The combination of four analytical methods to explore skeletal muscle metabolomics: Better coverage of metabolic pathways or a marketing argument?

OBJECTIVES: Metabolomics is an emerging science based on diverse high throughput methods that are rapidly evolving to improve metabolic coverage of biological fluids and tissues. Technical progress has led researchers to combine several analytical methods without reporting the impact on metabolic coverage of such a strategy. The objective of our study was to develop and validate several analytical techniques (mass spectrometry coupled to gas or liquid chromatography and nuclear magnetic resonance) for the metabolomic analysis of small muscle samples and evaluate the impact of combining methods for more exhaustive metabolite covering. DESIGN AND METHODS: We evaluated the muscle metabolome from the same pool of mouse muscle samples after 2 metabolite extraction protocols. Four analytical methods were used: targeted flow injection analysis coupled with mass spectrometry (FIA-MS/MS), gas chromatography coupled with mass spectrometry (GC-MS), liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS), and nuclear magnetic resonance (NMR) analysis. We evaluated the global variability of each compound i.e., analytical (from quality controls) and extraction variability (from muscle extracts). We determined the best extraction method and we reported the common and distinct metabolites identified based on the number and identity of the compounds detected with low analytical variability (variation coefficient<30%) for each method. Finally, we assessed the coverage of muscle metabolic pathways obtained. RESULTS: Methanol/chloroform/water and water/methanol were the best extraction solvent for muscle metabolome analysis by NMR and MS, respectively. We identified 38 metabolites by nuclear magnetic resonance, 37 by FIA-MS/MS, 18 by GC-MS, and 80 by LC-HRMS. The combination led us to identify a total of 132 metabolites with low variability partitioned into 58 metabolic pathways, such as amino acid, nitrogen, purine, and pyrimidine metabolism, and the citric acid cycle. This combination also showed that the contribution of GC-MS was low when used in combination with other mass spectrometry methods and nuclear magnetic resonance to explore muscle samples. CONCLUSION: This study reports the validation of several analytical methods, based on nuclear magnetic resonance and several mass spectrometry methods, to explore the muscle metabolome from a small amount of tissue, comparable to that obtained during a clinical trial. The combination of several techniques may be relevant for the exploration of muscle metabolism, with acceptable analytical variability and overlap between methods However, the difficult and time-consuming data pre-processing, processing, and statistical analysis steps do not justify systematically combining analytical methods.

Lipidomics Reveals Cerebrospinal-Fluid Signatures of ALS.

Amyotrophic lateral sclerosis (ALS), the commonest adult-onset motor neuron disorder, is characterized by a survival span of only 2-5 years after onset. Relevant biomarkers or specific metabolic signatures would provide powerful tools for the management of ALS. The main objective of this study was to investigate the cerebrospinal fluid (CSF) lipidomic signature of ALS patients by mass spectrometry to evaluate the diagnostic and predictive values of the profile. We showed that ALS patients (n = 40) displayed a highly significant specific CSF lipidomic signature compared to controls (n = 45). Phosphatidylcholine PC(36:4), higher in ALS patients (p = 0.0003) was the most discriminant molecule, and ceramides and glucosylceramides were also highly relevant. Analysis of targeted lipids in the brain cortex of ALS model mice confirmed the role of some discriminant lipids such as PC. We also obtained good models for predicting the variation of the ALSFRS-r score from the lipidome baseline, with an accuracy of 71% in an independent set of patients. Significant predictions of clinical evolution were found to be correlated to sphingomyelins and triglycerides with long-chain fatty acids. Our study, which shows extensive lipid remodelling in the CSF of ALS patients, provides a new metabolic signature of the disease and its evolution with good predictive performance.

CLA reduces breast cancer cell growth and invasion through ERalpha and PI3K/Akt pathways.

