The use of high-performance liquid chromatography and diode array detection in fungal chemotaxonomy based on profiles of secondary metabolites

FRISVAD, J. C, 1989. The use of high-performance liquid chromatography and diode array detection in fungal chemotaxonomy based on profiles of secondary metabolites. Fungal chemotaxonomy (that part dealing with secondary metabolites) has often been based on thin layer chromatography (TLC) and visual or UV inspection of separated spots, before and after different chemical treatments. The identity of a small proportion of the spots can be suggested based on known internal and external standards. In most chemotaxonomical studies it is impossible to isolate, purify and identify all secondary metabolites produced, due to restraints of time and resources. High performance liquid chromatography (HPLC) of fungal extracts may have some advantages over TLC, but the problems mentioned above remain. These problems have been approached by using an alkylphenone retention time index in a reversed phase HPLC system combined with the use of a diode array UV-VIS detector. High performance thin layer chromatography is used for further confirmation of identity of the secondary metabolites. A particular advantage of this method is that the number of biosynthetic families or groups ('chemosyndromes') can be detected, as biosynthetically related metabolites usually have the same chromophores and UV-VIS spectra. Results obtained from Penicillium, Aspergillus and Fusanum species have shown that each species produces 5 to 15 different biosynthetic families of secondaiy metabolites, indicating that good chromatography data may be sufficient to identify species in the three genera. The use of the technique is exemplified by data on Aspergillus and Talaromyces species.     

Rapid Method To Estimate the Presence of Secondary Metabolites in Microbial Extracts

Screening microbial secondary metabolites is an established method to identify novel biologically active molecules. Preparation of biological screening samples from microbial fermentation extracts requires growth conditions that promote synthesis of secondary metabolites and extraction procedures that capture the secondary metabolites produced. High-performance liquid chromatography (HPLC) analysis of fermentation extracts can be used to estimate the number of secondary metabolites produced by microorganisms under various growth conditions but is slow. In this study we report on a rapid (approximately 1 min per assay) surrogate measure of secondary metabolite production based on a metabolite productivity index computed from the electrospray mass spectra of samples injected directly into a spectrometer. This surrogate measure of productivity was shown to correlate with an HPLC measure of productivity with a coefficient of 0.78 for a test set of extracts from 43 actinomycetes. This rapid measure of secondary metabolite productivity may be used to identify improved cultivation and extraction conditions by analyzing and ranking large sets of extracts. The same methods may also be used to survey large collections of extracts to identify subsets of highly productive organisms for biological screening or additional study.     

Improved protocols for quantitative determination of metabolites from biological samples using high performance ionic-exchange chromatography with conductimetric and pulsed amperometric detection

Simple and reliable protocols are described for an extensive analysis of metabolites in extracts from different biological sources. The separation was performed by high performance ionic-exchange chromatography (HPIC) at alkaline pH using two types of chromatography columns and two detection methods. Organic acids and inorganic anions were separated on an ionPac AS11 column using a 0.5 to 35 mM Na0H gradient. Detection limits in the range of milligrams per liter were achieved by use of a conductivity detector equipped with an anion self-regenerating suppressor. Twelve phosphorylated compounds belonging to the glycolytic and the pentose phosphate pathways could be resolved on a CarboPac PA1 column using a Na0H/Na-acetate gradient. Quantification was achieved by pulsed amperometry with detection limits in the micromolar range. Cell extracts obtained by extraction in boiling buffered ethanol described previously could be directly injected onto HPIC columns for the separation of metabolites because the extraction procedure affected neither the retention time nor the stability of most of the metabolites, and yielded very clean chromatograms. These improved protocols were applied for a dynamic analysis of intracellular metabolites in Saccharomyces cerevisiae in response to a glucose pulse.     

Secondary Metabolites from the Marine Tunicate “Phallusia nigra” and Some Biological Activities

Biology Bulletin - Different extracts of solitary marine tunicates have attracted attention as a source of amazing secondary metabolites with a wide range of promising potential biological effects....     

