Trivialities in metabolomics: Artifacts in extraction and analysis.

The aim of this review is to show the risks of artifact formation in metabolomics analyses. Metabolomics has developed in a major tool in system biology approaches to unravel the metabolic networks that are the basis of life. Presently TLC, LC-MS, GC-MS, MS-MS and nuclear magnetic resonance are applied to analyze the metabolome of all kind of biomaterials. These analytical methods require robust preanalytical protocols to extract the small molecules from the biomatrix. The quality of the metabolomics analyses depends on protocols for collecting and processing of the biomaterial, including the methods for drying, grinding and extraction. Also the final preparation of the samples for instrumental analysis is crucial for highly reproducible analyses. The risks of artifact formation in these steps are reviewed from the point of view of the commonly used solvents. Examples of various artifacts formed through chemical reactions between solvents or contaminations with functional groups in the analytes are discussed. These reactions involve, for example, the formation of esters, trans-esterifications, hemiacetal and acetal formation, N-oxidations, and the formation of carbinolamines. It concerns chemical reactions with hydroxyl-, aldehyde-, keto-, carboxyl-, ester-, and amine functional groups. In the analytical steps, artifacts in LC may come from the stationary phase or reactions of the eluent with analytes. Differences between the solvent of the injected sample and the LC-mobile phase may cause distortions of the retention of analytes. In all analytical methods, poorly soluble compounds will be in all samples at saturation level, thus hiding a potential marker function. Finally a full identification of compounds remains a major hurdle in metabolomics, it requires a full set of spectral data, including methods for confirming the absolute stereochemistry. The putative identifications found in supplemental data of many studies, unfortunately, often become "truly" identified compounds in papers citing these results. Proper validation of the protocols for preanalytical and analytical procedures is essential for reproducible analyses in metabolomics.

Chalcone synthase and its functions in plant resistance.

Chalcone synthase (CHS, EC 2.3.1.74) is a key enzyme of the flavonoid/isoflavonoid biosynthesis pathway. Besides being part of the plant developmental program the CHS gene expression is induced in plants under stress conditions such as UV light, bacterial or fungal infection. CHS expression causes accumulation of flavonoid and isoflavonoid phytoalexins and is involved in the salicylic acid defense pathway. This review will discuss CHS and its function in plant resistance.

Cannabis smoke condensate II: influence of tobacco on tetrahydrocannabinol levels.

Medicinal cannabis has attracted a lot of attention in recent times. Various forms of administration are used, of which smoking is very common but the least desirable. Smoking cannabis generates a large amount of unwanted side products, of which carcinogenic compounds are the most dangerous. A common practice among recreational drug users, and to a lesser degree patients who uses cannabis as medicine, is to mix the cannabis material with commercially available tobacco in order to increase the burning efficiency of the cigarette and to reduce the overall costs of the cigarette. In this study cannabis material has been mixed with tobacco in order to determine whether tobacco has an influence on the amount of and ratio between tetrahydrocannabinol (THC), cannabigerol (CBG), and cannabinol (CBN) administered while smoking. A small-scale smoking machine has been used and cannabis mixed with various ratios of tobacco was smoked. The trapped smoke was quantitatively analyzed by high-performance liquid chromatography (HPLC) and the amount of THC, CBG, and CBN was determined for each cigarette. We have found that tobacco increases the amount of THC inhaled per gram of cannabis from 32.70 +/- 2.29 mg/g for a 100% cannabis cigarette to 58.90 +/- 2.30 mg/g for a 25% cannabis cigarette. This indicates that tobacco increases the vaporization efficiency of THC by as much as 45% under the conditions tested.

Cannabis smoke condensate III: the cannabinoid content of vaporised Cannabis sativa.

