Biomedical subjects

Jørn Smedsgaard

Publications and source records attributed to Jørn Smedsgaard.

17 recordsLinked to original sources

PubMed2007

Elucidating the mode-of-action of compounds from metabolite profiling studies.

Metabolite profiling has been carried out for decades and is as such not a new research area. However, the field has attracted increasing attention in the last couple of years, and the term metabolome is now often used to describe the complete pool of metabolites associated with an organism at any given time. Mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy are the best candidates for comprehensive analysis of the metabolome and the application of these technologies is presented in this chapter. In this relation, the importance of efficient metabolite screening for discovery of novel drugs is discussed. Related to metabolite profiling, the principals underlying the application of labeled substrates to quantify in vivo metabolic fluxes are introduced, and the chapter is concluded by discussing the perspectives of metabolite measurements in systems biology.

Anti-Infective Agents↗

PubMed2006

Soyasaponins resist extrusion cooking and are not degraded during gut passage in Atlantic salmon (Salmo salar L.).

The stability of soyasaponins in fish feed formulations was investigated. The level of soyasaponin Ab, Bb, Bc, Ba-2,3-dihydro-2,5-dihydroxy-6-methyl-4H-pyran-4-one (Ba-DDMP), Bb-DDMP, and Bc-DDMP was quantified in 15 samples of defatted soybean meal, two full fat soybean meals, and two soybean protein concentrates by reverse phase high-performance liquid chromatography. The total level of saponins in the 15 samples of commercial defatted soybean meal ranged from 4.8-6.8 micromol/g (5.1-7.0 g/kg). The two full fat meals contained 4.4 and 4.7 micromol/g whereas no saponins could be detected in the alcohol-extracted soybean protein concentrates. Fifteen batches of fish feed containing 20% defatted soybean meal were produced by twin-screw extrusion from the 15 different samples of defatted soybean meal. Extrusion did not reduce the total level of group B saponins, but the ratio between DDMP-conjugated group B saponins and non-DDMP-conjugated group B saponins was slightly reduced. A soybean-containing diet was fed to seawater adapted Atlantic salmon for 9 weeks. Yttrium oxide was included in the feed as an inert marker in order to estimate the disappearance of saponins during gut passage. High levels of intact non-DDMP-conjugated group B soyasaponins were found in feces whereas only low levels of DDMP-conjugated saponins could be detected. The overall disappearance of saponins was close to zero, and the concentration of intact saponins in dry feces reached levels several fold higher than dietary levels. The present work demonstrates that non-DDMP-conjugated group B soyasaponins resist extrusion cooking and remain intact during gut passage in Atlantic salmon. The latter is contrary to earlier findings in endothermic animals.

Animal Feed↗

PubMed2006

Real-time PCR quantification of the AM-toxin gene and HPLC qualification of toxigenic metabolites from Alternaria species from apples.

Some Alternaria species are able to produce plant pathogenic as well as toxic metabolites. In both agriculture and the food industry it is important know if toxigenic Alternaria are present to rapidly employ the correct corrective actions. The purpose of this work was to establish a real-time PCR method, which can detect and quantify apple pathogenic and toxigenic Alternaria. An AM-toxin I primer set, which could recognize Alternaria DNA only, was designed by using primers complementary to the AM-toxin I gene. The method could detect small amounts of DNA (4 pg) and still obtain a large dynamic range (4 decades) without interference from apple material. Eight Alternaria isolates were analyzed for the presence of AM-toxin I gene and their production of secondary metabolites. Then analyses showed that all eight isolates contained the AM toxin gene and were able to produce the plant pathogenic tentoxin in addition to AM toxin I. The analyses also showed the production of tenuazonic acid, alternariols, Altenuene, altenusin and/or altertoxin I in pure culture. Analyses of inoculated apples showed that both the AM-toxin gene and alternariol monomethyl ether could be detected. Morphological analyses suggested that the eight Alternaria strains, though they all carried the AM toxin genes, probably belong to different but closely related un-described Alternaria taxa in the A. tenuissima species-group based on morphological and chemical differences.

