Quantification of Free Radicals from Vaping Electronic Cigarettes Containing Nicotine Salt Solutions with Different Organic Acid Types and Concentrations.

Electronic (e-) cigarette formulations containing nicotine salts from a range of organic acid conjugates and pH values have dominated the commercial market. The acids in the nicotine salt formulations may alter the redox environment in e-cigarettes, impacting free radical formation in e-cigarette aerosol. Here, the generation of aerosol mass and free radicals from a fourth-generation e-cigarette device was evaluated at 2 wt % nicotine salts (pH 7, 30:70 mixture propylene glycol to vegetable glycerin) across eight organic acids used in e-liquids: benzoic acid (BA), salicylic acid (SLA), lactic acid (LA), levulinic acid (LVA), succinic acid (SA), malic acid (MA), tartaric acid (TA), and citric acid (CA). Furthermore, 2 wt % BA nicotine salts were studied at the following nicotine to acid ratios: 1:2 (pH 4), 1:1 (pH 7), and 2:1 (pH 8), in comparison with freebase nicotine (pH 10). Radical yields were quantified by spin-trapping and electron paramagnetic resonance (EPR) spectroscopy. The EPR spectra of free radicals in the nicotine salt aerosol matched those generated from the Fenton reaction, which are primarily hydroxyl (OH) radicals and other reactive oxygen species (ROS). Although the aerosol mass formation was not significantly different for most of the tested nicotine salts and acid concentrations, notable ROS yields were observed only from BA, CA, and TA under the study conditions. The e-liquids with SLA, LA, LVA, SA, and MA produced less ROS than the 2 wt % freebase nicotine e-liquid, suggesting that organic acids may play dual roles in the production and scavenging of ROS. For BA nicotine salts, it was found that the ROS yield increased with a higher acid concentration (or a lower nicotine to acid ratio). The observation that BA nicotine salts produce the highest ROS yield in aerosol generated from a fourth-generation vape device, which increases with acid concentration, has important implications for ROS-mediated health outcomes that may be relevant to consumers, manufacturers, and regulatory agencies.

Quantification, regulation and production of 5-aminolevulinic acid by green fluorescent protein in recombinant Escherichia coli.

5-Aminolevulinic acid (5-ALA) is an unnatural amino acid and has been approved as a biodegradable, non-toxic pesticide and herbicide with applications in sustainable agriculture. 5-ALA can also be applied for cancer targeting via tumor localization and photodynamic therapy. Herein, we developed a feasible quantification, regulation and production method of 5-ALA in Escherichia coli is based on the chimera of 5-ALA synthetase from Rhodobacter sphaeroides (RshemA) and super-fold green fluorescent protein (sfGFP) under the control of dual promoters/double plasmids. 5-ALA production based on quantification with the reporter sfGFP was unsuccessfully for the RshemA-sfGFP fusion protein owing to a steric hindrance effect, but was effective using dual constitutive promoters (i.e., J23100 and PLacI) for RshemA and sfGFP independently. Moreover, a simple quantification method based on the linear relationship between 5-ALA concentration and the change in sfGFP intensity was calculated with the Hill equation according to the results of dual plasmids which composed of RshemA-threonine/homoserine exporter (RhtA) and the sensing plasmid pSU-T7-sfGFP. Compared with the conventional detection method for 5-ALA using Ehrlich's reagent, our proposed method is advantages in effectiveness, real-time detection, and outstanding sensitivity. Finally, the highest yield of 5-ALA was obtained in E. coli D2TT strain, reaching 2.46 g/L of 5-ALA produced in a 2.5-L baffle flask fermentation. Hence, this approach shows strong potential for improving 5-ALA production with appropriate regulation and detection based on the fluorescent signal.

A gas chromatography-mass spectrometry method for the determination of delta-aminolevulinic acid in plant leaves.

Delta-aminolevulinic (δ-ALA) acid is an important intermediate for tetrapyrroles biosynthesis and it has recently received great attention in plant physiology and human toxicology. However, the colorimetric method which is the most common method for determination of δ-ALA is time consuming and is not specific. In this study, a method for determination of δ-ALA in plant tissues was developed based on the trimethylsilyl (TMS) derivative of the pyrrole formed from the reaction of δ-ALA with ethyl acetoacetate via Knorr condensation. The δ-ALA in the HCl extract was reacted with ethyl acetoacetate to form a pyrrole. Then, the pyrrole compound was extracted using ethyl acetate and the solvent was evaporated to dryness. The dried sample was derivatized to its TMS ester and analyzed using GC-MS. The concentration of δ-ALA in citrus leaves incubated with levulinic acid was also determined by the conventional colorimetric method. The linear range was 10-200ppm in the full scan mode and 0.1-20ppm in the selected ion monitoring (SIM). The limit of detection was 6ppm in the full scan and 0.05ppm in SIM mode, representing a four-fold increase in sensitivity compared to the colorimetric method. The GC-MS method developed in this study is simple, accurate, sensitive, and could also be used to measure δ-ALA in other biological samples.