Cannabis sativa is a well-known recreational drug and, as such, a controlled substance of which possession and use are illegal in most countries of the world. Due to the legal constraints on the possession and use of C. sativa, relatively little research on the medicinal qualities of this plant has been conducted. Interest in the medicinal uses of this plant has, however, increased in the last decades. The methods of administration for medicinal purposes are mainly through oral ingestion, smoking, and nowadays also inhalation through vaporization. During this study the commercially available Volcano vaporizing device was compared with cannabis cigarette smoke. The cannabis smoke and vapor (obtained at different temperatures) were quantitatively analyzed by high-performance liquid chromatography (HPLC). In addition, different quantities of cannabis material were also tested with the vaporizer. The cannabinoids:by-products ratio in the vapor obtained at 200 degrees C and 230 degrees C was significantly higher than in the cigarette smoke. The worst ratio of cannabinoids:by-products was obtained from the vaporized cannabis sample at 170 degrees C.

Effect of intrapulmonary tetrahydrocannabinol administration in humans.

This randomised, double-blind, placebo-controlled, cross-over study was designed to identify which pharmacodynamic parameters most accurately quantify the effects of delta-9-Tetrahydrocannabinol (THC), the predominantly psychoactive component of cannabis. In addition, we investigated the acceptability and usefulness of a novel mode of intrapulmonary THC administration using a Volcano vaporizer and pure THC instead of cannabis. Rising doses of THC (2, 4, 6 and 8 mg) or vehicle were administered with 90 minutes intervals to twelve healthy males using a Volcano vaporizer. Very low between-subject variability was observed in THC plasma concentrations, characterising the Volcano vaporizer as a suitable method for the administration of THC. Heart rate showed a sharp increase and rapid decline after each THC administration (8 mg: 19.4 bpm: 95% CI 13.2, 25.5). By contrast, dose dependent effects of body sway (8 mg: 108.5%: 95% CI 72.2%, 152.4%) and different subjective parameters did not return to baseline between doses (Visual Analogue Scales of 'alertness' (8 mg: -33.6 mm: 95% CI -41.6, -25.7), 'feeling high' (8 mg: 1.09 U: 95% CI 0.85, 1.33), 'external perception' (8 mg: 0.62 U: 95% CI 0.37, 0.86)). PK/PD-modeling of heart rate displayed a relatively short equilibration half-life of 7.68 min. CNS parameters showed equilibration half-lives ranging between 39.4 - 84.2 min. Some EEG-frequency bands, and pupil size showed small changes following the highest dose of THC. No changes were seen in saccadic eye movements, smooth pursuit and adaptive tracking performance. These results may be applicable in the development of novel cannabinoid agonists and antagonists, and in studies of the pharmacology and physiology of cannabinoid systems in humans.

Cannabis smoke condensate I: the effect of different preparation methods on tetrahydrocannabinol levels.

Cannabis sativa contains more than 400 known compounds, of which the terpene chemicals, called cannabinoids, are unique to this species. The cannabinoids, which occur as the corresponding acids in the plant material, are the major psychoactive components in this species. The compounds are decarboxylated from the inactive acidic form into the active form by means of smoking. Previous research has made use of the tobacco industry's standard method and adaptations thereof to produce a cannabis smoke condensate. In this study the method of smoke production, which includes the puff frequency, puff length, and puff volume, was tested and the concentration of the major cannabinoid, Delta(9)-tetrahydrocannabinol (THC), and the amount of by-products produced under the different conditions were quantified. This study aimed at combining the existing methodology and at providing quantitative results on the influence of the preparation method on the concentration of THC in the smoke. The results indicate that the method of smoke production influences the amount of THC produced (e.g., longer puff length yielding a higher amount of THC). The THC concentration in the smoke condensate varied between 22.17 mg/g of cannabis and 54.00 mg/g, while the amount of by-products produced varied between 25.57 mg/g and 107.40 mg/g.

Overproduction of salicylic acid in plants by bacterial transgenes enhances pathogen resistance.

After a hypersensitive response to invading pathogens, plants show elevated accumulation of salicylic acid (SA), induced expression of plant defense genes, and systemic acquired resistance (SAR) to further infection by a broad range of pathogens. There is compelling evidence that SA plays a crucial role in triggering SAR. We have transformed tobacco with two bacterial genes coding for enzymes that convert chorismate into SA by a two-step process. When the two enzymes were targeted to the chloroplasts, the transgenic (CSA, constitutive SA biosynthesis) plants showed a 500- to 1,000-fold increased accumulation of SA and SA glucoside compared to control plants. Defense genes, particularly those encoding acidic pathogenesis-related (PR) proteins, were constitutively expressed in CSA plants. This expression did not affect the plant phenotype, but the CSA plants showed a resistance to viral and fungal infection resembling SAR in nontransgenic plants.