电热蒸发-催化热解-原子吸收法直接测定茶叶中镉

重金属镉（Cd）一直是茶叶产品质量安全关注的重点。本研究基于电热蒸发-催化热解-原子吸收光谱仪（SS-ETV-AAS）,使用镍材质样品舟,在300 mL/min空气条件下,350 ℃干燥20 s,350~725 ℃灰化55 s;引入300 mL/min氢气与空气反应形成氮氢混合气氛,在725~800 ℃（50 s）下完成Cd的蒸发;之后,在高岭土填料催化热解炉800 ℃和准直管700 ℃条件下,氮氢火焰原子吸收测定镉的含量。方法检出限（LOD）为0.3 ng/g、定量限（LOQ）为1.0 ng/g,R2>0.998,多次测定的相对标准偏差（RSD）为1.8%~8.6%,多种茶叶样品中Cd的测定值与微波消解石墨炉原子吸收光谱法（GFAAS）无显著性差异（P>0.05）,Cd的回收率在92%~107%之间。试验结果表明,该方法灵敏度高、稳定性好、简单高效,且无需消解处理,样品分析时间仅为3min,适用于茶叶中Cd的快速检测。     

Metabolic profiling, metabolomic and metabonomic procedures for NMR spectroscopy of urine, plasma, serum and tissue extracts

Metabolic profiling, metabolomic and metabonomic studies mainly involve the multicomponent analysis of biological fluids, tissue and cell extracts using NMR spectroscopy and/or mass spectrometry (MS). We summarize the main NMR spectroscopic applications in modern metabolic research, and provide detailed protocols for biofluid (urine, serum/plasma) and tissue sample collection and preparation, including the extraction of polar and lipophilic metabolites from tissues. 1H NMR spectroscopic techniques such as standard 1D spectroscopy, relaxation-edited, diffusion-edited and 2D J-resolved pulse sequences are widely used at the analysis stage to monitor different groups of metabolites and are described here. They are often followed by more detailed statistical analysis or additional 2D NMR analysis for biomarker discovery. The standard acquisition time per sample is 4-5 min for a simple 1D spectrum, and both preparation and analysis can be automated to allow application to high-throughput screening for clinical diagnostic and toxicological studies, as well as molecular phenotyping and functional genomics.     

Host plant secondary metabolite profiling shows a complex, strain-dependent response of maize to plant growth-promoting rhizobacteria of the genus Azospirillum

Most Azospirillum plant growth-promoting rhizobacteria (PGPR) benefit plant growth through source effects related to free nitrogen fixation and/or phytohormone production, but little is known about their potential effects on plant physiology. These effects were assessed by comparing the early impacts of three Azospirillum inoculant strains on secondary metabolite profiles of two different maize (Zea mays) cultivars. After 10d of growth in nonsterile soil, maize methanolic extracts were analyzed by reverse-phase high-performance liquid chromatography (RP-HPLC) and secondary metabolites identified by liquid chromatography/mass spectrometry (LC/MS) and nuclear magnetic resonance (NMR). Seed inoculation resulted in increased shoot biomass (and also root biomass with one strain) of hybrid PR37Y15 but had no stimulatory effect on hybrid DK315. In parallel, Azospirillum inoculation led to major qualitative and quantitative modifications of the contents of secondary metabolites, especially benzoxazinoids, in the maize plants. These modifications depended on the PGPR strain×plant cultivar combination. Thus, Azospirillum inoculation resulted in early, strain-dependent modifications in the biosynthetic pathways of benzoxazine derivatives in maize in compatible interactions. This is the first study documenting a PGPR effect on plant secondary metabolite profiles, and suggests the establishment of complex interactions between Azospirillum PGPR and maize.     

1H NMR spectroscopic investigations of tissue metabolite biomarker response to Cu II exposure in terrestrial invertebrates: identification of free histidine as a novel biomarker of exposure to copper in earthworms

High resolution 1H NMR spectroscopy of biofluids, cells and tissue extracts allows rapid, non destructive analysis for a wide range of metabolites and organic compounds with minimal sample pre treatment. We have applied high resolution 1H NMR spectroscopy to investigate the biochemical effects of Cu II in two earthworm species Eisenia andrei n =78 and Lumbricus rubellus n =45 exposed under laboratory and semi field conditions respectively. The most marked metabolic response was the elevation of endogenous whole body free histidine in animals which positively correlated with increasing copper exposure and total copper burden in the semi field experiment. Histidine forms thermodynamically stable copper complexes under a wide range of physico chemical conditions and we proposed that the elevation of free histidine in response to copper challenge provides an energetically low cost detoxification mechanism. Histidine elevation may also provide a novel molecular biomarker of Cu II exposure in environmental situations.     

