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Quorum sensing is a process of bacterial cell-cell communication that uses small diffusible molecules to coordinate diverse behaviors in response to population density. The only quorum-sensing system shared by Gram-positive and Gram-negative bacteria involves the production of autoinducer-2 (AI-2). The AI-2 synthase LuxS is widely distributed among the Bacteria, which suggests that AI-2 is a language for interspecies communication. However, LuxS is also an integral component of the activated methyl cycle in bacteria. LuxS-based quorum sensing has been intensively studied in the past decade, mostly in relation to the AI-2 molecule and the downstream effects of luxS knockouts; few studies have focused on the gene and protein activity itself. Ongoing attempts to dissect the metabolic and signaling roles of LuxS leave little doubt that unraveling the regulation of luxS expression and cellular LuxS activity is the key to understanding LuxS-based quorum sensing.

We used a localized ozone (O3) fumigation (LOF) system to study acute and short-term O(3) effects on physiological leaf traits. The LOF system enabled investigation of primary and secondary metabolic responses of similarly and differently aged leaves on the same plant to three different O3 concentrations ([O3]), unconfounded by other influences on O3 sensitivity, such as genetic, meteorological and soil factors. To simulate the diurnal cycle of O3 formation, current-year and 1-year-old Quercus ilex (L.) and Quercus pubescens (L.) leaves were fumigated with O3 at different positions (and hence, different leaf ages) on the same branch over three consecutive days. The LOF system supplied a high [O3] (300+/-50 ppb) on leaves appressed to the vents, and an intermediate, super-ambient [O3] (varying between 120 and 280 ppb) on leaves less than 30 cm from the vent. Leaves more than 60 cm from the O3 vent were exposed to an [O3] comparable with the ambient concentration, with a 100 ppb peak during the hottest hours of the day. Only leaves exposed to the high [O3] were affected by the 3-day treatment, confirming that Mediterranean oak are tolerant to ambient and super-ambient [O3], but may be damaged by acute exposure to high [O3]. Stomatal and mesophyll conductance and photosynthesis were all reduced immediately after fumigation with high [O3], but recovered to control values within 72 h. Both the intercellular and chloroplast CO2 concentrations ([CO2]) remained constant throughout the experiment. Thus, although treatment with a high [O3] may have induced stomatal closure and consequent down-regulation of photosynthesis, we found no evidence that photosynthesis was limited by low [CO2] at the site of fixation. One-year-old leaves of Q. ilex were much less sensitive to O3 than current-year leaves, suggesting that the low stomatal conductance observed in aging leaves limited O3 uptake. No similar effect of leaf age was found in Q. pubescens. Dark respiration decreased during the treatment period, but a similar decrease was observed in leaves exposed to low [O3], and therefore may not be an effect of O3 treatment. Light respiration, on the other hand, was mostly constant in ozone-treated leaves and increased only in leaves in which photosynthesis was temporarily inhibited by high [O3], preventing them from acting as strong sinks that recycle respiratory CO2 in the leaves. There was no evidence of photochemical damage in Q. ilex leaves, whereas Q. pubescens leaves exposed to a high [O3] showed limited photochemical damage, but recovered rapidly. Biochemical markers were affected by the high [O3], indicating accumulation of reactive oxygen species (ROS) and increased denaturation of lipid membranes, followed by activation of isoprene biosynthesis in Q. pubescens leaves. We speculate that the high isoprene emissions helped quench ROS and normalize membrane stability in leaves recovering from O3 stress.

1,3-Butadiene and isoprene (2-methyl-1,3-butadiene) are chemically related substances that are carcinogenic to rodents. The overall aim of this work is to elucidate the role of the genotoxic action of diepoxide metabolites in the carcinogenesis of the dialkenes. In vivo doses of the diepoxide metabolites were measured through reaction products with hemoglobin (Hb adducts) in studies of induced micronuclei (MN) in rodents. In the reaction with N-terminal valine in Hb, diepoxybutane and isoprenediepoxide form ring-closed adducts, pyrrolidines [N,N-(2,3-dihydroxy-1,4-butadiyl)valine and N,N-(2,3-dihydroxy-2-methyl-1,4-butadiyl)valine, respectively]. The method applied for Hb-adduct measurement is based on tryptic degradation of the protein and liquid chromatography electrospray ionisation tandem mass spectrometry (LC-ESI-MS/MS) analysis. Mice were given single i.p. injections of the monoepoxides of butadiene and isoprene, 1,2-epoxy-3-butene or 1,2-epoxy-2-methyl-3-butene, respectively. Rats were treated in the same way with 1,2-epoxy-3-butene. In mice pyrrolidine adduct levels increased with increasing administered doses of the monoepoxides. The in vivo dose of diepoxybutane was on average twice as high (0.29+/-0.059 mMh) as the in vivo dose of isoprenediepoxide (0.15+/-0.053 mMh) per administered dose (mmol/kg body weight) of the monoepoxides. In mice the genotoxic effects of the two monoepoxides, measured as the increase in the frequencies of micronuclei (MN), were approximately linearly correlated to the in vivo doses of the diepoxides (except at the highest dose of diepoxybutane). In rats the pyrrolidine-adduct levels from diepoxybutane were below the limit of quantification at all administered doses of 1,2-epoxy-3-butene and no significant increase was observed in the frequency of MN. Measurement of the ring-closed adducts to N-termini in Hb by the applied method permits analysis of in vivo doses of diepoxybutane and isoprenediepoxide, which may be further used for the elucidation of the mechanisms of carcinogenesis of butadiene and isoprene.

Foliar emission of volatile organic compounds (VOC) from common Indian plant species was measured. Dynamic flow enclosure technique was used and the gas samples were collected onto Tenax-GC/Carboseive cartridges. The Tenax-GC/Carboseive cartridges were attached to the thermal disorber sample injection system and the gas sample was analysed using gas chromatography (GC) with flame ionisation detection (FID). Fifty-one local plant species were screened, out of which 36 species were found to emit VOC (4 high emitter; 28 moderate emitter; and 4 low-emitter), while in the remaining 15 species no VOC emission was detected or the levels of emission were below detection limit (BDL). VOC emission was found to vary from one species to another. There was a marked seasonal and diurnal variation in VOC emission. The minimum and maximum VOC emission values were < 0.1 and 87 microgg(-1) dry leaf h(-1) in Ficus infectoria and Lantana camara respectively. Out of the 51 plant species studied, 13 species are reported here for the first time. Among the nine tree species (which were selected for detailed study), the highest average hourly emission (9.69+/-8.39 microgg(-1) dry leaf) was observed in Eucalyptus species and the minimum in Syzygium jambolanum (1.89+/-2.48 microgg(-1) dry leaf). An attempt has been made to compare VOC emission from different plant species between present study and the literature (tropical and other regions).

We describe an original, short, and convenient chemical synthesis of enantiopure (S)-4,5-dihydroxy-2,3-pentanedione (DPD), starting from commercial methyl (S)-(-)-2,2-dimethyl-1,3-dioxolane-4-carboxylate. DPD is the precursor of autoinducer (AI)-2, the proposed signal for bacterial interspecies communication. AI-2 is synthesized by many bacterial species in three enzymatic steps. The last step, a LuxS-catalyzed reaction, leads to the formation of DPD, which spontaneously cyclizes into AI-2. AI-2-like activity of the synthesized molecule was ascertained by the Vibrio harveyi bioassay. To further validate the biological activity of synthetic DPD and to explore its potential in studying DPD (AI-2)-mediated signaling, a Salmonella typhimurium luxS mutant was constructed. Expression of the AI-2 regulated lsr operon can be rescued in this luxS mutant by addition of synthetic DPD or genetic complementation. Biofilm formation by S. typhimurium has been reported to be defective in a luxS mutant, and this was confirmed in this study to test DPD for chemical complementation. However, biofilm formation of the luxS mutant cannot be restored by addition of DPD. In contrast, introduction of luxS under control of its own promoter complemented biofilm formation. Further results demonstrated that biofilm formation of the luxS mutant cannot be restored with luxS under control of the strong nptII promoter. This indicates that altering the intrinsic promoter activity of luxS affects Salmonella biofilm formation. Conclusively, we synthesized biologically active DPD. Using this chemical compound in combination with genetic approaches opens new avenues in studying AI-2-mediated signaling.