Identification of phenylpropanoids in methyl jasmonate treated Brassica rapa leaves using two-dimensional nuclear magnetic resonance spectroscopy.

Metabolic analysis showed a clear increase in phenylpropanoid levels in Brassica rapa leaves after treatment with methyl jasmonate. A fraction of phenylpropanoids was prepared by Diaion HP-20 and Sephadex LH-20 column chromatography after MeOH-water extraction. Even with these purification steps, isolation of each phenylpropanoid for structure elucidation is not easy due to the low levels in the plants (ca. 0.004%). A mixture was analyzed without further purification using HPLC-electrospray ionization mass spectrometry and NMR spectroscopy. Based on the NMR results including (1)H NMR, J-resolved, correlated spectroscopy (COSY), heteronuclear single quantum coherence (HSQC), and heteronuclear multiple bond correlation (HMBC) spectra, both (1)H and (13)C resonances of the compounds were obtained. Using these NMR data, five phenylpropanoids conjugated with malate were identified: 5-hydroxyferuloyl-, caffeoyl-, coumaroyl-, feruloyl-, and sinapoyl malate. Of the compounds, 5-hydroxyferuloyl malate is a new phenylpropanoid. In addition to the five constitutive phenylpropanoids bearing trans-configuration, their cis forms, which are believed to be artifacts formed in the course of extraction steps, were also identified in the fraction.

Two new pimarane diterpenoids from Strychnos vanprukii Craib.

Two new pimarane diterpenoids, 7beta,12beta-dihydroxypimara-8,15-dien-14-one (1) and 14-hydroxy-15,16-dinorpimara-8,11,13-trien-7-one (2), were isolated from the n-hexane extract of the stem of Strychnos vanprukii Craib and their structures were elucidated based on spectroscopic evidences.

ORCAnization of jasmonate-responsive gene expression in alkaloid metabolism.

Jasmonic acid is an important plant stress signalling molecule. It induces the biosynthesis of defence proteins and protective secondary metabolites. In alkaloid metabolism, jasmonate acts by coordinate activation of the expression of multiple biosynthesis genes. In terpenoid indole alkaloid metabolism and primary precursor pathways, jasmonate induces gene expression and metabolism via ORCAs, which are members of the AP2/ERF-domain family of plant transcription factors. Other jasmonate-regulated (secondary) metabolic pathways might also be controlled by ORCA-like AP2/ERF-domain transcription factors. If so, such regulators could be used to improve plant fitness or metabolite productivity of plants or cell cultures.

Influence of Precursor Availability on Alkaloid Accumulation by Transgenic Cell Line of Catharanthus roseus

We have used a transgenic cell line of Catharanthus roseus (L.) G. Don to study the relative importance of the supply of biosynthetic precursors for the synthesis of terpenoid indole alkaloids. Line S10 carries a recombinant, constitutively overexpressed version of the endogenous strictosidine synthase (Str) gene. Various concentrations and combinations of the substrate tryptamine and of loganin, the immediate precursor of secologanin, were added to suspension cultures of S10. Our results indicate that high rates of tryptamine synthesis can take place under conditions of low tryptophan decarboxylase activity, and that high rates of strictosidine synthesis are possible in the presence of a small tryptamine pool. It appears that the utilization of tryptamine for alkaloid biosynthesis enhances metabolic flux through the indole pathway. However, a deficiency in the supply of either the iridoid or the indole precursor can limit flux through the step catalyzed by strictosidine synthase. Precursor utilization for the synthesis of strictosidine depends on the availability of the cosubstrate; the relative abundance of these precursors is a cell-line-specific trait that reflects the metabolic status of the cultures.

Engineering secondary metabolite production in plants.