1H NMR spectroscopic investigations of tissue metabolite biomarker response to Cu II exposure in terrestrial invertebrates: identification of free histidine as a novel biomarker of exposure to copper in earthworms

High resolution 1H NMR spectroscopy of biofluids, cells and tissue extracts allows rapid, non destructive analysis for a wide range of metabolites and organic compounds with minimal sample pre treatment. We have applied high resolution 1H NMR spectroscopy to investigate the biochemical effects of Cu II in two earthworm species Eisenia andrei n =78 and Lumbricus rubellus n =45 exposed under laboratory and semi field conditions respectively. The most marked metabolic response was the elevation of endogenous whole body free histidine in animals which positively correlated with increasing copper exposure and total copper burden in the semi field experiment. Histidine forms thermodynamically stable copper complexes under a wide range of physico chemical conditions and we proposed that the elevation of free histidine in response to copper challenge provides an energetically low cost detoxification mechanism. Histidine elevation may also provide a novel molecular biomarker of Cu II exposure in environmental situations.     

Secondary metabolites of soil <Emphasis Type="Italic">Bacillus</Emphasis> spp.

Bacillus species produce secondary metabolites that are the object of natural product chemistry studies. The wide structural variability of these compounds has attracted the curiosity of chemists and their biological activities have inspired the pharmaceutical industry to search for lead structures in microbial extracts. Screening of microbial extracts reveals the large structural diversity of natural compounds with broad biological activities, such as antimicrobial, antiviral, immunosuppressive, and antitumor activities, that enable the bacterium to survive in its natural environment. These findings widen the potential industrial importance of Bacillus spp., particularly of B. thuringiensis, beyond insecticidal usage and may help explain the role of Bacillus spp. in the soil ecosystem.     

The biological interpretation of metabolomic data can be misled by the extraction method used

The field of metabolomics is getting more and more popular and a wide range of different sample preparation procedures are in use by different laboratories. Chemical extraction methods using one or more organic solvents as the extraction agent are the most commonly used approach to extract intracellular metabolites and generate metabolite profiles. Metabolite profiles are the scaffold supporting the biological interpretation in metabolomics. Therefore, we aimed to address the following fundamental question: can we obtain similar metabolomic results and, consequently, reach the same biological interpretation by using different protocols for extraction of intracellular metabolites? We have used four different methods for extraction of intracellular metabolites using four different microbial cell types (Gram negative bacterium, Gram positive bacterium, yeast, and a filamentous fungus). All the quenched samples were pooled together before extraction, and, therefore, they were identical. After extraction and GC?CMS analysis of metabolites, we did not only detect different numbers of compounds depending on the extraction method used and regardless of the cell type tested, but we also obtained distinct metabolite levels for the compounds commonly detected by all methods (P-value?<?0.001). These differences between methods resulted in contradictory biological interpretation regarding the activity of different metabolic pathways. Therefore, our results show that different solvent-based extraction methods can yield significantly different metabolite profiles, which impact substantially in the biological interpretation of metabolomics data. Thus, development of alternative extraction protocols and, most importantly, standardization of sample preparation methods for metabolomics should be seriously pursued by the scientific community.     

Proteohistography--direct analysis of tissue with high sensitivity and high spatial resolution using ProteinChip technology.

On the proteomic level, all tissues, tissue constituents, or even single cells are heterogeneous, but the biological relevance of this cannot be adequately investigated with any currently available technique. The analysis of proteins of small tissue areas by any proteomic approach is limited by the number of required cells. Increasing the number of cells only serves to lower the spatial resolution of expressed proteins. To enhance sensitivity and spatial resolution we developed Proteohistography. Laser microdissection was used to mark special areas of interest on tissue sections attached to glass slides. These areas were positioned under microscopic control directly on an affinity chromatographic ProteinChip Array so that cells were lysed and their released proteins bound on a spatially defined point. The ProteinChip System, surface enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS), allows the laser to be steered to up to 215 distinct positions across the surface of the spot, enabling a high spatial resolution of measured protein profiles for the analyzed tissue area. Protein profiles of the single positions were visually plotted over the used tissue section to visualize distribution proteohistologically. Results show that the spatial distribution of detectable proteins could be used as a Proteohistogram for a given tissue area. Consequently, this procedure can provide additional information to both a matrix-assisted laser desorption/ionization (MALDI)-based approach and immunohistochemistry, as it is more sensitive, highly quantitative, and no specific antibody is needed. Hence, proteomic heterogeneity can be visualized even if proteins are not known or identified.     

