[Agar plate electrophoresis for separation and bioautographic determination of vitamin B complex and mixtures of vitamin antagonists and antibiotics].
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Future potential of targeted component analysis by multidimensional liquid chromatography-mass spectrometry.
Multidimensional liquid chromatography (MDLC) may be used in either (i) the profiling mode where it is the objective to fractionate all components in a mixture or (ii) the targeted component mode in which it is the objective to determine specific analytes. This paper focuses on targeted component analysis from complex mixtures, addressing the critical operations of analyte selection and transport from the first to the second dimension. Although the physical operation of switching a component into the second dimension with computer controlled valving is simple, it is shown that changes in analyte retention time and peak width with column age and fouling are a serious problem. The analyte moves out of the preselected time window for valve switching and quantitation is compromised in the second dimension. It is proposed that a solution to the "drifting peak" phenomenon in targeted component analysis is to use binary mobility elution in the first dimension. Binary mobility refers to those systems, such as affinity chromatography, in which analyte mobility is generally either 0 or 1 relative to mobile phase velocity. Coupling these binary changes in analyte mobility in the first dimension with valve switching eliminates the "drifting peak" phenomenon. In addition, it is shown that a wide time window may be used in affinity separations without compromising the separation or accumulating contaminants. Several cases are described in which immunosorbents were used with reversed phase columns to provide quantitative targeted component analyses from complex mixtures.
Animals↗
Method for evaluating the bioconversion of radioactive polyunsaturated fatty acids by use of reversed-phase liquid chromatography.
Reversed-phase high-performance liquid chromatography on a thermostatted octadecylsilyl column was used to separate mixtures of labelled polyunsaturated fatty acids (as their methyl esters) formed by successive desaturations and elongations of labelled linoleic (18:2 n - 6) or linolenic (18:3 n - 3) acid by rat liver microsomes. Acetonitrile-water mixtures were used for elution of the esters. Unsaturated and saturated esters were detected by their refractive indices. The order of elution of fatty acid methyl esters in complex mixtures varies as a function of the chain length and unsaturation, analysis temperature, water concentration and solvent flow-rate. The peak areas vary as a function of the unsaturation. Specific radioactivities of 14C-labelled fatty acids and the percentage distribution of radioactivity among fatty acids from complex mixtures can be efficiently determined by collection and direct measurement of the radioactivity in the solvent by liquid scintillation counting. The method can be applied to complete compositional analysis, but is especially useful for determination of specific radioactivities during studies on the metabolic conversion of labelled polyunsaturated fatty acids.
Chromatography, Gas↗
Ecotoxicological evaluation of diesel-contaminated soil before and after a bioremediation process.
Evaluation of contaminated sites is usually performed by chemical analysis of pollutants in soil. This is not enough either to evaluate the environmental risk of contaminated soil nor to evaluate the efficiency of soil cleanup techniques. Information on the bioavailability of complex mixtures of xenobiotics and degradation products cannot be totally provided by chemical analytical data, but results from bioassays can integrate the effects of pollutants in complex mixtures. In the preservation of human health and environment quality, it is important to assess the ecotoxicological effects of contaminated soils to obtain a better evaluation of the healthiness of this system. The monitoring of a diesel-contaminated soil and the evaluation of a bioremediation technique conducted on a microcosm scale were performed by a battery of ecotoxicological tests including phytotoxicity, Daphnia magna, and nematode assays. In this study we biostimulated the native microflora of soil contaminated with diesel by adding nutrients and crop residue (corn straw) as a bulking agent and as a source of microorganisms and nutrients; in addition, moisture was adjusted to enhance diesel removal. The bioremediation process efficiency was evaluated directly by an innovative, simple phytotoxicity test system and the diesel extracts by Daphnia magna and nematode assays. Contaminated soil samples were revealed to have toxic effects on seed germination, seedling growth, and Daphnia survival. After biostimulation, the diesel concentration was reduced by 50.6%, and the soil samples showed a significant reduction in phytotoxicity (9%-15%) and Daphnia assays (3-fold), confirming the effectiveness of the bioremediation process. Results from our microcosm study suggest that in addition to the evaluation of the bioremediation processes efficiency, toxicity testing is different with organisms representative of diverse phylogenic levels. The integration of analytical, toxicological and bioremediation data is necessary to properly assess the ecological risk of bioremediation processes.
