metabolites from the mother to the infant, ultimately affecting the health of the newborn.A pilot study was conducted using gas chromatography coupled with mass spectrometry (GC‐MS) to determine the milk metabolite profile under different storage temperatures over a 24‐hour period. Milk expressed from 5 lactating mothers, between 3 to 7 months post‐partum was collected, aliquoted, then stored immediately at room temperature, 4°C, −20°C or −80°C, before sample processing and analysis by GC‐MS.Levels of specific metabolites in stored milk samples were found to have changed at room temperature and/or 4°C but not at −20°C or −80°C in the first 24 hours. These results established the need for breastmilk storage at &lt; −20°C for both scientific and/or domestic use. Moreover, changes in metabolite levels could potentially serve as biomarkers/indicators for breast milk sample integrity.Funded by Medela AG. Switzerland.

Characterization of metabolic differences between embryogenic and non-embryogenic cells in forest trees

Somatic embryogenesis (SE) in forest trees is considered as one powerful approach in cloning elite genotypes. However, SE is difficult to achieve in tissue or cell beyond the mature embryo phase in trees and its physiological process involved are also poorly understood. Recently metabolic profiling studies have been contributed to understand the mechanism involved in SE process of plant [1-3]. In addition, there are also a number of reports indicating that polyamines (PAs) play a crucial role in SE process [4,5]. As part of an ongoing study of the transition of somatic cells to an embryogenic state in adult trees, embryogenic (EC) and non-embryogenic cells (NEC) in various forest species were investigated for its metabolic compositions including PAs. A comparison of metabolic compositions of NEC and EC using gas chromatography/mass spectrometry (GC/MS) identified around 50 compounds, partly displaying significant changes in metabolite levels, e.g., highly elevated levels of xanthosine and methyloxazole in EC compared to NEC of broadleaves and conifer species (Fig.​(Fig.1).1). Changes in the polyamine content were also analyzed in both cell types. Free polyamine contents varied according to the species and cell types, the highest levels occurring in the NEC on proliferation medium, when putrescine and spermidine were most abundant. However, the putrescine/spermine+spermidine (put/spm+spd) ratio was higher in EC of yellow poplar and hybrid pine. Analysis of PAs in both cell types indicated that total polyamine concentration was always higher in NEC than in EC, and spermine was present in only minute quantities and showed only a small change. From this analysis, we have identified numerous compounds involved with embryogenic state, and could characterize its differences between broadleaves and conifer trees. Figure 1 Non-embryogenic (NEC; A, C) and embryogenic cells (EC; B, D) of yellow poplar and hybrid pine ‘rigida x taeda’ respectively, and its metabolic compositions (NEC: a, c and EC: b, d).

Abstract 4070: Treatment with the MEK inhibitor U0126 induces increased lactate production in prostate and breast cancer cell lines

Abstract Introduction. In this study we investigated the metabolic consequences of U0126, a highly selective MEK1/2 inhibitor, in human prostate and breast cancer cell lines using 1H and 13C magnetic resonance spectroscopy (MRS) and gene expression data. Methods. Prostate (PC3 and LNCaP) and breast (MCF7) human cancer cells were treated for 48 h with the IC50 dose of U0126. 1H MRS data were acquired from cell extracts and repeated univariate statistical testing with false discovery rate correction was performed to determine significant changes in metabolite levels. The rate of glycolysis was determined from dynamic studies in live cells using 13C MRS to monitor the metabolism of 1-13C-labeled glucose. Protein and gene expression levels were measured using Western blotting and microarray analysis. Results and Discussion. The effect of U0126 was confirmed by western blot analysis demonstrating a drop in p-ERK. The univariate statistical analysis revealed that the 48 h drug treatment induced several metabolic changes including depletion of intracellular aspartate, succinate and phosphocholine in the prostate cells. However, one of the most significant metabolic change in all three cell lines was a substantial increase in intracellular lactate levels following treatment, with lactate up 134±16% (p=0.002), 149±20% (p=0.001) and 213±43% (n=2) of control in PC3, LNCaP and MCF7 cells respectively. Dynamic studies in live MCF7 cells were in line with the extract data and showed that the rate of lactate production increased following treatment 169±43% from 260±98 fmol/cell/hour to 439±114 fmol/cell/hour (p=0.03). Preliminary studies in PC3 cells showed a similar trend. Consistent with the metabolic observations, overexpression of genes associated with glycolysis including PFKFB3, PDK1, HK2, and GLUT1 was observed in PC3 cells pointing to an increase in glycolytic enzymes induced by U0126 treatment. In an effort to explain this observation we assessed the expression of other signaling proteins and found that although U0126 inhibits its target kinase, increased Akt phosphorylation was also observed in the PC3 cells. Importantly Akt activation was previously reported to mediate an increase in glycolysis. We therefore hypothesize that the observed increases in lactate production and its intracellular accumulation following inhibition of MAPK kinase signaling result from activation of the PI3K/Akt pathway observed in our cells. Conclusion. Deregulation of the MAPK pathway is often involved in oncogenesis, and therefore inhibitors of the pathway are being investigated as targeted cancer therapeutics. Our results highlight the value of MRS in identifying metabolic consequences of treatment that may inform on molecular drug action. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4070. doi:10.1158/1538-7445.AM2011-4070

