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Subjective reactions of discomfort, impaired air quality, irritation of mucosal membranes, and impaired memory have been reported in chemically sensitive subjects during exposure to volatile organic compounds (VOCs) found in new buildings. Sixty-six normal healthy male subjects aged 18-39 were exposed for 2.75 hr to a complex VOC mixture at 0 and 25 mg/m3. Each subject completed control and exposure sessions at one-week intervals in counterbalanced order. Measurements included comfort ratings of eye, nose and throat irritation, symptom questionnaire and computerized behavioral tests. Subjects found the odor of VOCs unpleasantly strong and reported that VOC exposure degraded air quality, increased headache and produced general discomfort. VOC exposure did not affect performance on any behavioral tests.
Humans are exposed to mixtures of chemicals, rather than to individual chemicals. From a public health point of view, it is most relevant to answer the question of whether or not the components in a mixture interact in a way that results in an increase in their overall effect compared with the sum of the effects of the individual components. In this article, options for the hazard identification and risk assessment of simple and complex chemical mixtures will be discussed. In addition, key research needed to continue the development of hazard characterization of chemical mixtures will be described. Clearly, more collaboration among toxicologists, model developers and pharmacologists will be necessary.
Two-sector linked-scan analysis of an unpurified proteolytic digest of a pyruvate decarboxylase enzyme (60,000 Da) has allowed the discovery and assignment of an amino-terminal post-translational modification and processing event. A difference in amino acid sequence from that predicted by a recently published nucleotide sequence has also been found. These results illustrate both the use and considerable potential of linked-scan methods for the analysis of complex biopolymer mixtures.
We outline simple methodology for the rapid and selective analysis of 2-aminoacridone (2-AMAC) derivatised oligosaccharides by matrix-assisted laser desorption/ionization mass spectrometry. This involves the addition of small amounts of lithium chloride to the matrix before evaporation of solvent and crystallization. Signals mainly attributed to proton, sodium and potassium adducts are suppressed to a great extent, and a single signal due to (M + Li)+ is observed. This technique is rapid and is most useful for the direct analysis of complex glycan mixtures, after derivatization with 2-aminoacridone and without separation of the individual components.
The application of the traditional methods of multivariate statistics, such as the calculation of principle components, to the analysis of NMR spectra taken on sets of biofluid samples is one of the central approaches in the field of metabonomics. While this approach has proven to be a powerful and widely applicable technique, it has an inherent weakness, in that it tends to be dominated by those chemical species present at relatively higher concentrations. Using a set of commercial honey samples, a comparison of this classical metabonomics approach to one based on the use of the selective TOCSY experiment is presented. While the NMR spectrum of honey and its classical metabonomic analysis is completely dominated by a very few chemical species, specifically alpha-glucose and fructose, the statistical signal carried by minor honey components, such as amino acids, may be accessed using a selective TOCSY-based approach. This approach has the intrinsic virtue that it focuses the statistical analysis on a set of predefined chemical species, which might be chosen for their metabolic significance, and could be composed of either major or minor mixture constituents. Furthermore, the selective TOCSY method allows for more certain chemical identification, acquisition times of approximately 1 min, and accurate quantification of the species contributing to the statistical discriminatory signal.
An automated two-dimensional liquid chromatographic system was developed for systematic protein separations which could serve for analytical mapping and preparative separations of proteins. The system applies the principles of the column-switching technique, and consists of two different columns connected in tandem through an electrical column switching valve, two pumping systems to operate each column independently and a system controller to perform sequential chromatography on the two columns. A protein mixture is applied to the first-dimensional anion-exchange column and is separated by stepwise elution with an increasing sodium chloride concentration. The eluent is introduced directly to the second-dimensional reversed-phase column, and is further separated by gradient elution with an increasing acetonitrile concentration. The two elution stages are synchronized by a computer program. By this system, very complex protein mixtures such as crude cerebellar extracts were resolved reproducibly into ca. 200 peaks within 12 h. The method can be used for the total analysis of proteins in various tissues and cells without complicated premanupulation of samples, and allows the simultaneous analysis of a protein isolated by chromatography. The isolated protein is most suitable for use in the strategy of protein and gene sequence analysis.
The chick embryotoxicity screening test (CHEST) and the Salmonella/microsome bioassay were used to evaluate embryotoxic and mutagenic endpoints from crude coal tar (CT) and its fractionated polycyclic aromatic hydrocarbon (PAH) mixtures (designated as A, B, C, D and E). In the CHEST assay, CT and PAH mixtures were injected into the egg yolk. A dose-dependent increase in embryo mortality was observed for all fractions. The E fraction resulted in 47% embryo mortality at a dose of 0.125 mg/kg and was more toxic than CT. At a dose of 1 mg/kg, 85-100% embryonic deaths occurred in fractions C and D and these two fractions were more potent than fractions A and B. The main visual toxic manifestations were liver lesions, discoloration of the liver, and edema. Both CT and fractionated PAH mixtures were also tested in the Salmonella/microsome plate incorporation assay with Salmonella typhimurium strain TA98 and were evaluated with and without metabolic activation at five dose levels. In the presence of S9, the CT and fractions C, D and E induced a dose-dependent positive response. Results from the Salmonella/microsome assay were in good agreement with findings from the CHEST assay suggesting that these two bioassays in combination may facilitate the rapid detection and ranking of complex PAH mixtures.
