Identification of volatile organic compounds secreted from cancer tissues and bacterial cultures

The early cancer diagnosis increases the possibility of total recovery. The infection of Helicobacter pylori is associated with gastric cancer, the second most common cancer in the world. The determination of volatile organic compounds (VOCs) excreted by stomach tissue and bacteria culture has been investigated. Solid-phase microextraction (SPME) was used for preconcentration and the determination was accomplished by gas chromatography coupled with mass spectrometry (GC/MS). The samples of tissue were taken from five patients (ten samples) with stomach cancer and normal (non-cancerous) segments from other parts of the stomach were used as a control. Eighteen compounds were identified in stomach tissue and seven of them were present both in healthy and cancer tissue. These compounds assumed to be endogenous and acetone ratio (AR) was calculated for ethanol, butane, carbon disulfide, 1-propanol, 2-butanone and 2-pentanone. The data shows that amount of 1-propanol and carbon disulfide in the gaseous composition is higher in cancer tissue than in normal tissue. Eight compounds were identified both in bacteria and tissue. These data suggest that bacteria present in the stomach might cause the increase in the concentration of 1-propanol and carbon disulfide in emission from cancer tissue.     

Rapid determination of acetone in human plasma by gas chromatography-mass spectrometry and solid-phase microextraction with on-fiber derivatization

Acetone is an important volatile disease marker. Due to its nature of activity and volatility, it is a difficult task to measure the concentration of acetone in biological samples with accuracy. In this paper, we developed a novel method for determination of trace amount acetone in human plasma by solid-phase microextraction technique with on-fiber derivatization. In this method, the poly(dimethylsiloxane)/divinylbenzene (PDMS/DVB) fiber was used and O-2,3,4,5,6-(pentafluorobenzyl) hydroxylamine hydrochloride (PFBHA) was first loaded on the fiber. Acetone in plasma sample was agitated into headspace and extracted by solid-phase microextraction (SPME) fiber and subsequently derivatized with PFBHA on the fiber. Acetone oxime was analyzed by gas chromatography-mass spectrometry (GC-MS). Quantitative analysis of acetone in plasma was carried out by using external standard method. The SPME conditions (extraction temperature and time) and the method validation were studied. The present method was tested by determination of acetone in diabetes plasma and normal plasma. Acetone concentration in diabetes plasma was found to be higher than 1.8mM, while in normal plasma was lower than 0.017 mM. The results show that the present method is a potential tool for diagnosis of diabetes.     

Optimization of solid-phase microextraction conditions for gas chromatographic determination of ethanol and other volatile compounds in blood

A procedure for the determination of acetaldehyde, acetone, methanol, ethanol, 1-propanol and 2-propanol in blood was developed. Separation of analytes was carried out on DB-wax capillary column (l = 30 m, I.D. = 0.32 mm, dF = 0.5 microm) at 40 degrees C, hydrogen was used as a carrier gas (at 30 kPa) and FID as a detector. Quantification was performed with the use of 2-butanol as an internal standard. Headspace solid-phase microextraction was applied as the sample preparation technique. The usefulness of most commercially available fiber coatings was checked and 65 microm Carbowax/DVB proved most effective. Microextraction was carried out from the headspace at 60 degrees C for 10 min. The sample was stirred at 750 rpm. In order to improve the extraction efficiency of analytes, salting-out agents were also applied. Potassium carbonate turned out to be the most efficient. A 1.0-g amount of this salt and 0.1 ml of I.S. were added to 0.5 ml of sample. Validation of the worked-out method was performed. For each analyte, the limits of detection and quantification, linearity, working range, accuracy and precision were determined or tested.     