We previously reported that conjugated linoleic acid (CLA), a naturally occurring fatty acid, inhibits the growth of ERalpha(+) MCF-7 and ERalpha(-) MDA-MB-231 human breast cancer cells by negative modulation of the ERK/MAPK pathway and apoptosis induction. Here we show that in these cell lines CLA also down-regulates the PI3K/Akt cascade. In MCF-7 cells CLA also triggers ERalpha/PP2A complex formation reducing the phosphorylation state and transcriptional activity of Eralpha whereas in MDA-MB-231 cells CLA does not induce PP2A activation. Moreover, CLA induces the expression of proteins involved in cell adhesion and inhibits cell migration and MMP-2 activity. These findings suggest that CLA may induce the down-regulation of ERalpha signalling and the reduction of cell invasion through the modulation of balancing between phosphatases and kinases.

Cytoskeleton-interacting activity of geiparvarin, diethylstilbestrol and conjugates.

Geiparvarin, a natural compound isolated from the leaves of Geijera parviflora, inhibits the growth of various tumor cell lines with a mode of action which may be attributed to its anti-microtubular activity. Our previous findings indicated that geiparvarin is able to inhibit the in vitro polymerization of tubulin and to derange the microtubular network in fibroblasts more effectively in the presence of paclitaxel. To further explore its biological activity here we have studied the effects exerted on the other components of the cytoskeleton by geiparvarin and two derivatives obtained by conjugating the 3(2H)-furanone ring of geiparvarin with diethylstilbestrol (DES). Firstly, observations by electron microscopy confirmed anti-microtubular properties, a near-total absence of microtubules is detected when tubulin is incubated with drugs in the presence of paclitaxel, whereas microtubule formation is not inhibited by drugs when assembly is induced by guanosine 5'-triphosphate (GTP). Immunofluorescence assays demonstrated that geiparvarin and DES act in a vinblastine-like fashion, causing a marked depletion of intermediate filaments while the network of microfilaments is not affected. Both the conjugates alter the 'stress fibers' organization of actin and disrupt the vimentin pattern; generally they derange cytoskeleton more markedly than the parent compounds. The cell growth inhibiting effects of geiparvarin and derivatives are dose-dependent; they vary according to the cell line used, when compounds were administered either alone or simultaneously with paclitaxel. Unlike other anti-microtubule agents, they do not exhibit cell-cycle compartment specificity and do not influence thymidine uptake in the cell.

Antimicrotubular and cytotoxic activity of geiparvarin analogues, alone and in combination with paclitaxel.

Geiparvarin is an antiproliferative compound isolated from the leaves of Geijera parviflora, and may represent a new drug which targets tubulin. To better explore the potential use of this agent, we investigated the antimicrotubular and cytotoxic effects of new synthetic aromatic derivatives of geiparvarin. These drugs inhibited polymerization of microtubular protein, particularly when the assembly was induced by paclitaxel. The microtubular network organization of fibroblasts was altered more effectively by some drugs. Normal microtubule architecture completely disappeared when the cells were treated simultaneously with drugs and paclitaxel: microtubules depolymerized or were reorganized into bundles, in a similar but more disarrayed fashion than that observed after treatment with paclitaxel alone. Cytotoxicity studies showed a dose-dependent effect, whereas combined administration of drugs and paclitaxel increased cytotoxicity, more effectively in paclitaxel versus derivatives administration alone.

Cellular uptake and cytotoxicity of solid lipid nanospheres (SLN) incorporating doxorubicin or paclitaxel.

Solid Lipid Nanospheres (SLN) are colloidal therapeutic systems proposed for several administration routes and obtained by dispersing warm microemulsions in cold water. SLN as carriers of doxorubicin and paclitaxel have been previously studied. In this study, the cellular uptake of SLN and the cytotoxicity of doxorubicin and paclitaxel incorporated into SLN were investigated on two cell-lines, human promyelocytic leukemia (HL60) and human breast carcinoma (MCF-7). Cellular uptake of SLN was determined by incorporating 6-coumarin as fluorescent marker. The cellular uptake of fluorescent SLN was clearly evidenced by fluorescence microscopy. The cytotoxicity of doxorubicin incorporated in SLN was higher compared to the conventional doxorubicin solution, even at the lower concentrations. Paclitaxel in SLN was about 100-fold more effective than free paclitaxel on MCF-7 cells, while on HL60 cells a lower sensitivity was achieved with paclitaxel in SLN. Unloaded SLN had no cytotoxic effect on HL60 and MCF-7 cells.