Monitoring/characterization of stickies contaminants coming from a papermaking plant--Toward an innovative exploitation of the screen rejects to levulinic acid.

Recycled paper needs a lot of mechanical/chemical treatments for its re-use in the papermaking process. Some of these ones produce considerable rejected waste fractions, such as "screen rejects", which include both cellulose fibers and non-fibrous organic contaminants, or "stickies", these last representing a shortcoming both for the papermaking process and for the quality of the final product. Instead, the accepted fractions coming from these unit operations become progressively poorer in contaminants and richer in cellulose. Here, input and output streams coming from mechanical screening systems of a papermaking plant using recycled paper for cardboard production were sampled and analyzed directly and after solvent extraction, thus confirming the abundant presence of styrene-butadiene rubber (SBR) and ethylene vinyl acetate (EVA) copolymers in the output rejected stream and cellulose in the output accepted one. Despite some significant drawbacks, the "screen reject" fraction could be traditionally used as fuel for energy recovery within the paper mill, in agreement with the integrated recycled paper mill approach. The waste, which still contains a cellulose fraction, can be also exploited by means of the hydrothermal route to give levulinic acid, a platform chemical of very high value added.

Analysis of the lignocellulosic components of biomass residues for biorefinery opportunities.

The present study aims to identify the renewable resources available in Brazil such as açai seed, coconut husks, coffee husks, rice husks, eucalyptus sawdust, grass, soy peel, bamboo, banana stems and banana stalks. To identify such renewable energy sources, samples were examined for their physical and chemical characteristics using X-ray diffraction (XRD), proximate and ultimate analyses, thermogravimetric analysis (TGA), calorific value determination, near-infrared (NIR) spectroscopy, UV spectroscopy, high-pH anion-exchange chromatography (HPAEC-PAD) and accelerated solvent extraction (ASE). Among the biomasses, açai and coffee exhibited higher total sugar content, 67.70% and 62.55%, respectively. Sawdust exhibited low ash, along with the highest calorific value and lignin content. The highest glucose contents were observed in bamboo (44.65%) and sawdust (38.80%). The maximum yield for the bioproducts levulinic acid (LA), formic acid (FA) and furfural were estimated; açai exhibited the highest yield of LA and FA, while coffee exhibited the best furfural yield. All of these properties indicate that the residues are potential candidates for bioenergy production.

[Process analysis for levulinic acid generated in waste wood liquefaction by non-development thin-layer chromatography based on oblique projection].

A rapid and quantitative method is presented for multi-component process analysis, based on multi-wavelength thin-layer chromatography (TLC) scanning but without the routine development. The samples from the waste wood liquefaction process are applied on silica plates, and just the last sample of spot need to be developed for getting separated spectra. These spectra are divided into two parts of production (levulinic acid) and background, respectively, to build an oblique projection operator. The other process samples do not need to be developed repeatedly, and are scanned to collect hybrid spectra immediately. The pure production spectrum can be separated from the process spectrum by the oblique projection algorithms to realize the production quantification. It was showed that the relative errors between the determination results by this method and those by high performance liquid chromatography (HPLC) were less than 3.27%, and so the consistency is perfect.

Acidic processing of hemicellulosic saccharides from pine wood: product distribution and kinetic modeling.

Water soluble compounds were removed from Pinus pinaster wood by a mild aqueous extraction, and the treated wood was subjected to hydrothermal processing to convert most hemicelluloses into soluble saccharides (including low molecular weight polymers, oligomers and monosaccharides). The liquid phase containing hemicellulose-derived saccharides was acidified with sulfuric acid and heated up to 130-250°C to obtain furans and levulinic acid as major products. The concentration profiles of the major compounds participating in the reactions were interpreted by a kinetic model. A maximum conversion of pentoses into furfural near 80% was predicted at high temperature and short time, conditions leading to 24% conversion of hexoses into HMF. Production of levulinic acid was favored at low temperatures. Maximum molar conversion of hexoses into levulinic acid (66.7% at 130°C) needed a long reaction time (235 h). A value of 53.0% can be achieved at 170°C after 5 h.

Rapid method for the determination of 5-hydroxymethylfurfural and levulinic acid using a double-wavelength UV spectroscopy.