Metabolic profiling of strawberry (Fragaria×ananassa Duch.) during fruit development and maturation

Strawberry (Fragaria × ananassa Duch), a fruit of economic and nutritional importance, is also a model species for fleshy fruits and genomics in Rosaceae. Strawberry fruit quality at different harvest stages is a function of the fruit's metabolite content, which results from physiological changes during fruit growth and ripening. In order to investigate strawberry fruit development, untargeted (GC-MS) and targeted (HPLC) metabolic profiling analyses were conducted. Principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) were employed to explore the non-polar and polar metabolite profiles from fruit samples at seven developmental stages. Different cluster patterns and a broad range of metabolites that exerted influence on cluster formation of metabolite profiles were observed. Significant changes in metabolite levels were found in both fruits turning red and fruits over-ripening in comparison with red-ripening fruits. The levels of free amino acids decreased gradually before the red-ripening stage, but increased significantly in the over-ripening stage. Metabolite correlation and network analysis revealed the interdependencies of individual metabolites and metabolic pathways. Activities of several metabolic pathways, including ester biosynthesis, the tricarboxylic acid cycle, the shikimate pathway, and amino acid metabolism, shifted during fruit growth and ripening. These results not only confirmed published metabolic data but also revealed new insights into strawberry fruit composition and metabolite changes, thus demonstrating the value of metabolomics as a functional genomics tool in characterizing the mechanism of fruit quality formation, a key developmental stage in most economically important fruit crops.

Detection of Leukemia in MLL-AF9 Transgenic Mice Using Magnetic Resonance Imaging and Magnetic Resonance Spectroscopy

Standard methods to assess tumor progression and treatment response for leukemia patients rely on repeat sampling of bone marrow. These methods evaluate overall disease status, but are not sensitive to early biochemical or cellular responses to therapies. Identification and validation of additional pharmacodynamic endpoints that are useful for accurate prediction of therapeutic efficacy would allow for earlier assessment of clinical response and thereby facilitate the development of effective individualized treatment regimens. Ideally, these markers would be assessed using non-invasive methods. Toward this end, we used in vivo magnetic resonance imaging (MRI) and ex vivo magnetic resonance spectroscopy (MRS) to investigate changes in the bone marrow, spleen, and/or blood of leukemic MLL-AF9 transgenic (Tg) mice relative to wild-type littermates.MLL-AF9 Tg mice with leukemia exhibited a statistically significant 1.5-fold increase in bone marrow T1-weighted MRI signal intensity. Increased signal intensity preceded development of leukemia and is therefore likely to be due, at least in part, to increased bone marrow cellularity, initially as a result of pre-neoplastic myeloproliferation and later as a result of marrow infiltration with leukemic blasts. We are currently determining microvessel density in the bone marrow of leukemic MLL-AF9 Tg mice and littermate controls to determine whether an increase in blood supply may also contribute to changes in MRI signal intensity in leukemic mice. These studies suggest that T1-weighted MRI signal intensity may be useful as an indicator of bone marrow tumor burden.Leukemic MLL-AF9 Tg mice also exhibited statistically significant changes in metabolite levels in spleen, blood, and bone marrow. The Warburg effect, whereby cancer cells utilize aerobic glycolysis to meet their increased energy demands, was evident in all tissues examined as indicated by increased glycolysis rates, increased glucose utilization, and increased levels of lactate and alanine, the end-products of glycolysis. Additional changes in metabolite levels were observed in the bone marrow and/or spleen of leukemic mice. Absolute levels of glutathione were increased. Glutathione reduces reactive oxygen species that are generated as a result of increased glycolysis and high levels of glutathione are associated with chemoresistance and poor prognosis in patients with acute leukemia (Maung et al., 1994, Leukemia 8:1487–91; Kearns et al., 2001, Blood 97:393–8). Increased glycine levels were also observed and may be associated with increased pyridine and DNA synthesis in leukemia cells. Decreased glutamate and glutamine levels may reflect a decrease in utilization of the mitochondrial Krebs cycle in tumor cells. Decreased levels of myo-inositol and taurine, which function as osmoregulators, may occur as a result of osmotic stress due to increased cellularity in leukemic organs. Levels of aromatic acids, lysine and arginine, and creatine and phosphocreatine were also decreased. Many of these changes in metabolism are recapitulated in immunocompetent mice orthotopically transplanted with MLL-AF9 Tg leukemias.This study represents the first description of a comprehensive analysis of the “metabolomic” profile associated with development of acute leukemia and is, to our knowledge, the first description of changes in metabolism in an animal model of acute leukemia. The data presented here suggest novel metabolic targets for therapeutic intervention. In addition, because metabolic changes often precede detectable changes in tumor burden, they may be particularly useful as early indicators of therapeutic efficacy and may thereby allow for more rapid determination of clinical response, decreased exposure to toxic therapies in resistant patients, and more expedient conversion to effective therapies. Because changes in glucose metabolism are a central feature of tumorigenesis and changes in glutathione levels have been associated with clinical outcome in patients with acute leukemia, these metabolites are of particular interest. Future studies will use MLL-AF9 transgenic mice to investigate the roles of metabolic changes during de novo development of leukemia. Mice transplanted with MLL-AF9 Tg leukemias will also be used for studies investigating the utility of metabolic changes as indicators of therapeutic efficacy.