Defining the metabolic capabilities and regulatory mechanisms controlling gene expression is a valuable step in understanding the pathogenic properties of infectious agents such as Borrelia burgdorferi. The present studies demonstrated that B. burgdorferi encodes functional Pfs and LuxS enzymes for the breakdown of toxic products of methylation reactions. Consistent with those observations, B. burgdorferi was shown to synthesize the end product 4,5-dihydroxy-2,3-pentanedione (DPD) during laboratory cultivation. DPD undergoes spontaneous rearrangements to produce a class of pheromones collectively named autoinducer 2 (AI-2). Addition of in vitro-synthesized DPD to cultured B. burgdorferi resulted in differential expression of a distinct subset of proteins, including the outer surface lipoprotein VlsE. Although many bacteria can utilize the other LuxS product, homocysteine, for regeneration of methionine, B. burgdorferi was found to lack such ability. It is hypothesized that B. burgdorferi produces LuxS for the express purpose of synthesizing DPD and utilizes a form of that molecule as an AI-2 pheromone to control gene expression.

Isoprene reduces visible damage (necrosis) of leaves caused by exposure to ozone but the mechanism is not known. Here we show that in Phragmites leaves isoprene emission was stimulated after a 3-h exposure to high ozone levels. The photosynthetic apparatus of leaves in which isoprene emission was inhibited by fosmidomycin became more susceptible to damage by ozone than in isoprene-emitting leaves. Three days after ozone fumigation, the necrotic leaf area was significantly higher in isoprene-inhibited leaves than in isoprene-emitting leaves. Isoprene-inhibited leaves also accumulated high amounts of nitric oxide (NO), as detected by epifluorescence light microscopy. Our results confirm that oxidative stresses activate biosynthesis and emission of chloroplastic isoprenoid, bringing further evidence in support of an antioxidant role for these compounds. It is suggested that, in nature, the simultaneous quenching of NO and reactive oxygen species by isoprene may be a very effective mechanism to control dangerous compounds formed under abiotic stress conditions, while simultaneously attenuating the induction of the hypersensitive response leading to cellular damage and death.

The aim of the study was to assess oxidative stress biomarkers considering the simplicity of their determination, invasiveness of material collection and their specificity. The following oxidative stress markers were evaluated: malonyldialdehyde, thiobarbituric acid reactive lipid peroxidation products, prostaglandin F2alpha, nitric oxide derivatives, nucleic acid damage products, advanced oxidation protein products and gas components in exhaled air: nitric oxide, hydrocarbons, isoprene, ethane. Analysis of the selected biomarkers indicates the usefulness of the determination of prostaglandin F2alpha, nucleic acid damage products, advanced oxidation protein products and gas components in the exhaled air as oxidative stress biomarkers in patients with chronic renal failure.

The increasing production of several plastics such as expanded polystyrene, widely used as packaging and building materials, has caused the release of considerable amounts of pentane employed as an expanding agent. Today many microorganisms are used to degrade hydrocarbons in order to minimize contamination caused by several industrial activities. The aim of our work was to identify a suitable microorganism to degrade pentane. We focused our attention on a strain of Arthrobacter sp. which in a shake-flask culture produced 95% degradation of a 10% mixture of pentane in a minimal medium after 42 days of incubation at 20 degrees C. Arthrobacter sp. cells were immobilized on a macroporous polystyrene particle matrix that provides a promising novel support for cell immobilization. The method involved culturing cells with the expanded polystyrene in shake-flasks, followed by in situ growth within the column. Scanning electron microscopy analysis showed extensive growth of Arthrobacter sp. on the polymeric surface. The immobilized microorganism was able to actively degrade a 10% mixture of pentane, allowing us to obtain a bioconversion yield of 90% after 36 h. Moreover, in repeated-batch operations, immobilized Arthrobacter sp. cells were able to maintain 85-95% pentane degradation during a 2 month period. Our results suggest that this type of bioreactor could be used in pentane environmental decontamination.

Twenty-three kinds of typical plants in Xishuangbanna, the tropical area of southwestern China, were screened to estimate the emission rates of isoprene and monoterpenes by adopting bag-enclosure and curette sampling methods followed by a GC-FID analysis. It was found that the Ficus species were mainly emitting isoprene and most tropical vegetations were mainly releasing monoterpenes. The results also showed that the emissions of isoprene were affected by both temperature and PAR(Photosynthetic Active Radiation), while monoterpene emissions were mainly temperature-dependent.

An extracellular protein with strong absorption at 406 nm was purified from cell-free culture fluid of latex-grown Xanthomonas sp. strain 35Y. This protein was identical to the gene product of a recently characterized gene cloned from Xanthomonas sp., as revealed by determination of m/z values and sequencing of selected isolated peptides obtained after trypsin fingerprint analysis. The purified protein degraded both natural rubber latex and chemosynthetic poly(cis-1,4-isoprene) in vitro by oxidative cleavage of the double bonds of poly(cis-1,4-isoprene). 12-oxo-4,8-dimethyltrideca-4,8-diene-1-al (m/z 236) was identified and unequivocally characterized as the major cleavage product, and there was a homologous series of minor metabolites that differed from the major degradation product only in the number of repetitive isoprene units between terminal functions, CHO-CH2--and--H2-COCH3. An in vitro enzyme assay for oxidative rubber degradation was developed based on high-performance liquid chromatography analysis and spectroscopic detection of product carbonyl functions after derivatization with dinitrophenylhydrazone. Enzymatic cleavage of rubber by the purified protein was strictly dependent on the presence of oxygen; it did not require addition of any soluble cofactors or metal ions and was optimal around pH 7.0 at 40 degrees C. Carbon monoxide and cyanide inhibited the reaction; addition of catalase had no effect, and peroxidase activity could not be detected. The purified protein was specific for natural rubber latex and chemosynthetic poly(cis-1,4-isoprene). Analysis of the amino acid sequence deduced from the cloned gene (roxA [rubber oxygenase]) revealed the presence of two heme-binding motifs (CXXCH) for covalent attachment of heme to the protein. Spectroscopic analysis confirmed the presence of heme, and approximately 2 mol of heme per mol of RoxA was found.

In the present study, important components of carbon metabolism of mature leaves of young poplar trees (Populus x canescens) were determined. Carbohydrate concentrations in leaves and xylem sap were quantified at five different times during the day and compared with photosynthetic gas exchange measurements (net assimilation, transpiration and rates of isoprene emission). Continuously measured xylem sap flow rates, with a time resolution of 15 min, were used to calculate diurnal balances of carbon metabolism of whole mature poplar leaves on different days. Loss of photosynthetically fixed carbon by isoprene emission and dark respiration amounted to 1% and 20%. The most abundant soluble carbohydrates in leaves and xylem sap were glucose, fructose and sucrose, with amounts of approx. 2 to 12 mmol m(-2) leaf area in leaves and about 0.2 to 15 mM in xylem sap. Clear diurnal patterns of carbohydrate concentration in xylem sap and leaves, however, were not observed. Calculations of the carbon transport rates in the xylem to the leaves were based on carbohydrate concentrations in xylem sap and xylem sap flow rates. This carbon delivery amounted to about 3 micromol C m(-2) s(-1) during the day and approx. 1 micromol C m(-2) s(-1) at night. The data demonstrated that between 9 and 28 % of total carbon delivered to poplar leaves during 24 h resulted from xylem transport and, hence, provide a strong indication for a significant rate of carbon cycling within young trees.