Recent achievements have been made in the metabolic engineering of plant secondary metabolism. Various pathways have been altered using genes encoding biosynthetic enzymes or genes encoding regulatory proteins. In addition, antisense genes have been used to block competitive pathways, thereby increasing the flux towards the desired secondary metabolites.

Ethnopharmacology and systems biology: a perfect holistic match.

Traditional medical doctors often apply a holistic approach in prescribing medicines to the patient. Each individual patient gets his own optimalized medicine, usually a mixture of different ingredients. The present day paradigm of drug development of , is based on a super reductionist approach which involves mostly tests of compounds at the molecular level in, e.g., receptor binding assays. This approach is not the best for studies on traditional medicines. A more holistic approach using systems biology seems much more suited to proof efficacy and to obtain information that might lead to understanding the mode of action. Synergy, prodrugs, novel targets, all these might be detected by a systems biology approach whereas the reductioinist approach only will recognize activity on already known targets, and will not detect synergism and prodrugs. Metabolomics will be a major tool in recognizing compounds connected with activity in the traditional medicines, and will also be very useful in recognizing the effect on the test organism, which can be the patient in case of clinical trials with well established traditional medicines.

The iridoid glucoside secologanin is derived from the novel triose phosphate/pyruvate pathway in a Catharanthus roseus cell culture.

Secologanin is the iridoid building block of the majority of the terpenoid indole alkaloids. In the biosynthesis of secologanin, mevalonate was considered to be the exclusive precursor of isopentenyl diphosphate. After [1-(13)C]glucose feeding to a cell culture of Catharanthus roseus, its incorporation into secologanin was studied by 13C NMR spectroscopy. The data showed that the novel triose phosphate/pyruvate and not the mevalonate pathway was the major route for the biosynthesis of secologanin.

Geraniol 10-hydroxylase, a cytochrome P450 enzyme involved in terpenoid indole alkaloid biosynthesis.

Geraniol 10-hydroxylase (G10H) is a cytochrome P450 monooxygenase involved in the biosynthesis of iridoid monoterpenoids and several classes of monoterpenoid alkaloids found in a diverse range of plant species. Catharanthus roseus (Madagascar periwinkle) contains monoterpenoid indole alkaloids, several of which are pharmaceutically important. Vinblastine and vincristine, for example, find widespread use as anti-cancer drugs. G10H is thought to play a key regulatory role in terpenoid indole alkaloid biosynthesis. We purified G10H from C. roseus cells. Using degenerate PCR primers based on amino acid sequence information we cloned the corresponding cDNA. The encoded CYP76B6 protein has G10H activity when expressed in C. roseus and yeast cells. The stress hormone methyljasmonate strongly induced G10h gene expression coordinately with other terpenoid indole alkaloid biosynthesis genes in a C. roseus cell culture.

4-Hydroxy-2-pyrone formation by chalcone and stilbene synthase with nonphysiological substrates.

Valerophenone synthase (VPS) is a polyketide synthase that catalyzes the formation of the phloroglucinol derivatives in the synthesis of the bitter acids in hop (Humulus lupulus). The reaction uses isovaleryl-CoA or isobutyryl-CoA, but otherwise it is identical to that of the chalcone synthase in flavonoid biosynthesis. Our study showed that chalcone synthase can perform the function of VPS, but not perfectly, because the majority of the reactions terminated after two condensation reactions (products: 4-hydroxy-2-pyrone derivatives). The same experiments with stilbene synthase yielded exclusively the 4-hydroxy-2-pyrone derivatives, not the products expected from three condensation reactions. The results are discussed in the context of the functional diversity and evolution in the family of CHS-related polyketide synthases.

Screening for acetylcholinesterase inhibitors from Amaryllidaceae using silica gel thin-layer chromatography in combination with bioactivity staining.