This study reports on a rapid method for the determination of levulinic acid (LA) and 5-hydroxymethylfurfural (HMF) in acid hydrolyze system of glucose based on UV spectroscopy. It was found that HMF and LA have a maximum absorption at the wavelengths of 284 nm and 266 nm, respectively, in a water medium, and the absorptions of HMF and LA at 284 nm and 266 nm follow Beer's law very well. However, it was found that a major spectral interference species will arise in the quantification of HMF and LA; nonetheless, this interference can be eliminated through the absorption treatment of charcoal. Therefore, both HMF and LA can be quantified with a double-wavelength technique. The repeatability of the method had a relative standard deviation of less than 4.47% for HMF and 2.25% for LA; the limit of quantification (LOQ) was 0.017 mmol/L for HMF and 4.68 mmol/L for LA, and the recovery ranged from 88% to 116% for HMF and from 94% to 105% for LA. The present method is simple, rapid, and accurate. It is suitable to use in the research of the preparation of HMF and LA in biorefinery area.

Protoporphyrin fluorescence induced by methyl-ALA in skin healing.

BACKGROUND: Estimation of the time period that precedes an injury is critical in forensic medicine. However, there is no reliable method that can be used to evaluate the oldness of a lesion. The aim of this work is to develop a fluorimetric method that can be used to follow the aging process of lesions by applying methyl-ALA (MAL) on wounds and by quantifying protoporphyrin IX (PPIX) fluorescence during the healing process. We also aim to understand the changes in PPIX fluorescence by establishing a correlation with histological evaluations during the healing process. METHODS: Standardized linear wounds were made on the dorsum of 72 mice, which were divided in control (MAL -) and experimental (MAL +) groups. In vivo fluorescence spectra (FS) were collected from normal and wound skin sites of control and experimental groups, corresponding to four groups of FS spectra: (a) FS of skin wound after MAL (+/+); (b) FS of normal skin after MAL (-/+); (c) FS of skin wound without MAL (+/-) and (d) FS of normal skin without MAL (-/-). Animals were monitored periodically for 3 months and euthanized. Tissue specimens were processed for histological analysis using design-based stereological methods. Serial cross-sections were analyzed to evaluate the organization of the dermis and epidermis, collagen deposition and cellular proliferation. RESULTS: FS of skin wound with MAL (+/+) showed an expressive intensity increase from the beginning of the experiment to the 34th day, with maximum fluorescence being observed on the ≈ 11 th day after wounding. There was preferential PPIX accumulation in healing sites as compared to adjacent normal skin (+/-) in the early stage of healing. Histological findings allowed correlation of the fluorescence increase mainly with cell proliferation. The drastic decrease in the FS intensity observed in the end of the healing process was correlated with the decrease in the proliferation rate as well as with the presence of new extracellular fibrous materials. CONCLUSIONS: In the mice wound-healing model tested here, it was possible to distinguish whether the injury was in early or advanced stages by using PPIX fluorescence induced by MAL. We conclude that this method is a promising approach to evaluate the age of skin wounding and we hope this work will stimulate human studies to allow this technique to become standardized in forensic medicine.

A continuous spectrophotometric assay for adenosine 5'-phosphosulfate reductase activity with sulfite-selective probes.

Mycobacterium tuberculosis (Mtb) adenosine 5'-phosphosulfate (APS) reductase (APR) catalyzes the first committed step in sulfate reduction for the biosynthesis of essential reduced sulfur-containing biomolecules, such as cysteine, and is essential for survival in the latent phase of tuberculosis (TB) infection. Despite the importance of APR to Mtb and other bacterial pathogens, current assay methods depend on the use of (35)S-labeled APS or shunt adenosine 5'-monophosphate (AMP) to a coupled-enzyme system. Both methods are cumbersome and require the use of expensive reagents. Here, we report the development of a continuous spectrophotometric method for measuring APR activity by using novel sulfite-selective colorimetric or "off-on" fluorescent levulinate-based probes. Thus, the APR activity can be followed by monitoring the increase in absorbance or fluorescence of the resulting phenolate product. Using this assay, we determined Michaelis-Menten kinetic constants (K(m), k(cat), and k(cat)/K(m)) and the apparent inhibition constant (Ki) for adenosine 5'-diphosphate (ADP), which compared favorably with values obtained in the "gold standard" radioactive assay. The newly developed assay is robust and easy to perform with a simple spectrophotometer.

Acid-catalyzed hydrothermal severity on the fractionation of agricultural residues for xylose-rich hydrolyzates.