The cannabis plant (Cannabis sativa L.) and products thereof (such as marijuana, hashish and hash oil) have a long history of use both as a medicinal agent and intoxicant. Over the last few years there have been an active debate regarding the medicinal aspects of cannabis. Currently cannabis products are classified as Schedule I drugs under the Drug Enforcement Administration (DEA) Controlled Substances act, which means that the drug is only available for human use as an investigational drug. In addition to the social aspects of the use of the drug and its abuse potential, the issue of approving it as a medicine is further complicated by the complexity of the chemical make up of the plant. This manuscript discusses the chemical constituents of the plant with particular emphasis on the cannabinoids as the class of compounds responsible for the drug's psychological properties.
RATIONALE: The competition ELISA assay is used to determine the potency of US standardized allergen extracts. We have been concerned that the competition ELISA is not sensitive to changes in individual allergen levels. This study was designed to determine the sensitivity of the competition ELISA to detect the specific loss of Bla g 1 and Bla g 2 in cockroach extracts. METHODS: German cockroach extract E3Cg was made from defatted German cockroaches. New Zealand White rabbits were immunized with rBla g 1 or rBla g 2. Optimal dilutions of anti-Bla g 1 and anti-Bla g 2 sera were established by ELISA. E3Cg was selectively depleted of Bla g 1 or Bla g 2 by immunoabsorption with anti-Bla g 1 or anti-Bla g 2 attached to Protein G agarose beads. Competition ELISA using pooled human sera, or mixed anti-Bla g 1 and anti-Bla g 2 serum, was performed on the depleted extracts, and on depleted extracts reconstituted with rBla g 1 or rBla g 2. RESULTS: Unlike pooled human-allergic IgE sera, anti-Bla g 1 and anti-Bla g 2 IgG -- in dilutions as low as 10(-6), could be used in the competition ELISA to measure the loss of allergen in depleted E3Cg. As little as 0.001 microg/mL of added rBla g 1 and 0.1 microg/mL of added rBla g 2, could be detected. CONCLUSION: The competition ELISA can be highly sensitive to compositional differences in complex allergen mixtures, even when the specific detecting antibody is present in relatively small amounts.
Canister methodology is applicable to 150 polar and nonpolar VOCs found in ambient air from parts-per-billion by volume (ppbv) to parts-per-million (ppmv) levels, and has been validated at parts-per-trillion (pptv) levels for a subset of these analytes. This article is a detailed review of techniques related to the collection of volatile organic compounds (VOCs) in evacuated Summa and fused-silica-lined canisters, and their analysis by gas chromatography/mass spectrometry (GC/MS). Emphasis is placed on canister cleaning, VOC stability in canisters, sample dilution, water management, and VOC cryogenic and sorbent preconcentration methods. A wide range of VOC preconcentration and water management methods are identified from the literature, and their relative merits and disadvantages are discussed. Examples of difficulties that commonly arise when processing canister samples are illustrated, and solutions to these problems are provided.
Recently, multidimensional shotgun proteomics has proven to be an alternative technology able to identify hundreds of proteins from single samples. Two major limitations of the technology are the presence of high abundance proteins (e.g. RUBISCO in plant leaf tissue) and the enormous number of co-eluting peptides that overstrain the loading and resolving capacity of conventional particle-packed columns as well as the capacity of electrospray ionisation due to ion suppression. Here, the coupling of fast performance liquid chromatography (FPLC) pre-fractionation of an Arabidopsis leaf protein extract and subsequent two-dimensional liquid chromatography/mass spectrometry with improved resolution using a monolithic silica C18 capillary column allowed the identification of 1032 unique proteins in a single 4 mg total protein plant leaf tissue sample. The reassignment of peptide IDs to distinct FPLC protein fractions enhances the identification procedure, especially in the case of present protein isoforms. The proposed strategy is useful to detect proteins otherwise not seen in conventional multidimensional chromatography/mass spectrometry approaches.
Mass spectrometry is now firmly established as a powerful technique for the identification and characterization of proteins when used in conjunction with sequence databases. Various approaches involving stable-isotope labeling have been developed for quantitative comparisons between paired samples in proteomic expression analysis by mass spectrometry. However, interpretation of such mass spectra is far from being fully automated, mainly due to the difficulty of analyzing complex patterns resulting from the overlap of multiple peaks arising from the assortment of natural isotopes. In order to facilitate the interpretation of a complex mass spectrum of such a mixture, such as an MS spectrum of a stable-isotope-enriched ion species, we report on the development of a software application, 'Matching' (web accessible), that enables the automatic matching of theoretical isotope envelopes to multiple ion peaks in a raw spectrum. It is particularly useful for resolving the relative abundances of narrow-split paired peaks caused by enrichment with a stable isotope, such as 18O, 13C, 2H, or 15N.