Thin-layer chromatography (TLC) was used to screen for acetylcholinesterase inhibitors from Amaryllidaceae extracts. The TLC plate was developed and then stained using Ellman's reagent, 5,5'-dithiobis-(2-nitrobenzoic acid), to detect acetylcholinesterase activity. The advantages of this TLC assay method were that we could dereplicate the known inhibitor galanthamine, widely occurring in Amaryllidaceae, at an early stage of the isolation procedure. Moreover, there is no disturbance from sample dissolving solvents as in the microplate assay, and it is a very simple method. The detection limits were 10-200 ng for several known acetylcholinesterase inhibitors tested, and it is thus more sensitive than UV or Dragendorff's reagent detection. Also the minimal detectable amount for an acetylcholinesterase inhibitor tested was much less than that needed for the microplate assay. We screened 15 Amaryllidaceae extracts using this TLC method, and chose candidates for acetylcholinesterase inhibitor isolation.

Assay of 2,3-dihydroxybenzoic acid and related compounds in plant materials by high-performance liquid chromatography.

Salicylic acid and its putative biosynthetic precursors were assayed isocratically by RP-HPLC with UV detection at 280 nm. Optimum resolution was provided by an HPLC mobile phase consisting of MeOH-1% aqueous HOAc (40:60, v/v), at pH 4. Furthermore, for the analysis of 2,3-dihydroxybenzoic acid (2,3-DHBA) in Catharanthus roseus cell cultures after elicitation, a mobile phase consisting of acetonitrile-1% aqueous HCOOH containing 0.25% trichloroacetic acid (1:5, v/v), at pH 2, was used. The recovery for the free form of 2,3-DHBA was about 80% after a one-step extraction of the cells. The detection limit of 2,3-DHBA was 3 microg by using saligenin as an internal standard.

High-performance liquid chromatography with on-line coupled UV, mass spectrometric and biochemical detection for identification of acetylcholinesterase inhibitors from natural products.

A high-performance liquid chromatography (HPLC) method with on-line coupled ultraviolet (UV), mass spectrometry (MS) and biochemical detection for acetylcholinesterase (AChE) inhibitory activity has been developed. By combining the separation power of HPLC, the high selectivity of biochemical detection, and the ability to provide molecular mass and structural information of MS, AChE inhibitors can be rapidly identified. The biochemical detection was based on a colorimetric method using Ellman's reagent. The detection limit of galanthamine, an AChE inhibitor, in the HPLC-biochemical detection is 0.3 nmol. The three detector lines used, i.e., UV, MS and Vis for the biochemical detection were recorded simultaneously and the delay times of the peaks obtained were found to be consistent. This on-line post-column detection technique can be used for the identification of AChE inhibitors in plant extracts and other complex mixtures such as combinatorial libraries.

Growth and alkaloid contents in leaves of Tabernaemontana pachysiphon Stapf (Apocynaceae) as influenced by light intensity, water and nutrient supply.

The growth of Tabernaemontana pachysiphon (Apocynaceae) plants and the alkaloid content of leaves were investigated in the greenhouse at three levels of nutrient supply under two contrasting water and light regimes. We determined height increment, above-ground biomass production, leaf size, specific leaf weight and the content of the alkaloids apparicine, A2, isovoacangine, tubotaiwine and tubotaiwine-N-oxide. The effects of major controlling factors such as light, water and nutrient supply could be directly correlated with growth and were largely independent of each other. In contrast, leaf-alkaloid contents were influenced by interdependencies among the main factors and individually affected in a synergistic or antagonistic manner which deviated from the effects on growth. The following general trends could be identified with respect to the quantitatively predominant alkaloids apparicine, tubotaiwine and isovoacangine. Increasing nutrient supply had a positive effect on both growth and alkaloid content. Drought increased alkaloid content, but retarded growth. High light intensity lowered alkaloid content but promoted growth. We investigated the relationship between primary production and the production of secondary metabolites with respect to relative and total alkaloid content as well as in relation to the leaves' nitrogen status. Our results showed that under conditions of low nutrient supply, higher proportions of leaf nitrogen were allocated to alkaloids than at moderate or high nutrient supply. Under conditions of drought and low light, all plants allocated almost equal proportions of leaf nitrogen to alkaloids, regardless of fertiliser. Total alkaloid content per plant, however, increased with fertilisation. With respect to the N-allocation strategy, we found no indication of a trade-off between primary production and the production of secondary metabolites in this species. Rather, our results are in accordance with the carbon nutrient balance hypothesis.