Production of Methyl Ketones from Secondary Alcohols by Cell Suspensions of C(2) to C(4)n-Alkane-Grown Bacteria

Nineteen new C(2) to C(4)n-alkane-grown cultures were isolated from lake water from Warinanco Park, Linden, N.J., and from lake and soil samples from Bayway Refinery, Linden, N.J. Fifteen known liquid alkane-utilizing cultures were also found to be able to grow on C(2) to C(4)n-alkanes. Cell suspensions of these C(2) to C(4)n-alkane-grown bacteria oxidized 2-alcohols (2-propanol, 2-butanol, 2-pentanol, and 2-hexanol) to their corresponding methyl ketones. The product methyl ketones accumulated extracellularly. Cells grown on 1-propanol or 2-propanol oxidized both primary and secondary alcohols. In addition, the activity for production of methyl ketones from secondary alcohols was found in cells grown on either alkanes, alcohols, or alkylamines, indicating that the enzyme(s) responsible for this reaction is constitutive. The optimum conditions for in vivo methyl ketone formation from secondary alcohols were compared among selected strains: Brevibacterium sp. strain CRL56, Nocardia paraffinica ATCC 21198, and Pseudomonas fluorescens NRRL B-1244. The rates for the oxidation of secondary alcohols were linear for the first 3 h of incubation. Among secondary alcohols, 2-propanol and 2-butanol were oxidized at the highest rate. A pH around 8.0 to 9.0 was found to be the optimum for acetone or 2-butanone formation from 2-alcohols. The temperature optimum for the production of acetone or 2-butanone from 2-propanol or 2-butanol was rather high at 60 degrees C, indicating that the enzyme involved in the reaction is relatively thermally stable. Metal-chelating agents inhibit the production of methyl ketones, suggesting the involvement of a metal(s) in the oxidation of secondary alcohols. Secondary alcohol dehydrogenase activity was found in the cell-free soluble fraction; this activity requires a cofactor, specifically NAD. Propane monooxygenase activity was also found in the cell-free soluble fraction. It is a nonspecific enzyme catalyzing both terminal and subterminal oxidation of n-alkanes.     

Enhanced activity and stability of immobilized lipases by treatment with polar solvents prior to lyophilization

Lipases from Candida rugosa, Mucor javanicus and Rhizopus oryzae were respectively adsorbed on Amberlite XAD-7 followed by incubation in 2-propanol and then lyophilization. The activities of the immobilized enzymes were 1.6–3.4 times higher than those of the immobilized enzymes without incubation in the organic solvent before lyophilization for esterification of lauric acid (0.1 M) and 1-propanol (0.1 M) in isooctane at 37 °C. The immobilized C. rugosa lipase (Sigma) without the incubation did not show any activity but displayed considerable activity (19.8 μmol h⁻¹ mg⁻¹) after the incubation before lyophilization. Besides 2-propanol, acetone, 1-propanol and ethyl acetate were also found to be good solvents for treating M. javanicus lipase immobilized on Amberlite XAD-7 and acetone was the best among them. When incubated in isooctane at 25 °C for 120 h, the immobilized M. javanicus lipase prepared by incubation in acetone for 1 h before lyophilization retained 70% of its initial activity while the immobilized enzyme without the solvent treatment kept only 50% of its initial activity.     

A Medium for the Isolation of Capsular Bacteria from Kefir Grains

Secondary alcohols (C₃ to C₁₀) were oxidized to the corresponding methylketones by resting mycelia of Scedosporium sp. A-4 grown on propane, but 3-pentanol and 3-hexanol were not oxidized. The oxidation of 2-propanol to acetone was inhibited by pyrazole, potassium cyanide, sodium azide and Hg^{2 +}. Alcohol dehydrogenase activity was found in the cell-free soluble fraction and this activity requires a cofactor, specifically NAD⁺. The oxidation of both 1-propanol and 2-propanol may be catalyzed by the same alcohol dehydrogenase.     