Transcriptional and metabolic profiles of stress-induced, embryogenic tobacco microspores

Higher plant microspores, when subjected to various stress treatments in vitro, are able to reprogram their regular gametophytic development towards the sporophytic pathway to form haploid embryos and plants. Suppression subtractive hybridization (SSH) and metabolic profiling were used to characterize this developmental switch. Following differential reverse Northern hybridizations 90 distinct up-regulated sequences were identified in stressed, embryogenic microspores (accessible at www.univie.ac.at/ntsm). Sequence analyses allowed the classification of these genes into functional clusters such as metabolism, chromosome remodeling, signaling, transcription and translation, while the putative functions of half of the sequences remained unknown. A comparison of metabolic profiles of non-stressed and stressed microspores using gas chromatography/mass spectrometry (GC/MS) identified 70 compounds, partly displaying significant changes in metabolite levels, e.g., highly elevated levels of isocitrate and isomaltose in stressed microspores compared to non-stressed microspores. The formation of embryogenic microspores is discussed on the basis of the identified transcriptional and metabolic profiles.

Efficacy of recombinant human Hb by 31P-NMR during isovolemic total exchange transfusion.

The ability of recombinant human Hb (rHb1.1), which is being developed as an oxygen therapeutic, to support metabolism was measured by in vivo 31P-NMR surface coil spectroscopy of the rat abdomen in control animals and in animals subjected to isovolemic exchange transfusion to hematocrit of <3% with human serum albumin or 5 g/dl rHb1.1. No significant changes in metabolite levels were observed in control animals for up to 6 h. The albumin-exchange experiments, however, resulted in a more than eightfold increase in Pi and a 50% drop in phosphocreatine and ATP within 40 min. The tissue pH dropped from 7.4 to 6.8. The decrease in high-energy phosphates obeyed Michaelis-Menten kinetics, with a Michaelis-Menten constant of 3% as the hematocrit at which a 50% drop in high-energy phosphates was observed. Exchange transfusion with rHb1.1 resulted in no significant drop in high-energy phosphates, no rise in Pi, and no change in tissue pH from 7.35 +/- 0.15 for up to 5 h after exchange. By these criteria, rHb1.1 at a plasma Hb concentration of approximately 5 g/dl after total exchange transfusion was able to sustain energy metabolism of gut tissue at levels indistinguishable from control rats with a threefold higher total Hb level in erythrocytes.

Cerebrospinal Fluid Biogenic Amine Metabolites in Children during Treatment for Acute Lymphocytic Leukemia

To learn more about the impact of intrathecal methotrexate and cytosine arabinoside therapy on neuronal metabolism, we measured serial cerebrospinal fluid concentrations of homovanillic acid and 5-hydroxyindoleacetic acid, major metabolites of the neurotransmitters dopamine and serotonin, in children with acute lymphocytic leukemia. Multiple sequential cerebrospinal fluids were obtained from 30 children with acute lymphocytic leukemia evaluated prospectively from the time of diagnosis. We focused on the period of induction and intensification when children received weekly intrathecal chemotherapy. Paired cerebrospinal fluid specimens were also obtained at 3-month intervals from 60 children with acute lymphocytic leukemia in remission. Homovanillic acid and 5-hydroxyindoleacetic acid were measured using high performance liquid chromatography with electrochemical detection. We found that pretreatment metabolite values were no different from those in age-matched subjects in remission. In the first 5 wk of treatment, there were no significant changes in metabolite levels in patients treated exclusively with methotrexate. There was a transient decrease in homovanillic acid (-28 +/- 10%, p less than 0.001, Student's t test) and 5-hydroxyindoleacetic acid (-28 +/- 12%, p less than 0.05) in five of six patients after a single intrathecal dose of cytosine arabinoside. In the next 4 wk there was a gradual rise in levels of homovanillic acid (p = 0.001, by analysis of variance) and 5-hydroxyindoleacetic acid (p = 0.029, analysis of variance); this pattern did not correlate with administration of cranial irradiation. In children in remission, there were no significant changes in metabolite levels over a 3-month period.(ABSTRACT TRUNCATED AT 250 WORDS)