Because of potential exposure both in the workplace and from ambient air, the known carcinogen 1,3-butadiene (BD) is considered a priority hazardous air pollutant. BD and its 2-methyl analog, isoprene (ISO), are chemically similar but have very different toxicities, with ISO showing no significant carcinogenesis. Once released into the atmosphere, reactions with species induced by sunlight and nitrogen oxides convert BD and ISO into several photochemical reaction products. In this study, we determined the relative toxicity and inflammatory gene expression induced by exposure of A549 cells to BD, ISO, and their photochemical degradation products in the presence of nitric oxide. Gas chromatography and mass spectrometry analyses indicate the initial and major photochemical products produced during these experiments for BD are acrolein, acetaldehyde, and formaldehyde, and products for ISO are methacrolein, methyl vinyl ketone, and formaldehyde; both formed < 200 ppb of ozone. After exposure the cells were examined for cytotoxicity and interleukin-8 (IL-8) gene expression, as a marker for inflammation. These results indicate that although BD and ISO alone caused similar cytotoxicity and IL-8 responses compared with the air control, their photochemical products significantly enhanced cytotoxicity and IL-8 gene expression. This suggests that once ISO and BD are released into the environment, reactions occurring in the atmosphere transform these hydrocarbons into products that induce potentially greater adverse health effects than the emitted hydrocarbons by themselves. In addition, the data suggest that based on the carbon concentration or per carbon basis, biogenic ISO transforms into products with proinflammatory potential similar to that of BD products.

S-Ribosylhomocysteinase (LuxS) cleaves the thioether bond in S-ribosylhomocysteine to produce homocysteine and 4,5-dihydroxy-2,3-pentanedione. This reaction serves the dual purposes of detoxification of S-adenosylhomocysteine and production of type 2 quorum sensing molecule. Recent research has shown that LuxS uses Fe(2+) to catalyze an internal redox reaction, shifting the ribose carbonyl group from its C1 to C3 position. Subsequent beta-elimination completes this highly unusual reaction. LuxS and other enzymes on the same pathway may provide a novel class of antibacterial drug targets.

Feeding experiments were independently performed with [1-13C]deoxy-D-xylulose triacetate and (RS)-[2-13C]mevalonolactone in the tobacco plant. The labeling pattern for solanesol was elucidated to reveal that the isoprene moiety of solanesol would be derived from deoxy-xylulose. The result strongly suggests that tobacco solanesol is biosynthesized via the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway.

More volatile organic carbon is lost from plants as isoprene than any other molecule. This flux of carbon to the atmosphere affects atmospheric chemistry and can serve as a substrate for ozone production in polluted air. Isoprene synthesis may help leaves cope with heatflecks and active oxygen species. Isoprene synthase, an enzyme related to monoterpene synthases, converts dimethylallyl diphosphate derived from the methylerythritol 4-phosphate pathway to isoprene. We used dideuterated deoxyxylulose (DOX-d(2)) to study the regulation of the isoprene biosynthetic pathway. Exogenous DOX-d(2) displaced endogenous sources of carbon for isoprene synthesis without increasing the overall rate of isoprene synthesis. However, at higher concentrations, DOX-d(2) completely suppressed isoprene synthesis from endogenous sources and increased the overall rate of isoprene synthesis. We interpret these results to indicate strong feedback control of deoxyxylulose-5-phosphate synthase. We related the emission of labeled isoprene to the concentration of labeled dimethylallyl diphosphate in order to estimate the in situ K(m) of isoprene synthase. The results confirm that isoprene synthase has a K(m) 10- to 100-fold higher for its allylic diphosphate substrate than related monoterpene synthases for geranyl diphosphate.

Isoprene emitted from plants is made in chloroplasts from dimethylallyl pyrophosphate (DMAPP). Leaves of Populus nigra and Phragmites australis exposed to (13)CO(2) for 15 min emitted isoprene that was about 90% (13)C, but DMAPP isolated from those leaves was only 28% and 36% (13)C, respectively. The labeled DMAPP is likely to represent chloroplastic DMAPP contributing to isoprene emission. A substantial (13)C labeling was also found in both emission and DMAPP pool of low-emitting, young leaves of Phragmites. This confirms that low emission of young leaves is not caused by absence of chloroplastic DMAPP but rather by enzyme characteristics. A very low (13)C labeling was found in the DMAPP pool and in the residual isoprene emission of leaves previously fed with fosmidomycin to inhibit isoprene formation. This shows that fosmidomycin is a very effective inhibitor of the chloroplastic biosynthetic pathway of isoprene synthesis, that the residual isoprene is formed from extra-chloroplastic sources, and that chloroplastic and extrachloroplastic pathways are not cross-linked, at least following inhibition of the chloroplastic pathway. Refixation of unlabeled respiratory CO(2) in the light may explain incomplete labeling of isoprene emission, as we found a good association between these two parameters.

Volatile organic compound (VOC) emissions from Sitka spruce (Picea sitchensis Bong.) growing in a range of controlled light and temperature regimes were monitored online with a proton transfer reaction-mass spectrometer (PTR-MS) operating at a temporal resolution of approximately 1 min. Isoprene emissions accounted for an average of more than 70% of measured VOCs and up to 3.5% of assimilated carbon. Emission rates (E) for isoprene correlated closely with photosynthetic photon flux (PPF) and temperature, showing saturation at a PPF of between 300 and 400 micromol m(-2) s(-1) and a maximum between 35 and 38 degrees C. Under standard conditions of 30 degrees C and 1000 micromol m(-2) s(-1) PPF, the mean isoprene E was 13 microg gdm(-1) h(-1), considerably higher than previously observed in this species. Mean E for acetaldehyde, methanol and monoterpenes at 30 degrees C were 0.37, 0.78 and 2.97 microg gdm(-1) h(-1), respectively. In response to a sudden light to dark transition, isoprene E decreased exponentially by > 98% over about 3 h; however, during the first 7 min, this otherwise steady decay was temporarily but immediately depressed to approximately 40% of the pre-darkness rate, before rallying during the following 7 min to rejoin the general downward trajectory of the exponential decay. The sudden sharp fall in isoprene E was mirrored by a burst in acetaldehyde E. The acetaldehyde E maximum coincided with the isoprene E minimum (7 min post-illumination), and ceased when isoprene emissions resumed their exponential decay. The causes of, and linkages between, these phenomena were investigated., (Copyright 2004 Heron Publishing)

This review focuses on the biosynthesis of pigments in the unicellular alga Chlamydomonas reinhardtii and their physiological and regulatory functions in the context of information gathered from studies of other photosynthetic organisms. C. reinhardtii is serving as an important model organism for studies of photosynthesis and the pigments associated with the photosynthetic apparatus. Despite extensive information pertaining to the biosynthetic pathways critical for making chlorophylls and carotenoids, we are just beginning to understand the control of these pathways, the coordination between pigment and apoprotein synthesis, and the interactions between the activities of these pathways and those for other important cellular metabolites branching from these pathways. Other exciting areas relating to pigment function are also emerging: the role of intermediates of pigment biosynthesis as messengers that coordinate metabolism in the chloroplast with nuclear gene activity, and the identification of photoreceptors and their participation in critical cellular processes including phototaxis, gametogenesis, and the biogenesis of the photosynthetic machinery. These areas of research have become especially attractive for intensive development with the application of potent molecular and genomic tools currently being applied to studies of C. reinhardtii.