The limitations of 2-D gels for global proteomics have encouraged the development of alternative approaches for identifying proteins in complicated mixtures, and determining their modification state. In this work, we describe the application of multidimensional liquid chromatography (SCX-RPLC) coupled with electrospray time-of-flight mass spectrometry and off-line fraction collection to analyze complex intact protein mixtures. Methods were developed using both standard proteins and an enriched yeast ribosomal fraction sample containing approximately 100 proteins, which permitted assessment of the effectiveness of the individual separation dimensions, as well as investigation of the interplay between separation capacity and electrospray MS performance.
To simplify the complex 1H NMR spectrum of a fulvic acid sample and gain structural and molecular size information, spectral editing techniques were used in conjunction with a general PFG NMR pulse sequence. These editing techniques exploit differences in T1 and T2 relaxation times as well as differences in coupling constants. The experiments were initially performed on a model mixture of glutamic acid and ethyl acetate in order to validate the method as a technique for measurement of diffusion coefficients. The editing experiments were then applied to the International Humic Substances Society Suwannee River fulvic acid standard. These editing techniques allowed more selective measurement of diffusion coefficients for broad classes of components within regions of the 1H NMR spectrum of the fulvic acid solution. The average radii of gyration calculated for the Suwannee River fulvic acid sample from the diffusion coefficients are in good agreement with literature values.
Numerous approaches have been employed for testing the biological activity of cigarette smoke in vitro. None of them has managed to expose cultured lung cells in a realistic manner to the complex gaseous and particulate mixture that constitutes cigarette smoke. We have devised a system that makes this possible. The system presented here enables the direct exposure of human lung cells to native, unmodified cigarette mainstream smoke. It consists of a smoking machine, a dilution device for the smoke, analytical devices for online monitoring and a specially adapted exposure module based on the Cultex** cell cultivation system that is equipped with a gas-exposure top. Due to the special design of the exposure device and the optimised exposure conditions, this equipment allows cultured human lung cells to be exposed to freshly generated cigarette mainstream smoke. Exploratory experiments revealed that the smoke could be diluted over a wide concentration range in a reproducible way with respect to gas and particulate phases, and also demonstrated reproducible particle deposition depending on smoke concentration. Furthermore, it was shown that the exposed cells maintained their viability. Native cigarette mainstream smoke induced dose-dependent cellular effects in exposed cells with respect to cellular viability (viable cell number monitored by tetrazolium salt cleavage) and intracellular parameters (ATP and glutathione content). Therefore, fresh, physically and chemically unmodified cigarette mainstream smoke can be tested using this novel system.
The paper describes a method for determining homologous classes of compounds in a multicomponent complex chromatogram obtained under programming elution conditions. The method is based on the computation of the autocovariance function of the experimental chromatogram (EACVF). The EACVF plot, if properly interpreted, can be regarded as a "class chromatogram" i.e., a virtual chromatogram formed by peaks whose positions and heights allow identification and quantification of the different homologous series, even if they are embedded in a random complex chromatogram. Theoretical models were developed to describe complex chromatograms displaying random retention pattern, ordered sequences or a combination of them. On the basis of theoretical autocovariance function, the properties of the chromatogram can be experimentally evaluated, under well-defined conditions: in particular, the two components of the chromatogram, ordered and random, can be identified. Moreover, the total number of single components (SCs) and the separated number of the SCs belonging to the random and ordered components can be determined, when the two components display the same concentration. If the mixture contains several homologous series with common frequency and different phase values, the number and identity of the different homologous series as well as the number of SCs belonging to each of them can be evaluated. Moreover, the power of the EACVF method can be magnified by applying it to the single ion monitoring (SIM) signals to selectively detect specific compound classes in order to identify the different homologous series. By this way, a full "decoding" of the complex multicomponent chromatogram is achieved. The method was validated on synthetic mixtures containing known amount of SCs belonging to homologous series of hydrocarbon, alcohols, ketones, and aromatic compounds in addition to other not structurally related SCs. The method was applied to both the total ion monitoring (TIC) and the SIM signals, to describe step by step the essence of the procedure. Moreover, the systematic use of both SIM and TIC can simplify the decoding procedure of complex chromatograms by singling out only specific compound classes or by confirming the identification of the different homologous series. The method was further applied to a sample containing unknown number of compounds and homologous series (a petroleum benzin, bp 140-160 degrees C): the results obtained were meaningful in terms of both the identified number of components and identified homologous series.
Today, 2D online or offline liquid chromatography/mass spectrometry is state of the art for the identification of proteins from complex proteome samples in many laboratories. Both 2D liquid chromatography methods use two orthogonal liquid chromatography separation techniques. The most commonly used techniques are strong cation exchange chromatography for the first dimension and reversed phase separation for the second dimension. In order to improve sensitivity the reversed phase separation is usually performed in the nanoflow scale and mass spectrometry is used as the final detection method. The high-performance liquid chromatography techniques complement the 2D-gel techniques supporting their weaknesses. This is especially true for the gel separation of hydrophobic membrane proteins, which play an important role in living cells as well as being important targets for future pharmaceutical drugs.