Animals↗
Application of several modified peak purity assays to real complex multicomponent mixtures by high-performance liquid chromatography with diode-array detection.
Simple to use interactive self-modelling mixture analysis (SIMPLISMA), orthogonal projection (OPA) and Needle Search (NS) approaches have been applied to the determination of a number of compounds present in a complex multicomponent system. None of these three approaches succeeded completely when they were tested using the whole data matrix. When OPA and NS were applied to three simpler submatrices, obtained by dividing the total data matrix, and where a smaller number of compounds were present, better performance was achieved.
Chromatography, High Pressure Liquid↗
Developing liquid chromatography ion mobility mass spectometry techniques.
When a packet of ions in a buffer gas is exposed to a weak electric field, the ions will separate according to differences in their mobilities through the gas. This separation forms the basis of the analytical method known as ion mobility spectroscopy and is highly efficient, in that it can be carried out in a very short time frame (micro- to milliseconds). Recently, efforts have been made to couple the approach with liquid-phase separations and mass spectrometry in order to create a high-throughput and high-coverage approach for analyzing complex mixtures. This article reviews recent work to develop this approach for proteomics analyses. The instrumentation is described briefly. Several multidimensional data sets obtained upon analyzing complex mixtures are shown in order to illustrate the approach as well as provide a view of the limitations and required future work.
Animals↗
Development and validation of in vitro induction assays for toxic halogenated aromatic mixtures: a review.
Halogenated aromatic industrial compounds, typified by the polychlorinated dibenzo-p-dioxins (PCDDs), dibenzofurans (PCDFs) and biphenyls (PCBs) have been identified as residues in almost every component of the global ecosystem. Risk assessment of the complex mixtures of halogenated aromatics found in environmental samples is complicated by analytical problems and the lack of toxicological information on individual compounds and mixtures. Research in our laboratory has focused on the development and vadidation of the in vitro aryl hydrocarbon hydroxylase (AHH) induction assay in rat hepatoma H-4-II E cells in culture for quantitating individual toxic halogenated aryl hydrocarbons and their mixtures. For several PCB, PCDD, PCDF congeners, their mixed bromo/chloro analogs and reconstituted mixtures there was an excellent linear correlation between their -log ED50 values for AHH induction in rat hepatoma cells and their -log ED50 values for in vivo hepatic microsomal AHH induction, inhibition of body weight gain and thymic atrophy in the rat. It has also been shown for selected compounds that there was a good correlation between their in vitro AHH induction potencies and their effects in guinea pigs (AHH induction, inhibition of body weight gain) and mice (immunotoxicity). This assay system has been utilized to quantitative the "2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) equivalents" present in extracts from diverse sources including fly ash from a municipal incinerator and pyrolyzed brominated flame retardants which contain a complex mixture of halogenated dibenzo-p-dioxins and dibenzofurans.
Animals↗
Reproducibility of quantitative proteomic analyses of complex biological mixtures by multidimensional protein identification technology.
If quantitative proteomic technologies are to be of widespread use to the biological community, the reproducibility of each method must be investigated and determined. We have analyzed the reproducibility of complex quantitative proteomic analyses of metabolically labeled S. cerevisiae analyzed via multidimensional protein identification technology (MudPIT). Three independent cell growths of S. cerevisiae grown in rich and minimal media and independent MudPIT analyses of each were compared and contrasted. Quantitative MudPIT was found to be intra- and interexperimentally reproducible at both the peptide and protein levels. Proteins of potential low abundance were detected, identified, and quantified by identical peptides from three independent samples. In addition, when multiple peptides were matched to a protein, the relative abundance of each peptide was in agreement across the three samples. Despite the reproducibility, errors in the experimental determination of protein expression levels occurred, but the impact of the variation was minimized by replicate experiments. Last, quantitative MudPIT analyses will likely be improved by increasing the number of peptide hits per protein in a given analysis, which will provide for greater intraexperimental reproducibility.
Amino Acid Sequence↗
Synaptic interactions due to convergent input from gustatory afferent fibers in the rostral nucleus of the solitary tract.