The aim of this study was to determine whether the administration of a lipid emulsion containing less polyunsaturated fatty acids but rich in monounsaturated fatty acids causes less in vivo lipid peroxidation in preterm infants. The prospective intervention study included 13 infants with birth weights and gestational ages ranging between 1,100 and 2,660 g and from 28.4 to 32.9 weeks. All were in a stable condition and randomly allocated for a 3-hour infusion (0.16 g/kg/h) of an olive oil-based and a soybean oil + medium chain fatty acid (MCT) emulsion on 2 consecutive days. Expired pentane and plasma triglycerides (TGs) were measured before, during, and after the 3-hour infusion. Basal exhaled pentane averaged 9.4 +/- 7.0 pmol/kg/min (mean +/- SD). During the olive oil-based emulsion, exhaled pentane increased to 95.2 +/- 56.7, and during soybean oil + MCT it increased to 110 +/- 93.9 pmol/kg/min (p < 0.05 both from basal, n.s. between preparations). One hour after discontinuation of the infusion, exhaled pentane returned to 21.1 +/- 12.6 pmol/kg/min (p < 0.05 vs. basal). Combined data on expired pentane measurements demonstrated that on day 1 pentane peaked at 124 +/- 87.0 pmol/kg/min which was significantly attenuated to 57.5 +/- 24.4 pmol/kg/min after an identical dose of lipid on day 2 (p < 0.05). No difference in peak TGs was detected between the two preparations or the study days. Infusion of a constant dose of intravenous lipids on 2 subsequent days to the newborn infants is associated with a reduction in lipid peroxidation. This finding may be dependent on normal postnatal maturation or may represent an appropriate adaptive response aiming at a reduction in oxidative stress. Peroxidation of soybean oil + MCT and olive oil-based lipid emulsions was similar in the newborn infants., (Copyright 2004 S. Karger AG, Basel)

The complete sequence of the circular 101,016-bp megaplasmid pKB1 from the cis-1,4-polyisoprene-degrading bacterium Gordonia westfalica Kb1, which represents the first described extrachromosomal DNA of a member of this genus, was determined. Plasmid pKB1 harbors 105 open reading frames. The predicted products of 46 of these are significantly related to proteins of known function. Plasmid pKB1 is organized into three functional regions that are flanked by insertion sequence (IS) elements: (i) a replication and putative partitioning region, (ii) a putative metabolic region, and (iii) a large putative conjugative transfer region, which is interrupted by an additional IS element. Southern hybridization experiments revealed the presence of another copy of this conjugational transfer region on the bacterial chromosome. The origin of replication (oriV) of pKB1 was identified and used for construction of Escherichia coli-Gordonia shuttle vectors, which was also suitable for several other Gordonia species and related genera. The metabolic region included the heavy-metal resistance gene cadA, encoding a P-type ATPase. Expression of cadA in E. coli mediated resistance to cadmium, but not to zinc, and decreased the cellular content of cadmium in this host. When G. westfalica strain Kb1 was cured of plasmid pKB1, the resulting derivative strains exhibited slightly decreased cadmium resistance. Furthermore, they had lost the ability to use isoprene rubber as a sole source of carbon and energy, suggesting that genes essential for rubber degradation are encoded by pKB1.

Ozone reacts with terpenes, common hydrocarbons in cleaning and consumer products as well as the ambient environment, to form particle- and gas-phase products; these have been shown to cause sensory irritation and airflow limitation in mice during single exposures. Isoprene, a hemiterpene emitted from both plants and animals as a bioeffluent, induces the largest effects. This study evaluated the effects of repeated exposures to isoprene oxidation products (OPs) on airway irritation, airflow limitation, and airway responsiveness. A secondary objective was to evaluate a genetic influence by examining multiple murine strains. Six strains (A/J, AKR/J, BALB/c, Swiss Webster, C57Bl/6, and C3H/HeN; total n = 35) were exposed to isoprene oxidation products (1080 +/- 155 ppb isoprene + 3227 +/- 157 ppb ozone at admixing) for 3 h/day for 4 consecutive days. Respiratory parameters were monitored on days 1 and 4 via head-out plethysmography, and airway responsiveness to aerosolized methacholine was evaluated 24 h before the first exposure and immediately after the fourth exposure. Sensory irritation was observed during exposure, as evidenced by a reduction in respiratory frequency (f). A reduction in peak expiratory flow normalized for tidal volume (PEF/VT) also occurred, indicating an airflow limitation effect. Marked enhancement of the effects on f and PEF/VT was observed with repeated exposures, suggesting a cumulative effect. Frequency was reduced from 46.0 +/- 2.3% of baseline during exposure 1 to 34.2 +/- 2.1% during exposure 4 (p =.00002; pooled values for all strains). Similarly, PEF/VT decreased from 75.6 +/- 3.9% of baseline during exposure 1 to 53.1 +/- 3.7% during exposure 4 (p <.00001). A significant reduction in airway responsiveness was observed following repeated exposures in most strains. Interstrain differences in responses were noted, indicating a genetic component. These findings have important implications for indoor environments, where isoprene concentrations can be high in buildings with high occupant densities. The findings are also relevant to outdoor environments, where isoprene emissions from vegetation lead to the formation of isoprene OPs, which may partition onto existing particulate matter (PM) in the atmosphere to contribute to secondary organic aerosol. Further, the genetic variability observed in the mouse strains examined suggests that interindividual differences in response may also exist in human populations, which may help to explain the high variability in symptom reporting in indoor environments.

Chemical-ionization mass spectrometry (CIMS) using flow reactors is an emerging method for on-line monitoring of trace concentrations of organic compounds in the gas phase. In this study, a flow-reactor CIMS instrument, employing the H(3)O(+) cation as the ionizing reagent, was used to simultaneously monitor several volatile metabolic products as they are released into the headspace during bacterial growth in a bioreactor. Production of acetaldehyde, ethanol, acetone, butanol, acetoin, diacetyl, and isoprene by Bacillus subtilis is reported. Ion signal intensities were related to solution-phase concentrations using empirical calibrations and, in the case of isoprene, were compared with simultaneous gas chromatography measurements. Identification of volatile and semivolatile metabolites is discussed. Flow-reactor CIMS techniques should be useful for bioprocess monitoring applications because of their ability to sensitively and simultaneously monitor many volatile metabolites on-line.

Isoprene (2-methylbuta-1,3-diene) is a large scale petrochemical used principally in the manufacture of synthetic rubbers. It is also produced by plants and trees and is formed endogenously in mammals as a major endogenous hydrocarbon. Mammalian metabolism of isoprene involves cytochrome P450-dependent monooxygenases to give the regioisomeric monoepoxides, prop-2-enyloxirane and 2-ethenyl-2-methyloxirane. The isoprene monoepoxides are further oxidized to the mutagenic diepoxides, 2-methyl-2,2'-bioxiranes. The present studies have investigated the stereochemistry and comparative rates of the metabolic epoxidation in vitro of isoprene to mono- and diepoxides by liver microsomes from rat, mouse, and human in order to identify stereochemical and kinetic differences between species in the formation of these epoxide metabolites, which are key to understanding the toxicology of isoprene. The assignments of stereochemistry were based on comparisons with synthetic standards, the syntheses for which are described. Comparative enzyme kinetic parameters (apparent K(m) and apparent V(max) values) for the in vitro formation of all of the monoepoxide and diepoxide stereoisomers have been obtained. The rates of formation of both mono- and diepoxides were greater in the rodent systems as compared with the human in vitro system. The results provide comparative kinetic data that have potential for modeling and assessing the relevance of the animal carcinogenicity data for man. The possibility of human interindividual variation was also investigated with liver preparations from several individual humans, but significant differences between individuals were not observed in the formation of the monoepoxides from isoprene.