Alternaria↗

PubMed2006

Identification of an abscisic acid gene cluster in the grey mold Botrytis cinerea.

Like several other phytopathogenic fungi, the ascomycete Botrytis cinerea is known to produce the plant hormone abscisic acid (ABA) in axenic culture. Recently, bcaba1, the first fungal gene involved in ABA biosynthesis, was identified. Neighborhood analysis of bcaba1 revealed three further candidate genes of this pathway: a putative P450 monooxygenase-encoding gene (bcaba2), an open reading frame without significant similarities (bcaba3), and a gene probably coding for a short-chain dehydrogenase/reductase (bcaba4). Targeted inactivation of the genes proved the involvement of BcABA2 and BcABA3 in ABA biosynthesis and suggested a contribution of BcABA4. The close linkage of at least three ABA biosynthetic genes is strong evidence for the presence of an abscisic acid gene cluster in B. cinerea.

Abscisic Acid↗

PubMed2005

Phenotypic taxonomy and metabolite profiling in microbial drug discovery.

Microorganisms and in particular actinomycetes and microfungi are known to produce a vast number of bioactive secondary metabolites. For industrially important fungal genera such as Penicillium and Aspergillus the production of these compounds has been demonstrated to be very consistent at the species level. This means that direct metabolite profiling techniques such as direct injection mass spectrometry or NMR can easily be used for chemotyping/metabolomics of strains from both culture collections and natural samples using modern informatics tools. In this review we discuss chemotyping/metabolomics as part of intelligent screening and highlight how it can be used for identification and classification of filamentous fungi and for the discovery of novel compounds when used in combination with modern methods for dereplication. In our opinion such approaches will be important for future effective drug discovery strategies, especially for dereplication of culture collections in order to avoid redundancy in the selection of species. This will maximize the chemical diversity of the microbial natural product libraries that can be generated from fungal collections.

Aspergillus↗

PubMed2005

X-Hitting: an algorithm for novelty detection and dereplication by UV spectra of complex mixtures of natural products.

A major challenge in lead discovery is to detect well-known and trivial compounds rapidly, a process known as dereplication, so that isolation, structure elucidation, and pharmacological investigations can be focused on novel compounds. In this paper, we present a new algorithm, X-hitting, based on cross sample comparison of full UV spectra from HPLC analysis of highly complex natural product extracts/samples. X-Hitting allows automatic identification of known compounds but more important also allows finding of potentially new or similar compounds. We demonstrate this new algorithm by automatic identification of known structures, a task we call cross-hitting, and tentative identification of potentially new bioactive compounds, a task we call new-hitting, in HPLC data from analysis of fungal extracts. Both tasks are illustrated using 18 important reference compounds and complex fungal extracts obtained from isolates in the IBT Culture Collection held at BioCentrum-DTU, Technical University of Denmark. The receiver operating characteristics statistic is used to evaluate the performance of the compound predictor, and it was found that compounds could be identified with high confidence (AUC approximately 0.98). Based on high confidence in retrieving identical spectra, the method is extended to include similar but still different spectra.

Algorithms↗

PubMed2005

Global metabolite analysis of yeast: evaluation of sample preparation methods.

Sample preparation is considered one of the limiting steps in microbial metabolome analysis. Eukaryotes and prokaryotes behave very differently during the several steps of classical sample preparation methods for analysis of metabolites. Even within the eukaryote kingdom there is a vast diversity of cell structures that make it imprudent to blindly adopt protocols that were designed for a specific group of microorganisms. We have therefore reviewed and evaluated the whole sample preparation procedures for analysis of yeast metabolites. Our focus has been on the current needs in metabolome analysis, which is the analysis of a large number of metabolites with very diverse chemical and physical properties. This work reports the leakage of intracellular metabolites observed during quenching yeast cells with cold methanol solution, the efficacy of six different methods for the extraction of intracellular metabolites, and the losses noticed during sample concentration by lyophilization and solvent evaporation. A more reliable procedure is suggested for quenching yeast cells with cold methanol solution, followed by extraction of intracellular metabolites by pure methanol. The method can be combined with reduced pressure solvent evaporation and therefore represents an attractive sample preparation procedure for high-throughput metabolome analysis of yeasts.