Novel aspects of grape berry ripening and post-harvest withering revealed by untargeted LC-ESI-MS metabolomics analysis

We established a step-by-step, experiment-guided metabolomics procedure, based on LC-ESI-MS analysis, to generate a detailed picture of the changing metabolic profiles during late berry development in the important Italian grapevine cultivar Corvina. We sampled berries from four developmental time points and three post-harvest time points during the withering process, and used chromatograms of methanolic extracts to test the performance of the MetAlign and MZmine data mining programs. MZmine achieved a better resolution and therefore generated a more useful data matrix. Then both the quantitative performance of the analytical platform and the matrix effect were assessed, and the final dataset was investigated by multivariate data analysis. Our analysis confirmed the results of previous studies but also revealed some novel findings, including the prevalence of two specific flavonoids in unripe berries and important differences between the developmental profiles of flavones and flavanones, suggesting that specific individual metabolites could have different functions, and that flavones and flavanones probably play quite distinct biological roles. Moreover, the hypothesis-free multivariate analysis of subsets of the wide data matrix evidentiated the relationships between the various classes of metabolites, such as those between anthocyanins and hydroxycinnamic acids and between flavan-3-ols and anthocyanins.     

Construction and genetic analysis of anthocyanin-deficient mutants induced by T-DNA insertion in ‘Tsuda’ turnip (<Emphasis Type="Italic">Brassica rapa</Emphasis>)

Centella asiatica, a perennial herb renowned for its wide range of nutraceuticals properties, is a valuable source of plant secondary metabolites with various pharmacological activities. Novel approaches to develop alternatives for the production and enrichment of these secondary metabolites are receiving much attention, for example the biotechnological manipulation of undifferentiated cells cultures as a potential source of natural products. This study investigated the differences of the metabolite profiles between undifferentiated cells and differentiated leaf tissues of C. asiatica in comparison to a commercially available herbal medicine supplement. The secondary metabolites were extracted using methanol and analysed on an UHPLC-QTOF-MS platform. Metabolites were further identified and characterised based on their MS-fragmentation patterns and through comparison with authentic standards where available. Results revealed the similarities and dissimilarities and showed inter- and intra- relationships amid the different samples. A total of 18 metabolites including a number of hydroxycinnamic acid derivatives, flavonoids as well as the four centelloids (pentacyclic triterpenoids) were annotated in different sample groups across the different extracts. The results obtained verified that the different biological systems of C. asiatica are chemo-diverse, possibly related to regional and processing differences, and in the case of cells, to the level of cellular differentiation.     

The separation and direct detection of ceramides and sphingoid bases by normal-phase high-performance liquid chromatography and evaporative light-scattering detection

Sphingolipids are an important class of lipids due to their role as biologically active molecules and as intracellular second messengers. Sphingolipid metabolites are involved in a wide variety of important biological processes including signal transduction and growth regulation. Simple, quantitative analytical methods are needed to assay these complex lipids, in order to study their biological functions. The current methods used to quantify ceramides and long-chain sphingoid bases are primarily based on derivatization with uv or fluorescent tags and with radioactive-based enzymatic assays. A method was developed to separate ceramides and sphingoid bases by normal-phase high-performance liquid chromatography and detect them directly with evaporative light-scattering detection. Ceramides and the sphingoid bases phytosphingosine, dihydrosphingosine, sphingosine, and sphingosine 1-phosphate were resolved with a rapid and quantitative assay in the nanomole range. Yeast extracts grown to various time points were assayed for ceramide and sphingoid bases using a simple, isocratic HPLC system. Both ceramide and phytosphingosine, the primary sphingoid base present in yeast cell extracts, were detected in yeast cell extracts. Phytosphingosine was resolved as a sharp peak with the addition of triethylamine and formic acid modifiers to a chloroform/ethanol mobile phase. This method demonstrates the first direct assay of both ceramides and sphingoid bases.     