Background: Uraemic odour is a characteristic feature of patients with end-stage renal disease (ESRD). However, few investigations have been carried out into the composition of exhaled air in ESRD patients undergoing haemodialysis (HD). Increases of exhaled isoprene levels by a factor of up to 2.7 following HD have been reported., Methods: We attempted to confirm these findings in 50 patients undergoing HD using haemophan (n=23) or polysulphone (n=27) dialysis membranes. Parallel evaluation of ambient air, calorie intake, medication and haemodynamic variables was performed. Samples were analysed using proton transfer reaction-mass spectrometry (PTR-MS)., Results: Significant changes in breath isoprene concentration were observed when comparing patients before [39.14+/-14.96 parts per billion (ppbv)] and after (63.54+/-27.59 ppbv) dialysis (P<0.001). The quotient of values before and after dialysis was 1.84 (SD 1.41). No significant differences in isoprene kinetics were found between the use of haemophan and polysulphone membranes. No significant correlations were observed between isoprene quotients and variations in blood pressure during HD, calorie intake, ingestion of lipid-lowering drugs or serum lipid levels., Conclusions: Isoprene concentration was higher in the exhaled air of patients after HD as compared with values before HD. Large interindividual variability existed in isoprene kinetics. Oxidative stress appears to be an unlikely cause for this rise. An alternative hypothesis is an influence of respiratory variables on isoprene exhalation based upon Henry's law constant. We therefore propose to perform online monitoring of isoprene exhalation by PTR-MS during the HD session to investigate the possible influence of respiratory variables.

A high rate of cardiovascular morbidity and mortality in uremic patients was documented early in the 1970s, and this remained unchanged according to more recent European and U.S. American registry report. In large cross-section studies of uremic patients, traditional cardiovascular risk factors such as hypertension and hypercholesterolemic have been found to have low predictive power, while emerging factors such as markers of inflammation and malnutrition, are strongly correlated with cardiovascular mortality. In addition, retained uremic solute such as b-2-microglobulin, advanced glycosylated end product (AGE), homocysteine, cysteine, sulfate may contribute to the pro-atherogenic milieu of uremia. Exhaled alkanes such as isoprene, increased during and after hemodialysis sessions, have been identified as accelerating factors of the atherosclerotic process. In patients with renal disease, even when GFR is still normal, increased sympathetic activity is demonstrable. The rising catecholamines turnover is a risk factor for cardiovascular disease for the high sensitivity of the heart and the arteries to increased sympthetic activity.

A reaction of 13-hydroperoxide octadecadienoic acid (13-HPODE) with cytochrome c was analysed using ESR, HPLC-ESR and HPLC-ESR-MS by the combined use of the spin-trapping technique. The ESR, HPLC-ESR and HPLC-ESR-MS analyses showed that cytochrome c catalyses formation of pentyl and octanoic acid radicals from 13-HPODE. On the other hand, only the alpha-(4-pyridyl-1-oxide)-N-t-butylnitrone/octanoic acid radical adduct was detected in the elution profile of HPLC-ESR for a mixture of 13-HPODE with haematin, indicating that haematin catalyses the formation of octanoic acid radical. In addition, the reaction of 13-HPODE with cytochrome c was inhibited by chlorogenic acid, caffeic acid and ferulic acid via two possible mechanisms, i.e. reducing cytochrome c (chlorogenic acid and caffeic acid) and scavenging the radical intermediates (chlorogenic acid, caffeic acid and ferulic acid).

Background: Pentane in exhaled gas is often used as an index of lipoperoxidation, but today, there is no standardization for its measurement. In this study, with our technical experience, we determined basal production of pentane in healthy subjects, and we evaluated variability of pentane flow 1 month later., Methods: 18 subjects inhaled hydrocarbon-free air (HCFA) in order to realize a lung washout. Ambient air and three samples (at T0, T10, T30 min) of expired gas were concentrated using a "trap-and-purge" procedure. For the analysis of pentane, an Al(2)O(3)/KCl plot column contained in a gas chromatograph equipped with a flame ionization detector was used., Results: After 10 min of washout, mean (+/-SD) exhalation rate of pentane was 1+/-0.6 pmol min(-1) kg(-1). After 30 min of washout, mean (+/-SD) exhalation rate of pentane was 0.7+/-0.5 pmol min(-1) kg(-1). No significant difference in pentane flow was shown 1 month later for eight subjects who repeated the protocol., Conclusion: With our results and data of the literature, exhalation rates of pentane from healthy adults appear to range between 0.3 and 2 pmol min(-1) kg(-1). The variability of pentane flow 1 month later seems not very important.

In order to estimate cell damage caused by free radicals during oxygenotherapy, we investigated the time course of two markers of lipoperoxidation: pentane in breath and malondialdehyde (MDA) in blood during brief normobaric hyperoxia. Nine healthy subjects inhaled hydrocarbon-free air (HCFA) for 30 minutes, hydrocarbon-free 100% O2 (HCFO2) for 125 minutes and then HCFA for 70 minutes. After 15 minutes of washout with HCFA, ambient pentane was eliminated. After HCFO2, at T175 versus T30 (i.e., 145 min from the start of 100% HCFO2), pentane production increased (P< 0.05). MDA rose significantly at T155 min (i.e., 125 min from the start of HCFO2), versus T30 (P< 0.01). These results suggest that acute hyperoxia causes a moderate increase in lipid peroxidation in healthy subjects. The increase of pentane and MDA confirms that acute hyperoxia induces lipid peroxidation in healthy subjects.

Oxidant stress has been widely implicated as a mechanism of disease, yet clinical trials of antioxidants have not included a biochemical basis for dose selection or patient inclusion. Many of the indices traditionally employed to assess lipid peroxidation have relied on measurements performed in ex vivo systems of questionable relevance to events in vivo. Commonly employed in vivo indices of lipid peroxidation are constrained by such issues as the nonspecificity or instability of the target anylate, contamination of the anylate by events ex vivo, and nonspecificity of analytical methodology. More recently, specific methodology based on mass spectrometry has been applied to both 4-hydroxynonenal and a variety of isoprostanes in human biological fluids. Measurement of these compounds in urine reflects lipid peroxidation in vivo and offers a noninvasive approach that may be readily applied to clinical trials.

The clearance of seven different ligands from the deeply buried active-site of Torpedo californica acetylcholinesterase is investigated by combining multiple copy sampling molecular dynamics simulations, with the analysis of protein-ligand interactions, protein motion and the electrostatic potential sampled by the ligand copies along their journey outwards. The considered ligands are the cations ammonium, methylammonium, and tetramethylammonium, the hydrophobic methane and neopentane, and the anionic product acetate and its neutral form, acetic acid. We find that the pathways explored by the different ligands vary with ligand size and chemical properties. Very small ligands, such as ammonium and methane, exit through several routes. One involves the main exit through the mouth of the enzyme gorge, another is through the so-called back door near Trp84, and a third uses a side door at a direction of approximately 45 degrees to the main exit. The larger polar ligands, methylammonium and acetic acid, leave through the main exit, but the bulkiest, tetramethylammonium and neopentane, as well as the smaller acetate ion, remain trapped in the enzyme gorge during the time of the simulations. The pattern of protein-ligand contacts during the diffusion process is highly non-random and differs for different ligands. A majority is made with aromatic side-chains, but classical H-bonds are also formed. In the case of acetate, but not acetic acid, the anionic and neutral form, respectively, of one of the reaction products, specific electrostatic interactions with protein groups, seem to slow ligand motion and interfere with protein flexibility; protonation of the acetate ion is therefore suggested to facilitate clearance. The Poisson-Boltzmann formalism is used to compute the electrostatic potential of the thermally fluctuating acetylcholinesterase protein at positions actually visited by the diffusing ligand copies. Ligands of different charge and size are shown to sample somewhat different electrostatic potentials during their migration, because they explore different microscopic routes. The potential along the clearance route of a cation such as methylammonium displays two clear minima at the active and peripheral anionic site. We find moreover that the electrostatic energy barrier that the cation needs to overcome when moving between these two sites is small in both directions, being of the order of the ligand kinetic energy. The peripheral site thus appears to play a role in trapping inbound cationic ligands as well as in cation clearance, and hence in product release., (Copyright 2000 Academic Press.)