Geiparvarin and derivatives in combination with taxol: effect on microtubular organization in 3T3 fibroblasts.

Geiparvarin, a natural product that exhibits antiproliferative activity, inhibits the growth of various tumour cell lines with a mechanism of action so far unknown. Our preliminary findings showed that geiparvarin and some derivatives obtained from its conjugation with diethylstilboestrol and meso-hexestrol significantly inhibit taxol-induced in vitro polymerization of both tubulin and microtubular protein. In this study we investigated the effect of geiparvarin and of the oestrogen-combined derivatives on the cellular microtubular network of fibroblasts. Geiparvarin altered the microtubular organization of fibroblasts and strengthened the derangement of the microtubular pattern in cells exposed simultaneously to taxol. However, the microtubular network remained quite well organized in fibroblasts exposed to geiparvarin and preincubated with taxol, which in this case prevented the deranging effect of the former. The antimicrotubular activity of the oestrogen-combined derivatives was more similar to that of geiparvarin than to that of the oestrogens, and often this activity was stronger than that of each reference drug alone; the cytotoxic activity examined in the same experimental conditions generally confirmed the microscopic analysis.

Interaction of geiparvarin and related compounds with purified microtubular protein.

Geiparvarin is an antiproliferative compound whose mechanism of action has not yet been identified. We have investigated the biochemical action on purified microtubular protein, as well as on tubulin, of geiparvarin and of some derivatives resulting from its conjugation with two synthetic oestrogens, diethylstilboestrol and meso-hexoestrol, in comparison with the antimicrotubular action of the reference oestrogens. Geiparvarin and derivatives did not inhibit colchicine binding to tubulin nor did they significantly influence GTP-induced polymerization. On the contrary, they effectively counteracted the stimulating effect of taxol on microtubule formation, both in the presence and in the absence of microtubule-associated proteins. A competitive inhibition mechanism at the taxol binding site of tubulin may thus be proposed to explain the antimicrotubular action of geiparvarin.

Antimicrotubular effect of calvatic acid and of some related compounds.

A structure-activity relationship has been established between calvatic acid and some related synthetic compounds, and their ability to inhibit GTP-induced microtubular protein polymerization in vitro. These compounds were effective in a dose- and a time-dependent manner. The most active drug was the p-chloro substituted compound, which showed its inhibitory activity without any preincubation period, which the others needed. Since if cysteine was present, polymerization was no longer affected, an involvement of titratable -SH groups of tubulin could be suggested. In contrast, taxol-induced polymerization was only slightly inhibited by these compounds, and colchicine-binding activity was not generally impaired.

Cytoskeletal modifications induced by 4-hydroxynonenal.

The antiproliferative action of 4-hydroxynonenal (HNE) could be related to an interaction with cytoskeletal structures. In this paper the effects exerted by HNE on microtubules and on microfilaments are examined by immunofluorescence. HNE alters cell morphology causing both the depolymerization of the microtubular structures and the dissolution of the stress-fibres. Taxol protects microtubules, preventing the depolymerizing effect of the aldehyde. The action of HNE could be attributed to its affinity for sulphydryl groups, which are essential in maintaining tubulin and actin both in the polymerized form.

Cytoskeletal modifications induced by 4-hydroxynonenal.

The antiproliferative action of 4-hydroxynonenal (HNE) could be related to an interaction with cytoskeletal structures. In this paper the effects exerted by HNE on microtubules and on microfilaments are examined by immunofluorescence. HNE alters cell morphology causing both the depolymerization of the microtubular structures and the dissolution of the stress-fibres. Taxol protects microtubules, preventing the depolymerizing effect of the aldehyde. The action of HNE could be attributed to its affinity for sulphydryl groups, which are essential in maintaining tubulin and actin both in the polymerized form.