Secondary alcohol dehydrogenase catalyzes the reduction of exogenous acetone to 2-propanol in Trichomonas vaginalis

Secondary alcohols such as 2-propanol are readily produced by various anaerobic bacteria that possess secondary alcohol dehydrogenase (S-ADH), although production of 2-propanol is rare in eukaryotes. Specific bacterial-type S-ADH has been identified in a few unicellular eukaryotes, but its function is not known and the production of secondary alcohols has not been studied. We purified and characterized S-ADH from the human pathogen Trichomonas?vaginalis. The kinetic properties and thermostability of T.?vaginalis S-ADH were comparable with bacterial orthologues. The substantial activity of S-ADH in the parasite's cytosol was surprising, because only low amounts of ethanol and trace amounts of secondary alcohols were detected as metabolic end products. However, S-ADH provided the parasite with a high capacity to scavenge and reduce external acetone to 2-propanol. To maintain redox balance, the demand for reducing power to metabolize external acetone was compensated for by decreased cytosolic reduction of pyruvate to lactate and by hydrogenosomal metabolism of pyruvate. We speculate that hydrogen might be utilized to maintain cytosolic reducing power. The high activity of Tv-S-ADH together with the ability of T.?vaginalis to modulate the metabolic fluxes indicate efficacious metabolic responsiveness that could be advantageous for rapid adaptation of the parasite to changes in the host environment.     

Anaerobic 2-Propanol Degradation in Anoxic Paddy Soil and the Possible Role of Methanogens in Its Degradation

The anaerobic degradation of 2-propanol in anoxic paddy soil was studied with soil cultures and a 2-propanol-utilizing methanogen. Acetone was the first and the major intermediate involved in the methanogenic degradation of 2-propanol. Analyses with a methanogenesis inhibitor, bacteria antibiotics, and the addition of H₂ to the gas phase revealed that 2-propanol oxidation to acetone directly occurred using 2-propanol-utilizing methanogens, but not with H₂-producing syntrophic bacteria, for which the removal of acetone is required for complete 2-propanol oxidation. The 2-propanol-utilizing strain IIE1, which is phylogenetically closely related to Methanoculleus palmolei, was isolated from paddy soil, and the potential role of the strain in 2-propanol degradation was investigated. 2-Propanol is one of the representative fermentation intermediates in anaerobic environments. This is the first report on the anaerobic 2-propanol degradation process.     

Verrucomicrobial methanotrophs grow on diverse C3 compounds and use a homolog of particulate methane monooxygenase to oxidize acetone

Short-chain alkanes (SCA; C2-C4) emitted from geological sources contribute to photochemical pollution and ozone production in the atmosphere. Microorganisms that oxidize SCA and thereby mitigate their release from geothermal environments have rarely been studied. In this study, propane-oxidizing cultures could not be grown from acidic geothermal samples by enrichment on propane alone, but instead required methane addition, indicating that propane was co-oxidized by methanotrophs. “Methylacidiphilum” isolates from these enrichments did not grow on propane as a sole energy source but unexpectedly did grow on C3 compounds such as 2-propanol, acetone, and acetol. A gene cluster encoding the pathway of 2-propanol oxidation to pyruvate via acetol was upregulated during growth on 2-propanol. Surprisingly, this cluster included one of three genomic operons (pmoCAB3) encoding particulate methane monooxygenase (PMO), and several physiological tests indicated that the encoded PMO3 enzyme mediates the oxidation of acetone to acetol. Acetone-grown resting cells oxidized acetone and butanone but not methane or propane, implicating a strict substrate specificity of PMO3 to ketones instead of alkanes. Another PMO-encoding operon, pmoCAB2, was induced only in methane-grown cells, and the encoded PMO2 could be responsible for co-metabolic oxidation of propane to 2-propanol. In nature, propane probably serves primarily as a supplemental growth substrate for these bacteria when growing on methane.Subject terms: Environmental microbiology, Biogeochemistry, Microbial ecology     

Mutagenicity studies on complex environmental mixtures: selection of solvent system for extraction.