1. Whole cell recordings from neurons in the rostral, gustatory nucleus of the solitary tract (rNST) were made using the "blind" patch-clamp technique in horizontal brain stem slices of rats. 2. Postsynaptic potentials (PSP) were elicited in 71 rNST neurons by electrical stimulation of the solitary tract (ST). To investigate PSPs evoked by convergent input from the chorda tympani and glossopharyngeal nerves, the ST was stimulated at levels where these two nerves terminate. These are referred to as rostral (rST) and intermediate (iST) ST, respectively. 3. When the rST was stimulated 72% of the PSPs were depolarizing, and 28% were hyperpolarizing (n = 64). Stimulation of the intermediate ST resulted in 75% depolarizing and 25% hyperpolarizing PSPs (n = 56). 4. Application of gamma-aminobuturic acid-A (GABAA) and glutamate receptor blockers revealed that all PSPs recorded in the present study were a composite of summed excitatory and inhibitory PSPs. Application of the GABAA receptor blocker bicuculline, by eliminating the hyperpolarizing component of a PSP, revealed the excitatory postsynaptic potential (EPSP) component of the potential. Bicuculline also increased the amplitude and prolonged the decay time of the depolarizing potentials once the hyperpolarizing potential component had been eliminated. These pure EPSP revealed by GABAA receptor blockade reversed at approximately 0 mV. 5. Application of glutamate ionotropic receptor blockers effectively eliminated the initiation of the synaptic responses evoked by ST stimulation. If the stimulus strength was increased, an inhibitory postsynaptic potential (IPSP) was elicited, presumably by direct activation of interneurons close to the stimulating electrode. These IPSPs had a mean reversal potential of -88 mV. 6. When synaptic responses were initiated by stimulation of the projection areas of both the chorda tympani and glossopharyngeal nerves, all neurons tested (n = 49) responded to stimulation of both sites on the ST. The resulting synaptic potential was a sum of the two individual synaptic potentials. 7. If stimulation of the rostral and intermediate sites both elicited depolarizing potentials, the potential resulting from stimulation of both sites was the arithmetical sum of the two individual PSPs. The EPSPs summed even if the time between stimulation of the rostral and intermediate sites was separated by < or = 100 ms. 8. Inhibitory PSPs evoked by simultaneous stimulation of the rostral and intermediate ST also summed. The summation was not linear and saturated at a mean level of -66 mV. 9. When the PSP at one stimulation site was excitatory but inhibitory at the other site, the PSP wave form resulting from dual stimulation was a complex mixture of the two individual potentials. The inhibitory potential was capable of blocking action potentials resulting from the excitatory PSP. 10. These results indicate that synaptic responses in rNST are complex mixtures of excitatory and inhibitory potentials. The synaptic potentials result from excitatory afferent input mediated by glutamate and inhibitory input derived from interneurons. Inhibitory transmission is mediated primarily at GABAA synapses. Stimulation of the rNST afferent input derived from the chorda tympani and glossopharyngeal nerves reveals complex convergent input. The complexity of these synaptic interactions indicates that considerable processing of gustatory information occurs at the first central synapse in the taste pathway.
Animals↗
Proteome, transcriptome and genome: top down or bottom up analysis?
Biological systems are comprised of protein components found at a wide variety of abundances from millions of molecules of a single species per cell to less than one copy per cell. Because of this wide range of concentrations, measurement or a full accounting of each system is presently unavailable. Conventional separation and analytical methods (two-dimensional gel electrophoresis and mass spectrometry) allow identification and quantitation of many of the most abundant gene products (top down methods); and the majority of gene products, which are found at low abundance, can be neither identified nor measured in complex mixtures at present. The gene products that are found at low levels can be characterized and their properties analyzed by preparing ordered gene libraries of limited complexity from mRNA. When such preparations are expressed in cell free systems and analyzed by two-dimensional gel electrophoresis, the features of the gene products are available for analysis. This 'bottom up' approach allows identification of gene product properties so that analytical procedures can be devised and applied to complex mixtures.
Cell Line↗
DNA adduct measurements, cell proliferation and tumor mutation induction in relation to tumor formation in B6C3F1 mice fed coal tar or benzo[a]pyrene.