Background: Although physical exercise training is highly recommended, physical exercise causes oxidative stress, which is potentially injurious. This study evaluates this 'exercise paradox' by evaluating the effect of physical exercise on exercise-induced lipid peroxidation., Methods: Measurement of lipid peroxidation (ie, expired ethane and pentane and plasma malondealdehyde) taken during cardiopulmonary exercise stress testing were compared between a group of 10 cardiac patients who underwent physical exercise training in a cardiac rehabilitation setting and a group of 10 nonexercising cardiac patients., Results: Our findings indicate that physical exercise training increased physical work capacity without a concomitant increase in expired markers of lipid peroxidation (ethane and pentane) and decreased malondealdehyde levels., Conclusions: Because physical exercise-trained people can perform more intense physical work with less oxidative stress, we conclude that physical exercise training can reduce potential chronic health effects associated with daily activities by contributing to an overall reduction in exercise-induced free radical production.

A bacterial strain identified as Pseudomonas aeruginosa was isolated from a soil consortium able to mineralize pentane. P. aeruginosa could metabolize methyl t-butyl ether (MTBE) in the presence of pentane as the sole carbon and energy source. The carbon balance for this strain, grown on pentane, was established in order to determine the fate of pentane and the growth yield (0.9 g biomass/g pentane). An inhibition model for P. aeruginosa grown on pentane was proposed. Pentane had an inhibitory effect on growth of P. aeruginosa, even at a concentration as low as 85 micrograms/l. This resulted in the calculation of the following kinetic parameters (mumax = 0.19 h-1, Ks = 2.9 micrograms/l, Ki = 3.5 mg/l). Finally a simple model of MTBE degradation was derived in order to predict the quantity of MTBE able to be degraded in batch culture in the presence of pentane. This model depends only on two parameters: the concentrations of pentane and MTBE.

The complex formed by isopentane, as a model for the isoleucine residue present in the wild-type thymidylate synthase, with 4-mercaptopyridine as a fragment of the thymidylate synthase inhibitor Thymitaq (AG337) is investigated with ab initio quantum chemical calculations at Hartree-Fock and MP2 levels, using the 3-21G* basis set. The binding energy is compared with the binding energies of 4-mercaptopyridine with amino acid residues found in mutant thymidylate synthase enzymes. As compared with isoleucine, alanine and glycine do not show binding, in agreement with enzyme-inhibition results.

Previous studies have shown that cigarette smoke enhances lipid peroxidation. This study examined the effect of daily consumption of a tomato-based juice supplemented with vitamin C (600 mg), vitamin E (400 IU, or 400 mg), and beta-carotene (30 mg) on various indexes of lipid peroxidation (breath pentane excretion and susceptibility of LDL to copper-mediated oxidation) in smokers. In addition, plasma lycopene and vitamin concentrations and total peroxyl radical trapping potential, a measure of antioxidant defenses, were assessed. Relative to the placebo juice, the vitamin-supplemented juice resulted in a significant decrease in breath-pentane excretion as well as a significant improvement in the resistance of LDL to oxidation. The lag phase of conjugated diene formation lengthened and the propagation rate decreased, indicating a decreased susceptibility of LDL to oxidative modification. Increased concentrations of plasma vitamin C, beta-carotene, and lycopene were found to be significantly correlated with the conjugated diene lag phase and rate of formation. Vitamin E was highly correlated with beta-carotene. Plasma total peroxyl radical trapping potential values did not change in response to supplementation. This study thus indicates that an antioxidant-supplemented drink can reduce lipid peroxidation and susceptibility of LDL to oxidation in smokers and may ameliorate the oxidative stress of cigarette smoke.

The purpose of this study was to investigate in healthy humans the effect of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) intake, alone or in combination with dL-alpha-tocopherol acetate (vitamin E) supplements on lipid peroxidation. Eighty men were randomly assigned in a double-blind fashion to take daily for 6 wk either menhaden oil (6.26 g, n-3 fatty acids) or olive oil supplements with either vitamin E (900 IU) or its placebo. Antioxidant vitamins, phospholipid composition, malondialdehyde (MDA), and lipid peroxides were measured in the plasma at baseline and week 6. At the same time, breath alkane output was measured. Plasma alpha-tocopherol concentration increased in those receiving vitamin E (P < 0.0001). In those supplemented with n-3 fatty acids, EPA and DHA increased in plasma phospholipids (P < 0.0001) and plasma MDA and lipid peroxides increased (P < 0.001 and P < 0.05, respectively). Breath alkane output did not change significantly and vitamin E intake did not prevent the increase in lipid peroxidation during menhaden oil supplementation. The results demonstrate that supplementing the diet with n-3 fatty acids resulted in an increase in lipid peroxidation, as measured by plasma MDA release and lipid peroxide products, which was not suppressed by vitamin E supplementation.

Background: Exhaled pentane, a product of lipid peroxidation, has been proposed as an objective, nonspecific, and noninvasive marker of active inflammation. Reactive oxygen species, which elicit lipid peroxidation, are increased in asthma and contribute to airway dysfunction., Objective: To determine whether exhaled pentane levels are increased in acute asthma, and whether they decrease once acute asthma subsides., Methods: Expired air was collected through a mouthpiece into a pentane-impermeable collection bag from 12 patients (40+/-5 years; mean+/-SEM) presenting to the emergency department of the University of Illinois Hospital in Chicago with acute asthma. Exhaled air was also collected after discharge from the hospital once acute asthma subsided. Eleven patients with stable asthma (40+/-5 years) and 17 healthy volunteers (31+/-5 years) served as control subjects. Exhaled air and ambient room air were analyzed for pentane content by gas chromatography. Peak expiratory flow rates were determined in each subject., Results: Peak expiratory flow rates were 202+/-29 L/min during acute asthma and 327+/-26 L/min once acute asthma subsided (p<0.05). Exhaled pentane levels were 8.4+/-2.9 nmol/L during acute asthma and decreased significantly to 3.5+/-0.5 nmol/L once acute asthma subsided (p<0.05). Exhaled pentane levels were similar in patients with stable asthma and normal control subjects (3.6+/-0.4 nmol/L and 2.6+/-0.2 nmol/L, respectively; p>0.05). No pentane was detected in ambient air., Conclusion: Exhaled pentane levels are increased in patients with acute asthma and decrease significantly once acute asthma subsides.

The measurement of breath pentane as a marker of lipid peroxidation has recently been criticized. Problems encountered include the coelution of isoprene with pentane, contamination with exogenous pentane, and the influence of elevated oxygen concentration. The aim of this project was to investigate and evaluate the Chrompack 9001 Gas Chromatograph, using thermal desorption and cryofocussing and an Al2O3/KCl PLOT column with FID, for use in the measurement of breath pentane in ventilated preterm infants. We have clearly separated isoprene from n-pentane and used hydrocarbon free air to clear the airways and avoid contamination with exogenous pentane. Samples should be stored in Tedlar bags for a maximum of 48 h and on capped desorption tubes for no longer than 24 h. Patient variability was relatively high (mean 18%, n = 4); thus, all patients were sampled in duplicate. No correlation was found between fractional inspired oxygen concentration (FiO2) and exhaled pentane in preterm infants ventilated for respiratory distress syndrome. In conclusion, we feel that despite the pitfalls and technical difficulties, with careful attention to detail it is possible to reliably measure breath pentane in ventilated preterm infants as an index of lipid peroxidation.