The mutagenic activities in the Salmonella/microsome assay of dichloromethane (DCM) and acetone extracts of complex environmental mixtures were compared. The particulate samples used in the IPCS collaborative study were Soxhlet-extracted twice with DCM followed by a third extraction with acetone. Compared with the mutagenic activity of the first extract, the third (acetone) extract of the urban particulate matter showed a relatively high mutagenic activity. In contrast to this the third extract of the diesel particulate matter contributed very little additional mutagenic activity. Furthermore, 10 filter samples of air particulates from a suburban airport area were collected for comparison of the extraction efficiency of DCM and acetone. Each sample was divided into two samples of identical size followed by extraction with acetone and DCM, respectively. No clear difference in the mutagenic activity of these extracts was observed in strains TA98 and TA98NR. It is concluded that for ambient air particulates (but not emission samples) acetone may extract some mutagenic compounds which are not extracted by DCM. The amount of these additional extractable compounds seems to depend on the composition of the sample. As DCM extracts are better suited for further fractionation and chemical analysis DCM is considered to be the best choice for a general solvent system for extraction of complex environmental mixtures.     

Production of Methyl Ketones from Secondary Alcohols by Cell Suspensions of C2 to C4n-Alkane-Grown Bacteria

Nineteen new C₂ to C₄n-alkane-grown cultures were isolated from lake water from Warinanco Park, Linden, N.J., and from lake and soil samples from Bayway Refinery, Linden, N.J. Fifteen known liquid alkane-utilizing cultures were also found to be able to grow on C₂ to C₄n-alkanes. Cell suspensions of these C₂ to C₄n-alkane-grown bacteria oxidized 2-alcohols (2-propanol, 2-butanol, 2-pentanol, and 2-hexanol) to their corresponding methyl ketones. The product methyl ketones accumulated extracellularly. Cells grown on 1-propanol or 2-propanol oxidized both primary and secondary alcohols. In addition, the activity for production of methyl ketones from secondary alcohols was found in cells grown on either alkanes, alcohols, or alkylamines, indicating that the enzyme(s) responsible for this reaction is constitutive. The optimum conditions for in vivo methyl ketone formation from secondary alcohols were compared among selected strains: Brevibacterium sp. strain CRL56, Nocardia paraffinica ATCC 21198, and Pseudomonas fluorescens NRRL B-1244. The rates for the oxidation of secondary alcohols were linear for the first 3 h of incubation. Among secondary alcohols, 2-propanol and 2-butanol were oxidized at the highest rate. A pH around 8.0 to 9.0 was found to be the optimum for acetone or 2-butanone formation from 2-alcohols. The temperature optimum for the production of acetone or 2-butanone from 2-propanol or 2-butanol was rather high at 60°C, indicating that the enzyme involved in the reaction is relatively thermally stable. Metal-chelating agents inhibit the production of methyl ketones, suggesting the involvement of a metal(s) in the oxidation of secondary alcohols. Secondary alcohol dehydrogenase activity was found in the cell-free soluble fraction; this activity requires a cofactor, specifically NAD. Propane monooxygenase activity was also found in the cell-free soluble fraction. It is a nonspecific enzyme catalyzing both terminal and subterminal oxidation of n-alkanes.     

分光光度法测定地骨皮中牛磺酸含量

用分光光度法测定地骨皮中是否含有牛磺酸。在一定条件下,牛磺酸与乙酰丙酮和甲醛反应生成带色的配合物,建立了测定牛磺酸含量的分光光度法。结果表明,地骨皮中含有牛磺酸,已测定样品1中牛磺酸的质量分数为3.124 mg.g-1,样品2中牛磺酸的质量分数为6.203 mg.g-1,且样品2中的牛磺酸质量分数极显著高于样品1(p<0.01)。研究结果表明,地骨皮中含有牛磺酸,而且分光光度法成本低,干扰少,是测定地骨皮中牛磺酸质量分数的较好方法。     

Polymer membrane ion-selective electrodes as a convenient tool for lipases and esterases assays