The objective of this work was to investigate the feasibility of acid-catalyzed hydrothermal fractionation for maximum solubilization of the hemicellulosic portion of three agricultural residues. The fractionation conditions converted into combined severity factor (CS) in the range of 1.2-2.9. The highest hemicellulose yield of 87.88% was achieved when barley straw was fractionated at a CS of 2.19. However, the maximum glucose release of 15.29% was achieved for the case of rice straw. The maximum productions of various by-products were observed with the fractionation of rape straw: 0.88 g/L of 5-hydroxymethylfurfural (5-HMF), 2.16 g/L of furfural, 0.44 g/L of levulinic acid, 1.59 g/L of formic acid, and 3.06 g/L of acetic acid. The highest selectivities, a criterion for evaluating the fractionation of 21.55 for fractionated solid and 7.48 for liquid hydrolyzate were obtained from barley straw.

Synthesis and evaluation of 4-substituted semicarbazones of levulinic acid for anticonvulsant activity.

OBJECTIVE: A series of 4-aryl substituted semicarbazones of levulinic acid (4-oxo pentanoic acid) was designed and synthesized to meet the structural requirements essential for anticonvulsant activity. METHODS: All the compounds were evaluated for anticonvulsant activity. Anticonvulsant activity was determined after intraperitoneal (i.p.) administration to mice by maximal electroshock (MES) and subcutaneous metrazol (ScMet) induced seizure methods and minimal motor impairment was determined by rotorod test. RESULTS: A majority of the compounds exhibited significant anticonvulsant activity after intraperitoneal administration. In the present study 4-(4'-fluoro phenyl) levulinic acid semicarbazone emerged as the most active molecule, showing broad spectrum of activity with low neurotoxicity. Unsubstituted levulinic acid semicarbazone was found to be inactive in all the screens. CONCLUSION: The results obtained validate the hypothesis that presence of an aryl group near the semicarbazone moiety is essential for anticonvulsant activity. The results also indicate that the hydrophilic-hydrophobic site can accommodate hydrophilic groups.

Isolation, structural characterization, and immunological evaluation of a high-molecular-weight exopolysaccharide from Staphylococcus aureus.

Colonization of implanted medical devices by coagulase-negative staphylococci such as Staphylococcus epidermidis is mediated by the bacterial polysaccharide intercellular adhesin (PIA), a polymer of beta-(1-->6)-linked glucosamine substituted with N-acetyl and O-succinyl constituents. The icaADBC locus containing the biosynthetic genes for production of PIA has been identified in both S. epidermidis and S. aureus. Whereas it is clear that PIA is a constituent that contributes to the virulence of S. epidermidis, it is less clear what role PIA plays in infection with S. aureus. Recently, identification of a novel polysaccharide antigen from S. aureus termed poly N-succinyl beta-(1-->6)-glucosamine (PNSG) has been reported. This polymer was composed of the same glycan backbone as PIA but was reported to contain a high proportion of N-succinylation rather than acetylation. We have isolated a glucosamine-containing exopolysaccharide from the constitutive over-producing MN8m strain of S. aureus in order to prepare polysaccharide-protein conjugate vaccines. In this report we demonstrate that MN8m produced a high-molecular-weight (>300,000 Da) polymer of beta-(1-->6)-linked glucosamine containing 45-60% N-acetyl, and a small amount of O-succinyl (approx 10% mole ratio to monosaccharide units). By detailed NMR analyses of polysaccharide preparations, we show that the previous identification of N-succinyl was an analytical artifact. The exopolysaccharide we have isolated is active in in vitro hemagglutination assays and is immunogenic in mice when coupled to a protein carrier. We therefore conclude that S. aureus strain MN8m produces a polymer that is chemically and biologically closely related to the PIA produced by S. epidermidis.

GC/ITMS measurement of carbonyls and multifunctional carbonyls in PM2.5 particles emitted from motor vehicles.