Coal tar is a complex mixture containing hundreds of compounds, at least 30 of which are polycyclic aromatic hydrocarbons, including the carcinogen benzo[a]pyrene (BaP). Although humans are exposed to complex mixtures on a daily basis, the synergistic or individual effects of components within a mixture on the carcinogenic process remain unclear. We have compared DNA adduct formation and cell proliferation in mice fed coal tar or BaP for 4 weeks with tumor formation in a 2 year chronic feeding study. Additionally, we have analyzed tumor DNA for mutations in the K-ras, H-ras and p53 genes. In the forestomach of mice fed either coal tar or BaP an adduct indicative of BaP was detected, with adduct levels increasing in a dose-responsive manner. K-ras mutations were detected in the forestomach tumors, with the incidence being similar in mice fed coal tar or BaP. These results suggest that the BaP within coal tar is associated with forestomach tumor induction in coal tar-fed mice. DNA adduct levels in the small intestine were not predictive of tumor incidence in this tissue; instead, the tumors appeared to result from compound-induced cell proliferation at high doses of coal tar. K-ras mutations were detected in lung tumors. Since lung tumors were not increased by BaP, coal tar components other than BaP appear to be responsible for the tumors induced in this tissue. H-ras mutations, primarily occurring at codon 61, were the most common mutation observed in liver tumors induced by coal tar. Since this mutation profile is observed in spontaneous hepatic tumors, components in the coal tar may be promoting the expansion of pre-existing lesions.
Animals↗
UPLC/MS(E); a new approach for generating molecular fragment information for biomarker structure elucidation.
A new approach to obtain fragmentation information in liquid chromatography/mass spectrometry (LC/MS) studies of small molecules in complex mixtures is presented using simultaneous acquisition of exact mass at high and low collision energy, MS(E). LC/MS-TOF and LC/MS/MS-TOF are powerful tools for the analysis of complex mixtures, especially those for biological fluids allowing the elucidation of elemental composition and fragmentation information. In this example the composition of rat urine was studied using this new approach, allowing the structures of several endogenous components to be confirmed in one analytical run by the simultaneous acquisition of exact mass precursor and fragment ion data. The spectral data obtained using this new approach are comparable to those obtained by conventional LC/MS/MS as exemplified by the identification of endogenous metabolites present in rat urine.
Animals↗
Characterization of a new qQq-FTICR mass spectrometer for post-translational modification analysis and top-down tandem mass spectrometry of whole proteins.
The use of a new electrospray qQq Fourier transform ion cyclotron mass spectrometer (qQq-FTICR MS) instrument for biologic applications is described. This qQq-FTICR mass spectrometer was designed for the study of post-translationally modified proteins and for top-down analysis of biologically relevant protein samples. The utility of the instrument for the analysis of phosphorylation, a common and important post-translational modification, was investigated. Phosphorylation was chosen as an example because it is ubiquitous and challenging to analyze. In addition, the use of the instrument for top-down sequencing of proteins was explored since this instrument offers particular advantages to this approach. Top-down sequencing was performed on different proteins, including commercially available proteins and biologically derived samples such as the human E2 ubiquitin conjugating enzyme, UbCH10. A good sequence tag was obtained for the human UbCH10, allowing the unambiguous identification of the protein. The instrument was built with a commercially produced front end: a focusing rf-only quadrupole (Q0), followed by a resolving quadrupole (Q1), and a LINAC quadrupole collision cell (Q2), in combination with an FTICR mass analyzer. It has utility in the analysis of samples found in substoichiometric concentrations, as ions can be isolated in the mass resolving Q1 and accumulated in Q2 before analysis in the ICR cell. The speed and efficacy of the Q2 cooling and fragmentation was demonstrated on an LCMS-compatible time scale, and detection limits for phosphopeptides in the 10 amol/muL range (pM) were demonstrated. The instrument was designed to make several fragmentation methods available, including nozzle-skimmer fragmentation, Q2 collisionally activated dissociation (Q2 CAD), multipole storage assisted dissociation (MSAD), electron capture dissociation (ECD), infrared multiphoton induced dissociation (IRMPD), and sustained off resonance irradiation (SORI) CAD, thus allowing a variety of MS(n) experiments. A particularly useful aspect of the system was the use of Q1 to isolate ions from complex mixtures with narrow windows of isolation less than 1 m/z. These features enable top-down protein analysis experiments as well structural characterization of minor components of complex mixtures.