Tissue specialization at the metabolite level is perceived during the development of tomato fruit

Fruit maturation and tissue differentiation are important topics in plant physiology. These biological phenomena are accompanied by specific alterations in the biological system, such as differences in the type and concentration of metabolites. The secondary metabolism of tomato (Solanum lycopersicum) fruit was monitored by using liquid chromatography (LC) coupled to photo-diode array (PDA) detection, fluorescence detection (FD), and mass spectrometry (MS). Through this integrated approach different classes of compounds were analysed: carotenoids, xanthophylls, chlorophylls, tocopherols, ascorbic acid, flavonoids, phenolic acids, glycoalkaloids, saponins, and other glycosylated derivatives. Related metabolite profiles of peel and flesh were found between several commercial tomato cultivars indicating similar metabolite trends despite the genetic background. For a single tomato cultivar, metabolite profiles of different fruit tissues (vascular attachment region, columella and placenta, epidermis, pericarp, and jelly parenchyma) were examined at the green, breaker, turning, pink, and red stages of fruit development. Unrelated to the chemical nature of the metabolites, behavioural patterns could be assigned to specific ripening stages or tissues. These findings suggest spatio-temporal specificity in the accumulation of endogenous metabolites from tomato fruit.     

Stress associated protein 1 regulates the development of glandular trichomes in Artemisia annua

Undifferentiated plant cells in culture represent a renewable system conducive to understanding biological processes and a valuable alternative for secondary metabolite production. Additionally, manipulation of these systems by plant growth regulators (PGRs) may result in redifferentiation/organogenesis and hence changes in metabolic profiles. The aim of the study was to investigate the effects of combining auxin (2,4-dichlorophenoxyacetic acid) and cytokinin (kinetin) at concentrations of 2, 4, 6 and 9 µM on undifferentiated Moringa oleifera callus cells, at a metabolome level. Results indicated that the callus became habituated, i.e. developed the ability to grow without added stimulatory PGRs, and no organogenesis was observed on any of the different PGR combinations under investigation. Methanolic extracts were screened for total phenolic content (TPC) and anti-oxidant activity, and further analysed using liquid chromatography coupled to mass spectrometry combined with multivariate data analysis to facilitate analysis of the metabolite profiles. While the anti-oxidant capacity of extracts from the various treatments exhibited little variation, the TPC differed significantly. Despite the observed habituation phenomenon, the calli retained responsiveness towards external PGRs and each of the 25 conditions generated a unique metabolome as found by principal component analysis. This was also reflected by a number of phytochemicals that were annotated as biomarkers from PGR-treated calli. These findings demonstrate the differential influence of 2,4-D and kinetin on M. oleifera callus for the production of secondary metabolites.

     

Application of 2-D DIGE to formalin-fixed, paraffin-embedded tissues

The ability to investigate the proteome of formalin-fixed, paraffin-embedded (FFPE) tissues can be considered a major recent achievement in the field of clinical proteomics. However, gel-based approaches to the investigation of FFPE tissue proteomes have lagged behind, mainly because of insufficient quality of full-length protein extracts. Here, the 2-D DIGE technology was investigated for applicability to FFPE proteins, for internal reproducibility among replicate FFPE extracts, and for comparability between FFPE and fresh-frozen tissue profiles. The 2-D DIGE patterns obtained upon labeling and electrophoresis of replicate FFPE tissue extracts were highly reproducible, with satisfactory resolution and complexity. Moreover, the implementation of DIGE enabled to highlight and characterize the consistent differences found in the FFPE profiles compared with fresh-frozen profiles, represented by an acidic shift, directly correlated to the protein pI value, and by a reduction in spot signal intensity, directly correlated to molecular weight and percentage of lysine residues. Being constantly and reproducibly present in all FFPE tissue extract replicates at similar extents, these modifications do not appear to hinder the comparative analysis of FFPE tissue extracts by 2-D DIGE, opening the way to its application for the differential proteomic investigation of archival tissue repositories.     

Abdominal magnetic resonance imaging in small rodents using a clinical 1.5 T MR scanner

Because of superior soft-tissue contrast compared to other imaging techniques, non-invasive abdominal magnetic resonance imaging (MRI) is ideal for monitoring organ regeneration, tissue repair, cancer stage, and treatment effects in a wide variety of experimental animal models. Currently, sophisticated MR protocols, including technically demanding procedures for motion artefact compensation, achieve an MRI resolution limit of < 100 microm under ideal conditions. However, such a high spatial resolution is not required for most experimental rodent studies. This article describes both a detailed imaging protocol for MR data acquisition in a ubiquitously and commercially available 1.5 T MR unit and 3-dimensional volumetry of organs, tissue components, or tumors. Future developments in MR technology will allow in vivo investigation of physiological and pathological processes at the cellular and even the molecular levels. Experimental MRI is crucial for non-invasive monitoring of a broad range of biological processes and will further our general understanding of physiology and disease.