Freeze Drying↗

PubMed2005

Andrastins A-D, Penicillium roqueforti Metabolites consistently produced in blue-mold-ripened cheese.

This is the first finding of andrastins in blue cheese as well as any other sample type. Here, they were produced by the secondary starter culture Penicillium roqueforti. After purification by normal-phase chromatography followed by combined reverse-phase ion-exchange chromatography, the andrastins A-D were detected by liquid chromatography combined with UV and high-resolution mass spectrometry. In 23 representative samples of European blue cheeses, andrastin A was consistently found in quantities between 0.1 and 3.7 microg/g of cheese (median 2.4 microg/g). Assuming the same molar response factors as for andrastin A, the B, C, and D analogues were present in approximately 5-, 3-, and 5-20-fold lower amounts than andrastin A, respectively. The andrastins are protein farnesyltransferase inhibitors and are capable of inhibiting the efflux of anticancer drugs from multidrug-resistant cancer cells. Thus, their presence in common blue cheese suggests a potential for a positive or negative impact on human health.

Alkyl and Aryl Transferases↗

PubMed2005

Automated and unbiased classification of chemical profiles from fungi using high performance liquid chromatography.

In this paper we present a method for unbiased/unsupervised classification and identification of closely related fungi, using chemical analysis of secondary metabolite profiles created by HPLC with UV diode array detection. For two chromatographic data matrices a vector of locally aligned full spectral similarities is calculated along the retention time axis. The vector depicts the evaluating of the alikeness between two fungal extracts based upon eluted compounds and corresponding UV-absorbance spectra. For assessment of the chemotaxonomic grouping the vector is condensed to one similarity describing the overall degree of similarity between the profiles. Two sets of data were used in this study: One set was used in the method development and a second dataset used for method validation. First we developed a method for evaluating the secondary metabolite production from closely related Penicillium species. Then the algorithm was validated on fungal isolates belonging to the genus Alternaria. The results showed that the species may be segregated into taxa in full accordance with published taxonomy.

Algorithms↗

PubMed2004

Metabolite profiling of fungi and yeast: from phenotype to metabolome by MS and informatics.

Filamentous fungi and yeast from the genera Saccharomyces, Penicillium, Aspergillus, and Fusarium are well known for their impact on our life as pathogens, involved in food spoilage by degradation or toxin contamination, and also for their wide use in biotechnology for the production of beverages, chemicals, pharmaceuticals, and enzymes. The genomes of these eukaryotic micro-organisms range from about 6000 genes in yeasts (S. cerevisiae) to more than 10,000 genes in filamentous fungi (Aspergillus sp.). Yeast and filamentous fungi are expected to share much of their primary metabolism; therefore much understanding of the central metabolism and regulation in less-studied filamentous fungi can be learned from comparative metabolite profiling and metabolomics of yeast and filamentous fungi. Filamentous fungi also have a very active and diverse secondary metabolism in which many of the additional genes present in fungi, compared with yeast, are likely to be involved. Although the 'blueprint' of a given organism is represented by the genome, its behaviour is expressed as its phenotype, i.e. growth characteristics, cell differentiation, response to the environment, the production of secondary metabolites and enzymes. Therefore the profile of (secondary) metabolites--fungal chemodiversity--is important for functional genomics and in the search for new compounds that may serve as biotechnology products. Fungal chemodiversity is, however, equally efficient for identification and classification of fungi, and hence a powerful tool in fungal taxonomy. In this paper, the use of metabolite profiling is discussed for the identification and classification of yeasts and filamentous fungi, functional analysis or discovery by integration of high performance analytical methodology, efficient data handling techniques and core concepts of species, and intelligent screening. One very efficient approach is direct infusion Mass Spectrometry (diMS) integrated with automated data handling, but a full metabolic picture requires the combination of several different analytical techniques.