Equipment Design↗
Separation of 32P-postlabeled DNA adducts of polycyclic aromatic hydrocarbons and nitrated polycyclic aromatic hydrocarbons by HPLC.
The 32P-postlabeling assay, thin-layer chromatography, and reverse-phase high-pressure liquid chromatography (HPLC) were used to separate DNA adducts formed from 10 polycyclic aromatic hydrocarbons (PAHs) and 6 nitrated polycyclic aromatic hydrocarbons (NO2-PAHs). The PAHs included benzo[j]fluoranthene, benzo[k]fluoranthene, indeno[1,2,3-cd]pyrene, benzo[a]pyrene, chrysene, 6-methylchrysene, 5-methylchrysene, and benz[a]anthracene. The NO2-PAHs included 1-nitropyrene, 2-nitrofluoranthene, 3-nitrofluoranthene, 1,6-dinitropyrene, 1,3-dinitropyrene, and 1,8-dinitropyrene. Separation of seven of the major PAH-DNA adducts was achieved by an initial PAH HPLC gradient system. The major NO2-PAH-DNA adducts were not all separated from each other using the initial PAH HPLC gradient but were clearly separated from the PAH-DNA adducts. A second NO2-PAH HPLC gradient system was developed to separate NO2-PAH-DNA adducts following one-dimensional TLC and HPLC analysis. HPLC profiles of NO2-PAH-DNA adducts were compared using both adduct enhancement versions of the 32P-postlabeling assay to evaluate the use of this technique on HPLC to screen for the presence of NO2-PAH-DNA adducts. To demonstrate the application of these separation methods to a complex mixture of DNA adducts, the chromatographic mobilities of the 32P-postlabeled DNA adduct standards (PAHs and NO2-PAHs) were compared with those produced by a complex mixture of polycyclic organic matter (POM) extracted from diesel emission particles. The diesel-derived adducts did not elute with the identical retention time of any of the PAH or NO2-PAH standards used in this study. HPLC analyses of the NO2-PAH-derived adducts (butanol extracted) revealed the presence of multiple DNA adducts.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Comparison of DNA adduct formation in mice fed coal tar or benzo[a]pyrene.
Coal tar is a complex mixture containing hundreds of compounds, including the carcinogenic polycyclic aromatic hydrocarbon, benzo[a]pyrene. In order to compare the metabolic activation of a single carcinogen versus a complex mixture containing the carcinogen, we determined the DNA adduct profiles in B6C3F1 mice fed doses of coal tar or benzo[a]pyrene at concentrations corresponding to the amount of benzo[a]pyrene found in the respective coal tar treatments. DNA adduct formation was quantified in liver, lungs and forestomach by 32P-postlabeling and was found to increase as a function of dose in each tissue with both coal tar and benzo[a]pyrene. In mice fed benzo[a]pyrene, a major adduct was detected with the same elution characteristics by TLC and HPLC as the major adduct, 10 beta-(deoxyguanosin-N2-yl)-7 beta, 8 alpha, 9 alpha-trihydroxy-7,8,9,10- tetrahydrobenzo[a]-pyrene (dG-N2-BPDE), obtained from reacting (+/-)-antibenzo[a]pyrene-7,8- dihydrodiol-9,10-epoxide (BPDE) with DNA. DNA binding was in the order forestomach > or = liver > lung, except at the highest dose group where the order was liver > forestomach > lung. In mice fed coal tar, a diagonal zone of radioactivity with a number of discrete adducts was observed. One area of radioactivity contained the major BPDE adduct, dG-N2-BPDE, based on co-elution by TLC and HPLC with the synthesized adduct. Total DNA binding was greater in the coal tar-fed mice than in the mice fed benzo[a]pyrene, and the adduct levels were in the order lung > liver > forestomach. These results indicate that there are tissue-specific differences in the activation of coal tar components when compared to a representative carcinogen contained within the mixture.
7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
Bioflavonoid effects on the mitochondrial respiratory electron transport chain and cytochrome c redox state.