We have proposed a novel assay for lipases and esterases activity determination based on potentiometry with ion-selective electrodes (ISEs). Enzyme preparations, obtained from the living cells, are complex mixtures of various proteins, short peptides, lipids, carbohydrates, and other compounds. The most commonly used quantitative methods in enzyme studies are based on spectrophotometric or spectroflourimetric protocols which has significant limitations. They are not valid for samples that are turbid or strongly colored. To overcome those drawbacks we have proposed an assay based on potentiometry with ISEs for lipases and esterases activity determination. This electrochemical methodology represents an attractive tool for enzyme analysis, because of its low detection limit, independence from sample volume and from sample turbidity. The usefulness of this assay has been proven by the determination of the activity of various raw enzymes “acetone powders” isolated from animal tissues. Moreover, activities of fractions obtained during purification of one of those raw biocatalysts were also determined that way. The reliability of determination enzyme activity with ISE assay was proven by comparison with a classical spectrophotometric method.     

2-Propanol degradation by Sulfolobus solfataricus

Sulfolobus solfataricus used 2-propanol and 2-propanone (acetone) when grown in static cultures at 78 °C with or without glucose at 10 g l^–1. The presence of 3.92 g 2-propanol l^–1 in both cases inhibited growth. However, acetone accumulation following 2-propanol depletion suggested that 2-propanol was co-metabolized via the acetone metabolic pathway. Glucose at 10 g l^–1 increased 2-propanol and acetone utilization from 0.93 g l^–1 to 1.77 g l^–1 and from 0.11 g l^–1 to 1.62 g l^–1, respectively. Without glucose, immobilized S. solfataricus cells increased the 2-propanol removal rate to 0.035 g l^–1 h^–1, compared to 0.0012 g l^–1 h^–1 by its suspended counterpart. The results suggest the establishment of an immobilized reactor configuration is preferential for the treatment of high temperature solvent waste streams by this acidothermophilic Crenarchaeon.     

Influence of the solvent ability to form hydrogen bonds in the crystal structure of ([3β,5β,7α,12α]-3[(norbornyl-2-acetyl)-amino]-7,12-dihydroxycholan-24-oic acid (a norbornyl derivative of cholic acid)

A norbornyl-2-acetyl derivative of cholic acid ([3β,5β,7α,12α]-3[(norbornyl-2-acetyl)-amino]-7,12-dihydroxycholan-24-oic acid -NbCH₂CA-) was synthesized and recrystallized in two dipolar aprotic solvents (acetone, DMSO) and in one protic solvent (2-propanol). In DMSO and acetone the crystals are orthorhombic, P2₁2₁2₁ (all their parameters being very similar) while in 2-propanol the crystal is monoclinic, P2₁. The inclusion complexes with the solvent have a 1:1 stochiometry with DMSO and acetone and 1:2 with 2-propanol. All solvents are forming a hydrogen bond with the amide bond of the bridge between the norbornyl residue and the steroid nucleus of the bile acid. In DMSO and acetone the β side of the steroid groups lies in the same region facilitating hydrophobic interactions, and the molecules are disposed in an antiparallel orientation (the methyl groups having a β interdigitation) forming bilayers. The width of the bilayers is 9.231 Å and 8.859 Å in DMSO and acetone, respectively. A lamellar structure is also evident for the crystal in 2-propanol (the width being 11.908 Å), but the packing is different from the previous one since a sliding between the steroid groups is observed and the methyl groups are not interdigitated. Four different hydrogen bonds are established by every steroid molecule in the NbCH₂CA/DMSO (or acetone) crystal. This hydrogen bond network interconnects the hydrophilic regions of the lamellar structure. The hydrogen bond network of the NbCH₂CA:2-propanol crystal is different because of the different abilities of 2-propanol to form hydrogen bonds. The side chain has a ttti conformation in the two orthorhombic crystals, and a tgtg one in the monoclinic crystal.     