Biochemistry↗

PubMed2004

Penicillium expansum: consistent production of patulin, chaetoglobosins, and other secondary metabolites in culture and their natural occurrence in fruit products.

Penicillium expansum is known for its destructive rot and patulin production in apple juice. According to the literature, P. expansum can, among other compounds, produce citrinin, ochratoxin A, patulin, penitrem A, and rubratoxin B. In this study the qualitative production of metabolites was examined using TLC (260 isolates), HPLC (85 isolates), and MS (22 isolates). The results showed that none of the 260 isolates produced ochratoxin A, penitrem A, or rubratoxin B. However, chaetoglobosin A and communesin B were produced consistently by all 260 isolates. Patulin and roquefortine C were produced by 98% of the isolates. Expansolides A/B and citrinin were detected in 91 and 85% of the isolates, respectively. Chaetoglobosins and communesins were detected in naturally infected juices and potato pulp, whereas neither patulin nor citrinin was found. Because most P. expansum isolates produce patulin, citrinin, chaetoglobosins, communesins, roquefortine C, and expansolides A and B, foods contaminated with this fungus should ideally be examined for chaetoglobosin A as well as patulin.

Chromatography, High Pressure Liquid↗

PubMed2004

A new matching algorithm for high resolution mass spectra.

We present a new matching algorithm designed to compare high-resolution spectra. Whereas existing methods are bound to compare fixed intervals of ion masses, the accurate mass spectrum (AMS) distance method presented here is independent of any alignment. Based on the Jeffreys-Matusitas (JM) distance, a difference between observed peaks across pairs of spectra can be calculated, and used to find a unique correspondence between the peaks. The method takes into account that there may be differences in resolution of the spectra. The algorithm is used for indexing in a database containing 80 accurate mass spectra from an analysis of extracts of 80 isolates representing the nine closely related species in the Penicillium series Viridicata. Using this algorithm we can obtain a retrieval performance of approximately 97-98% that is comparable with the best of the existing methods (e.g., the dot-product distance). Furthermore, the presented method is independent of any variable alignment procedures or binning.

Algorithms↗

PubMed2004

A proposed framework for the description of plant metabolomics experiments and their results.

The study of the metabolite complement of biological samples, known as metabolomics, is creating large amounts of data, and support for handling these data sets is required to facilitate meaningful analyses that will answer biological questions. We present a data model for plant metabolomics known as ArMet (architecture for metabolomics). It encompasses the entire experimental time line from experiment definition and description of biological source material, through sample growth and preparation to the results of chemical analysis. Such formal data descriptions, which specify the full experimental context, enable principled comparison of data sets, allow proper interpretation of experimental results, permit the repetition of experiments and provide a basis for the design of systems for data storage and transmission. The current design and example implementations are freely available (http://www.armet.org/). We seek to advance discussion and community adoption of a standard for metabolomics, which would promote principled collection, storage and transmission of experiment data.

Database Management Systems↗

PubMed2004

The P450 monooxygenase BcABA1 is essential for abscisic acid biosynthesis in Botrytis cinerea.