The polyphenolic structure common to flavonoids enables them to donate electrons and exert antioxidant activity. Since the mitochondrial electron transport chain consists of a series of redox intermediates, the effect of flavonoids in a complex mixture of polyphenols, as well as related pure flavonoids, was evaluated on the rat liver mitochondrial electron transport chain. A French maritime pine bark extract (PBE), a complex mixture of polyphenols and related pure flavonoids, was able to reduce cytochrome c reversibly, possibly by donation of electrons to the iron of the heme group; the donated electrons can be utilized by cytochrome c oxidase. Among single flavonoids tested, (-)-epicatechin gallate had the greatest ability to reduce cytochrome c. In addition, PBE competitively inhibited electron chain activity in both whole mitochondria and submitochondrial particles. A 3.5-fold increase in the apparent Km value for succinate was calculated from reciprocal plots. Among the flavonoids tested, taxifolin and (-)-epicatechin gallate showed minor inhibitory effects, while (+/-)-catechin and (+)-epicatechin were ineffective. Activities of NADH-ubiquinone, succinate-ubiquinone, and ubiquinol-cytochrome c reductases were inhibited by low concentrations of PBE to a similar extent. However, inhibition of cytochrome c oxidase activity required 4-fold higher PBE concentrations. These results suggest that flavonoids reduce cytochrome c and that PBE inhibits electron transport chain activity mainly through NADH-ubiquinone, succinate-ubiquinone, and ubiquinol-cytochrome c reductases.
Animals↗
Screening of natural products extracts for the presence of phosphodiesterase inhibitors using liquid chromatography coupled online to parallel biochemical detection and chemical characterization.
The ability to rapidly identify active compounds in a complex mixture (e.g., natural products extract) is still one of the major problems in natural products screening programs. An elegant way to overcome this problem is to separate the complex mixture by gradient liquid chromatography followed by online biochemical detection parallel with chemical characterization, referred to as high-resolution screening (HRS). To find and identify phosphodiesterase (PDE) inhibitors in natural products extracts using the HRS technology, the authors developed a continuous-flow PDE enzymatic assay. The suitability of the continuous-flow PDE enzymatic assay for natural products screening was demonstrated. After optimization of the continuous-flow PDE assay, the limit of detection for 3-isobutyl-1-methyl-xanthine (IBMX) was 1 muM, with a dynamic range from 1 to 100 muM IBMX. The applicability of the HRS technology for the detection of PDE inhibitors in natural products extracts was demonstrated by the analysis of a plant extract spiked with 2 naturally occurring PDE inhibitors. The plant extract was analyzed with 2 assay lines in parallel, enabling background fluorescence correction of the sample. The simultaneous quantification of the active compounds using evaporative light-scattering detection allowed the estimation of the IC(50) value of the active compounds directly in the crude extract.
1-Methyl-3-isobutylxanthine↗
The use of mechanistically defined chemical mixtures (MDCM) to assess mixture component effects on the percutaneous absorption and cutaneous disposition of topically exposed chemicals. II. Development of a general dermatopharmacokinetic model for use in risk assessment.
We present a conceptual approach to a general comprehensive mathematical model to quantify percutaneous absorption of topically applied chemicals in complex mixtures on the basis of biophysical parameters estimated or measured using in vitro and ex vivo perfused skin preparations. This model addresses mechanistically defined chemical mixtures (MDCM) which consist of components selected because of their potential to modulate by various mechanisms the absorption of a marker toxic penetrant. This model accounts for observed toxicodynamic general and specific effects of chemicals, acting single or in concert, on the absorption of any or all components in a defined mixture. We have also included experimental data from an isolated perfused porcine skin flap study with topically applied parathion as the marker penetrant and acetone or DMSO as solvent, with methyl nicotinate as a potential rubefacient, sodium laurel sulfate as a surfactant, and stannous chloride as a reducing agent in order to provide an illustration of the application and performance of the model. This model supports the MDCM concept that defining and then simulating those components of a complex mixture that could have a significant impact on the absorption of a marker toxic compound would be a useful screening approach in the risk assessment of topical chemical mixtures. It may also be used to identify critical pathways where chemical mixture component interactions significantly modify the absorption of the penetrant of interest.
Animals↗