Lipid peroxidation is thought to be an important event in the pathogenesis of atherosclerosis. It has been suggested that pentane, which can be formed during the oxidation of omega-6 fatty acids, is a marker of lipid peroxidation. Previous studies have reported elevated breath pentane and serum markers of lipid peroxidation in smokers. However, chromatographic separation of pentane from isoprene in virtually all of these studies was incomplete and the methods used did not resolve pentane into its isomers, n-pentane and isopentane. Additionally, most current methods are complicated, requiring trapping and concentrating steps to obtain adequate sensitivity prior to hydrocarbon analysis. The purpose of the current study was to develop a gas chromatographic system to analyze breath pentane, that addresses the above technical problems and that would provide a simple in vivo method for measuring lipid. n-Pentane and isopentane standards were easily separated from isoprene with a Al2O3/KCI capillary column contained in a portable gas chromatograph equipped with a photoionization detector. The analysis of repeated measures showed a low coefficient of variation for measurements of n-pentane (10%) and isopentane (9%). We measured breath pentane in 27 subjects (15 smokers, 12 non-smokers). There were no significant difference between the baseline and 4 week interval measurements of n-pentane for smokers both before and after cigarette smoking. The within-subject variability data showed that the assay is highly reproducible for both low and high pentane levels in smokers. Smokers were found to have higher levels of both n-pentane and isopentane than non-smokers (P < 0.001). In addition, smokers had further significant elevation of pentane levels 10 min after smoking (P < 0.001), which returned to baseline by 1 h. These studies demonstrate that measurement of breath pentane, using a gas chromatograph with a photoionization detector, is simple and reproducible. Additionally, these results suggest that pentane elevation associated with smoking is secondary to the oxidant effects of cigarette smoke and an important temporal relationship exists between cigarette smoking and breath sample analysis.

Propofol has been reported to have antioxidant properties and to inhibit lipid peroxidation. In this study, we examined the ability of propofol to inhibit lipid peroxidation induced by three free radical systems (hydroxyl, ferryl, and oxo-ferryl radicals), and we compared the effect of propofol with that of vitamin E, an endogenous antioxidant. Lipid peroxidation was induced by exposing a linoleic acid emulsion to either water gamma radiation, a ferrous iron-ascorbate solution, or human hemoglobin, generating the hydroxyl, ferryl, and oxo-ferryl radicals, respectively. Each experiment was performed in triplicate with and without propofol or vitamin E at concentrations between 10(-5) and 10(-4) M. Lipid peroxidation was quantified by gas chromatography measurement of the pentane released (nmoles) from lipid decomposition. In each condition, a significant dose-response relationship was found between the release of pentane and the concentration of either propofol or vitamin E. The antioxidant activities of both agents were similar but significantly higher against the hydroxyl than the ferryl and oxo-ferryl radicals. The study suggests that propofol could be beneficial as an anesthetic or sedative drug in patients presenting pathologies associated with free radical reactions.

Victims of fires are sometimes discovered to have less-than-lethal levels of carbon monoxide (CO) in the blood and no significant antemortem fire damage. Such occurrences are often linked to flash fires involving volatile hydrocarbon fuels. In this study, the dynamics of hydrocarbon fuel fires are examined, and the results of fullscale room tests ignited with small (< 2 L) quantities of flammable liquid are found to confirm the theoretical predictions. These tests showed that flame plumes with temperatures of 500-975 degrees C were produced above flammable liquids. Ignition of their vapors in a carpeted room produced a very short-lived flash of fire throughout the room, followed by intense flames in a layer above the floor approximately 1 m deep, which quickly degenerated to isolated pools of low flames. Combustion of hydrocarbon vapors in a room caused oxygen levels to drop below 8.5% in < 100 s, while causing carbon dioxide (CO2) levels to increase to 12-16% whether the door to the room was open or closed. Production of CO trailed maximum CO2 production by 15-30 s. A victim exposed to such a fire may collapse from extreme heat (aided by the water vapor created by the combustion of hydrocarbons), weakened by oxygen deprivation, before CO inhalation becomes a significant factor.

The concentrations of acetone, isoprene, and pentane in alveolar breath were examined in 50 smokers and 50 nonsmokers by gas chromatography. The baseline pentane in smokers was 0.17 +/- 0.03 nmol/L (mean +/- SE), which was not different from pentane in nonsmokers (0.23 +/- 0.03 nmol/L). There were also no differences between smokers and nonsmokers in the concentrations of acetone and isoprene. Serial breath samples were obtained from 15 smokers before smoking and at 5, 15, and 60 min after smoking. Although acetone was not altered by smoking, isoprene increased by 86% +/- 26% 5 min after smoking (P <0.001) and returned to baseline 10 min later. Pentane increased by 456% +/- 156% 5 min after smoking (P <0.001) and remained increased 10 min later (204% +/- 73% of baseline, P <0.05). Isoprene concentrations in mainstream cigarette smoke were >5000 times greater than breath concentrations, whereas pentane could not be detected in mainstream smoke. Because pentane is produced from the peroxidation of n-6 polyunsaturated fatty acids, the results provide evidence that cigarette smoking causes an immediate increase in lipid peroxidation.

Exhalation of ethane and pentane was used to access lipid peroxidation in rats receiving the hepatocarcinogenic peroxisome proliferator Wy-14,643 at 0.1% in the diet. Wy-14,643 treatment from 23 to 345 days did not increase ethane or pentane exhalation. The lack of an increase in ethane exhalation from rats fed Wy-14,643 was not due to resistance to lipid peroxidation or increased metabolism of ethane since rats fed Wy-14,643 were sensitive to CCl4-induced increases in ethane exhalation and cleared exogenous ethane at rates similar to controls. Difference spectra of hepatic lipids extracted from control and Wy-14,643-treated rats showed peaks at 220 and 275 nm but no defined peak at 240 nm, the wavelength at which conjugated dienes in peroxidized lipids absorb. In contrast, injection of CCl4 ip into control rats produced dose-dependent increases in ethane exhalation at 6.25 to 100 microliters/kg and increases in hepatic conjugated dienes and serum liver enzyme activities at 200 to 1000 microliters/kg. The lack of increased ethane and pentane exhalation in combination with no detectable increase in hepatic conjugated dienes argues against increased hepatic lipid peroxidation in rats receiving a hepatocarcinogenic dose of Wy-14,643.

Objectives: To investigate the possibilities of personal ambient monitoring and biological monitoring for methylpentane isomers., Methods: The performance of activated carbon cloth to absorb 2- and 3-methylpentane was studied by experimental vapour exposure followed by solvent extraction and gas chromatography (GC). Urine from workers and rats exposed to 2- and 3-methylpentane was analysed by GC with or without acid or enzymatic hydrolysis., Results: Carbon cloth absorbed 2- and 3-methylpentane linearly to exposures up to eight hours and to 400 ppm, and was sensitive enough to detect a 15 minute peak of exposure. The two isomers were clearly separated from hexane on a DB-1 column. For analysis of the urine, enzymatic hydrolysis was superior to acid hydrolysis. Exposure of rats to methylpentane vapours showed that 2-methyl-2-pentanol and 3-methyl-2-pentanol were excreted in urine in proportion to the dose of 2-methylpentane and 3-methylpentane, respectively. 2-Methyl derivatives of 1-, 3-, and 4-propanol, 2-methylpentane-2,4-diol, and 3-methyl-2-pentanol were minor metabolites. Analysis of urine from the exposed workers showed that 2-methyl- and 3-methyl-2-pentanol are leading urinary metabolites after exposure to the corresponding methylpentane., Conclusions: Diffusive sampling is applicable to monitor 2- and 3-methylpentane vapours as is the case for hexane vapour. 2-Methyl-2-pentanol and 3-methyl-2-pentanol will be markers of occupational exposure to 2-methylpentane and 3-methylpentane, respectively. Also, 2-methylpentane-2,4-diol might be a marker of exposure to 2-methylpentane.