On the organic solvent and thermostability of the biocatalytic redox system of Rhodococcus ruber DSM 44541

The sec-alcohol dehydrogenase activity of whole cells of Rhodococcus ruber DSM 44541 has been employed as an efficient biocatalytic redox system due to the use of acetone and 2-propanol at elevated concentrations for cofactor regeneration in the oxidation and reduction mode, respectively, and external addition of NADH/NAD(+) can be omitted. The operational half-life time of the redox system is 29 hours in 20% v/v acetone and 37 hours in 30% v/v 2-propanol. The Redox system allows the enantioselective oxidation of sec-alcohols and the asymmetric reduction of ketones to furnish (S)-configurated alcohols in high optical purity. The stability of the cells towards further organic solvents was investigated. In addition, the system displays thermostability of up to 60 degrees C and pH stability of up to pH 11. The system represents a simple to handle tool for environmentally benign redox reactions.     

Characterization of a solvent resistant and thermostable aminopeptidase from the hyperthermophillic bacterium, Aquifex aeolicus

A leucine aminopeptidase gene of Aquifex aeolicus, a hyperthermophilic bacterium, was cloned and expressed in Escherichia coli, and its expression product was purified and characterized. The expressed protein was purified to homogeneity by using heat to denature contaminating proteins followed by ion-exchange chromatography to purify the heat-stable product. The purified enzyme gave a single band on SDS-PAGE with a molecular weight of 54 kDa. Kinetic studies on the purified enzyme confirmed that it was a leucine aminopeptidase. The optimum temperature for its activity was around 80 degrees C and the optimum pH was in the range from 8.0 to 8.5. It was stable at high temperatures and 27% of its activity was retained after heating at 115 degrees C for 30 min. The purified enzyme had a pH stability range between 4.0 and 11.0. This aminopeptidase was highly resistant to organic solvents such as methanol, ethanol, tetrahydrofuran, dimethyl sulfoxide, acetone, acetonitrile, dimethyl formamide, 1-propanol, 2-propanol, and dioxane.     

Optimizing lipase activity, enantioselectivity, and stability with medium engineering and immobilization for beta-blocker synthesis.

Lipase from Pseudomonas cepacia showed poor activity and moderate enantioselectivity (E) in pure aqueous systems for hydrolysis of a racemic mixture (+/-)-1-chloro-2-acetoxy-3-(1-naphthyloxy)-propane, which is a potential intermediate for beta-blocker synthesis. However, addition of polar organic solvents to the reaction can change both the activity and the enantioselectivity for this chiral reaction significantly. It was observed, in general, that the activity increases and the enantioselectivity decreases with the increase in the polarity of the organic solvent added to the medium. Among the six solvents chosen (i.e., dimethylsulfoxide [DMSO], 1, 4-dioxane, dimethylformamide [DMF], acetone, 1-propanol, and tetrahydrofuran [THF]), maximum activity and minimum enantioselectivity was obtained with DMSO, whereas minimum activity and maximum enantioselectivity was obtained with THF as the cosolvents. In the subsequent studies, native or polyethylene glycol (PEG)-modified lipase was immobilized by entrapping in Caalginate gel beads. In a fixed-bed continuous reactor containing these catalyst beads, the enzyme was found to be at least three times more enantioselective than the native form in a batch reactor. This fixed-bed reactor with the beads could be operated with high concentration of acetone (33% v/v) for about 1 month without a significant loss of enzyme activity and enantioselectivity.     

Simple and rapid measurement of platelet-activating factor (PAF) in whole blood.

A simple assay of platelet-activating factor (PAF) in whole blood was developed, employing acetone extraction and thin layer chromatography (TLC) purification of blood sample. The activity of acetylhydrolase present in blood sample was almost completely suppressed by ice-cold acetone extraction, and other inhibitory substances interfering the activity of PAF were effectively removed from the acetone extract by TLC. Then, the treated samples were subjected to a conventional PAF bioassay using rabbit platelets. The recovery rate of PAF by the above procedure was constant and feasible (46-48%). The lower limit of the present assay was estimated to be 1.0 x 10(-10) M. Employing the present method, it was able to determine the amount of PAF in blood (1.2-6.0 x 10(-10) M) of 6 out of 14 septic patients, while no significant PAF activity was detected in the samples from 6 healthy subjects. These results indicate a potential application of the present method in the clinical assay of PAF in blood.