The phytopathogenic ascomycete Botrytis cinerea is known to produce abscisic acid (ABA), which is thought to be involved in host-pathogen interaction. Biochemical analyses had previously shown that, in contrast to higher plants, the fungal ABA biosynthesis probably does not proceed via carotenoids but involves direct cyclization of farnesyl diphosphate and subsequent oxidation steps. We present here evidence that this "direct" pathway is indeed the only one used by an ABA-overproducing strain of B. cinerea. Targeted inactivation of the gene bccpr1 encoding a cytochrome P450 oxidoreductase reduced the ABA production significantly, proving the involvement of P450 monooxygenases in the pathway. Expression analysis of 28 different putative P450 monooxygenase genes revealed two that were induced under ABA biosynthesis conditions. Targeted inactivation showed that one of these, bcaba1, is essential for ABA biosynthesis: DeltaBcaba1 mutants contained no residual ABA. Thus, bcaba1 represents the first identified fungal ABA biosynthetic gene.

Abscisic Acid↗

PubMed2003

Fungal metabolite screening: database of 474 mycotoxins and fungal metabolites for dereplication by standardised liquid chromatography-UV-mass spectrometry methodology.

A standardised LC-UV-MS micro-scale method for screening of fungal metabolites and mycotoxins in culture extracts is presented. The paper includes data for detection and dereplication of > 400 fungal metabolites to facilitate detection and identification when standards are not available. The data also shows the types of components that can be analysed by positive electrospray (ESI+) mass spectrometry (MS) along with common fragments and adducts of these, as well as giving suggestions on whether UV or ESI+-MS methods should be used. Examples of dereplication of penitrems and macro-cyclic ichothecenes, and detection of several novel compounds are shown. This was done by UV spectroscopy combined with accurate mass determination of adduct and fragment ions obtained by high-resolution orthogonal time-of-flight MS.

Chromatography, Liquid↗

PubMed2003

Flavor release measurement by atmospheric pressure chemical ionization ion trap mass spectrometry, construction of interface and mathematical modeling of release profiles.

An instrumental on-line retronasal flavor analysis was developed to obtain information about the release of flavor compounds in expired air from humans during eating. The volatile flavor compounds were measured by ion trap mass spectrometry with an atmospheric pressure chemical ionization source (APCI). An interface was designed to sample the breath directly from the nose. The repeatability in vitro for seven different flavor compounds came out with relative standard derivation less than 10% in most cases, which is acceptable. In vitro quantification was carried out by a determination of the concentration in the gas phase over a flavor solution by GC/MS, followed by measurements of intensities by the APCI ion trap. Ion suppression by acetone in the breath was negligible at concentration levels relevant in these experiments. The instrumental limits of detection for menthone and menthol coincide with that of the flavor detection threshold. An application study on the release of menthone and menthol from chewing gum by a group of six test persons was performed. Flavored chewing gum was used as a model matrix because of the long chewing periods and the simplicity of the system. It is concluded that the interface and the method can be used to measure breath from the nose. A mathematical model of the data was developed to give a quantitative method for description and characterization of the release of flavor compounds. The release profiles consisted of two sequences, one for a chewing period, and one for a phasing out process. The proposed method for modeling provided a reasonable description of the release process. In addition to flavor compounds, this new interface and mathematical application could provide information on chemicals in the human breath, which could be interesting, for example, within medical diagnosis.

Journal Article↗

PubMedDate not supplied

Mass spectrometry in metabolome analysis.

In the post-genomic era, increasing efforts have been made to describe the relationship between the genome and the phenotype in cells and organisms. It has become clear that even a complete understanding of the state of the genes, messages, and proteins in a living system does not reveal its phenotype. Therefore, researchers have started to study the metabolome (or the metabolic complement of functional genomics). Within this context, mass spectrometry (MS) has increasingly occupied a central position in the methodologies developed for determination of the metabolic state. This review is mainly focused on the status of MS in the metabolome field, trying to direct the reader to the main approaches for analysis of metabolites, reviewing basic methodologies in sample preparation, and the most recent MS techniques introduced. Apart from the description of the different methods, this review will try to state a general comparison between the several different techniques that involve MS and metabolite analysis, and will highlight their limitations and preferred applicability.

Animals↗