Pentane and isoprene concentrations were analyzed in single end-expiratory breath samples using gas chromatography. Breath analysis was performed in 15 patients with acute myocardial infarction, 15 patients with stable angina, and 15 healthy control subjects. The two patient groups were well matched for age, sex, smoking habits, hypertension and serum cholesterol levels. There was no significant difference in breath pentane concentration in the acute myocardial infarction group (0.29 +/- 0.03 nmol/l) (mean +/- SEM) compared to the group with stable angina (0.31 +/- 0.03 nmol/l) or the control group (0.36 +/- 0.04 nmol/l). However, breath isoprene concentration was higher (p < 0.01) in the acute myocardial infarction group (11.4 +/- 1.2 nmol/l), compared to both the stable angina group (7.7 +/- 0.5 nmol/l) and the control group (7.1 +/- 1.0 nmol/l). There was no difference in either the pentane or isoprene concentrations between the control group and the group with stable angina. Since pentane is thought to be an index of lipid peroxidation, the results do not support the presence of enhanced lipid peroxidation in acute myocardial infarction in the absence of thrombolytic therapy or primary angioplasty. The mechanism responsible for isoprene elevation in acute myocardial infarction is unknown.

A new method designed to monitor lipid peroxidation in plants has been set up with soybean hypocotyl/radicles. The hydroperoxy fatty acids present in situ are converted by rapid thermal treatment (80 s and 210 J g-1) of the biological sample into ethane and n-pentane, which are analyzed by gas chromatography. The method has been directly calibrated by quantification of the hydroperoxy fatty acids by silica-phase HPLC analysis of their reduced hydroxy derivatives. Hypocotyl/radicles from the two soybean cultivars Argenta and Soriano were submitted to various chemical oxidative treatments and were analyzed for both thermally produced volatile alkanes and hydroperoxy fatty acid levels. Our results showed that ethane and n-pentane production are in both cases closely correlated with linolenic as well as linoleic acid hydroperoxide levels (P < 0.001). Within a given plant material, thermal conversion of both hydroperoxides into alkanes occurred with yields which were not dependent on the oxidative treatment. These yields are however functions of the biological material since in Soriano and Argenta cultivars they were around 6 and 25%, respectively. Taking into account the last point, the alkane test cannot be used to directly quantify the absolute lipid hydroperoxide levels of plant tissues but it is convenient to monitor the peroxidative phenomenon as it occurs. The assay is easy and rapid to perform (analysis of 50 samples per day) since no sample preparation is needed, and the low detection limit (20 pmol of alkane g-1) permits the analysis of small samples.

Objective: Reactive oxygen species (ROS) may be a major cause of tissue injury in ulcerative colitis (UC). One possible mechanism for ROS-mediated tissue injury is lipid peroxidation. Breath ethane and pentane excretion are noninvasive means for measuring peroxidation of omega-3 and omega-6 polyunsaturated fatty acid, respectively. Hence, we measured breath ethane in 17 subjects with active UC and correlated the results with disease severity., Methods: Breath samples for ethane and pentane analysis were collected every 2 weeks, and rectal biopsies were obtained monthly to assess for chemiluminescence, a marker of ROS. Ethane and pentane concentrations (nmol/L) were measured by gas chromatography, and mucosal chemiluminescence was measured spectrophotometrically. Data were compared to control values (C) from healthy subjects. Disease activity was assessed both clinically and endoscopically., Results: Ethane excretion was significantly elevated in patients (UC, 0.45 +/- 0.04; C, 0.33 +/- 0.06; p = 0.013). Ethane excretion was positively correlated with endoscopic score (r = 0.45; p < 0.05), symptom score (r = 0.34; p < 0.05), disease activity (r = 0.36, p < 0.05), and chemiluminescence (r = 0.65; p < 0.001). Pentane levels did not correlate with any of the clinical measurements. Chemiluminescence in the rectal tissue was positively correlated with endoscopic score (r = 0.71; p < 0.05) and disease activity (r = 0.61; p < 0.01)., Conclusions: Tissue damage in UC may be ROS-induced lipid peroxidation. Disease activity can be assessed noninvasively by breath ethane excretion.

Sevoflurane is a recently introduced volatile inhalation anaesthetic and is already used commonly in Japan. We investigated the potential of sevoflurane to cause lipid peroxidation in vivo and in vitro. For the in vitro study, pentane formation in a mixture of guinea pig liver microsomes and sevoflurane in the presence of nicotinamide adenine dinucleotide phosphate (NADPH) was analyzed by gas chromatography. Under anaerobic conditions, pentane formed without sevoflurane, but sevoflurane potentiated this anaerobic pentane formation. Two antioxidant agents, vitamine E and glutathione, reduced the pentane formation induced by sevoflurane. In the in vivo study, 18 guinea pigs were exposed to air (control), 0.5% halothane, or 1.2% sevoflurane. The extent of lipid peroxidation and liver damage was investigated by measuring the level of thiobarbituric acid reactive products and serum transaminase (alanine-aminotransferase: ALAT and aspartate-aminotransferase: ASAT) activity 12 hr after exposure. Both halothane and sevoflurane significantly increased thiobarbituric acid-reactive products. The increase in thiobarbituric acid-reactive products seen with sevoflurane administration was half that seen with halothane. Sevoflurane increased the ALAT activity to the same extent as did halothane but did not increase the ASAT activity. We conclude that sevoflurane potentiates lipid peroxidation in guinea pig liver microsomes in vivo and in vitro. However, because the degree of liver damage as measured by transaminase activity was minimal and the mechanism of sevoflurane-induced lipid peroxidation is still unknown, we must be cautious in applying these results to humans.

Previous studies have raised the question of whether pentane is a normal constituent of human breath, since its concentrations in inspired room air and expired breath are often similar. Using a highly sensitive assay for volatile organic compounds, we studied 37 normal subjects in order to determine the alveolar gradient of pentane in their breath (i.e. concentration in alveolar breath minus concentration in the inspired air). The chemical identity of pentane was confirmed by mass spectroscopy. The alveolar gradient of pentane was zero +/- 0.175 nmol/l in 54.1% of subjects, and distributed in an approximately bell-shaped curve. Determination of the alveolar gradient divided the normal subjects into three groups: the "passive equilibrators" who did not appear to excrete pentane in the breath (the majority), "metabolizers" who actively catabolized inhaled pentane, and "manufacturers" who excreted more pentane than they inhaled.

Bioefficiencies of alpha-, gamma- and delta-tocopherol in comparison with all-rac-alpha-tocopherol were established in broiler chickens. For this, 1-d-old male broiler chickens received a diet deficient in vitamin E and supplemented with increasing doses of the corresponding tocopheryl acetates. After 2 and 3 weeks of feeding, the animals were killed to obtain blood and liver samples. The ex vivo tests used were detergent-induced haemolysis and pentane production by liver microsomes. Bioefficiencies were calculated by comparison of the dose-response curves. It is concluded that haemolysis and pentane production are appropriate indicators of the bioefficiency of tocopherols in broiler chickens. The values obtained by both tests hardly differed and agree well with the figures previously obtained from rats and other species.