A method was developed and tested to identify and quantitate carbonyls and multifunctional carbonyls in fine particulate matter (PM2.5; <2.5 microm aerodynamic diameter). The method relies on ultrasonic extraction of particulate matter on filters at -8 degrees C; derivatization with O-(2,3,4,5,6-pentafluorobenzyl) hydroxylamine (PFBHA), and PFBHA along with bis (trimethylsilyl) trifluoroacetamide (BSTFA); and detection of the derivatives by gas chromatography/ion trap mass spectrometry. Ultrasonic extraction of model compounds from enriched particles was affected by solvent polarity (water > methylene chloride > toluene-isopropanol (2 + 1, v/v). Water provided the highest recovery for dihydroxy acetone, pyruvic acid, and hydroxy acetone, compared to methylene chloride, and toluene-isopropanol. Lowering the ultrasonication bath temperature from 0 degrees to -8 degrees C improved the recoveries of the less water soluble and more volatile species-methacrolein, methyl vinyl ketone, 2,3-butanedione, and tolualdehyde. The power of the method was demonstrated by identification and quantitation of carbonyls and multifunctional carbonyls in sample extracts of fine particulate matter (PM2.5) collected in the Caldecott tunnel, CA. The identities of crotonaldehyde, 2,3-butanedione, glyoxal, 9H-fluoren-9-one, glycolaldehyde, glyoxylic acid, levulinic acid, and 3-hydroxybenzaldehyde were confirmed by comparing the relative retention time and mass spectra of the analyte in the sample extract with an authentic standard. Quantitation of crotonaldehyde, glyoxal, 2,3-butanedione, glyoxylic acid, and levulinic acid was accomplished. This is the first report of glyoxylic acid, levulinic acid, and 3-hydroxybenzaldheyde in PM2.5 particles sampled in a roadway tunnel. It is also the first report of a C10 carbonyl with the molecular formula of C10H16O2, a hydroxy carbonyl with the molecular formula of C17H21NO2, and a hydroxy or dihydroxy carbonyl with the molecular formula of C16H14O2 or C9H10O3. The high-molecular weight hydroxy carbonyls, which were found only in the heavy-duty (diesel) bore, may be tracers of diesel emissions in air.

Simultaneous determination of delta-aminolevulinic acid, porphobilinogen, levulinic acid and glycine in culture broth by capillary electrophoresis.

Capillary electrophoretic simultaneous determination of a mixture containing delta-aminolevulinic acid, porphobilinogen, levulinic acid and glycine was investigated. With increases in the sodium tetraborate buffer concentration (5-70 mM), resolution of the four components was improved, but the migration time was increased. Alternatively, with increases in the applied voltage (5-22.5 kV), a shortened migration time was seen but this adversely affected resolution. The components were separated with high resolution by using a fused-silica capillary column (75 cm x 75 microm I.D.) filled with 30 mM sodium tetraborate buffer (pH 9.3-9.4) under the applied voltage of 20 kV (constant voltage mode). When the established method was applied to the culture broth of Rhodopseudomonas sphaeroides, a photosynthetic bacterium, the four components mentioned above were separated with good resolution. Furthermore, the use of this method would provide a fast, sensitive and specific method for monitoring the administration of delta-aminolevulinic acid in photodynamic cancer therapy, for the measurement of delta-aminolevulinic acid dehydratase activity in erythrocytes, and for testing the delta-aminolevulinic acid assay and for impurities in drug formulation.

Interference by methyl levulinate in determination of total fat in low-fat, high-sugar products by gas chromatographic fatty acid methyl ester (GC-FAME) analysis.

Gas chromatographic fatty acid methyl ester (GC-FAME) analyses of some acid-hydrolyzed foods revealed a large peak that did not correspond to any FAME standards. The unknown peak eluted just after the C12 FAME. If the fatty acid response factor and the conversion factor for the nearest calibrated peak (C12 FAME) were used to determine the total fat, the resulting total fat determination was much higher than expected. This peak was present only in acid-hydrolyzed samples and was absent in extracts obtained with supercritical CO2 or solvents without acid hydrolysis. The compound was isolated, analyzed by mass spectrometry and nuclear magnetic resonance spectroscopy, and proved by synthesis to be methyl-4-oxopentanoate (methyl levulinate). Its source was determined to be sugar in the product formula. Levulinic acid is produced by acid hydrolysis of sugar and is transesterified by BF3 in methanol to methyl levulinate. Although methyl levulinate may appear in the GC analyses of any acid-hydrolyzed products containing sugar, if the ratio of fat to sugar is high, the impact of methyl levulinate on fat determination would be small. On the other hand, the presence of methyl levulinate in analyses of low-fat, high-sugar products is potentially problematic if not recognized, although GC analysis can account for the presence of this compound.

Detection of delta-aminolevulinic acid, porphobilinogen and porphyrins related to heme biosynthesis by high-performance liquid chromatography.

Two new high-performance liquid chromatographic methods are described for the quantitative determination of porphyrins and their precursors. In our method, sub-nanomole quantities of porphyrins, delta-aminolevulinic acid and porphobilinogen derivatized with ophthalaldehyde were injected onto a C18 reversed-phase column and eluted with 0.1 M monobasic sodium phosphate-methanol-tetrahydrofuran (4:6:3) and detected with a spectrofluorometer. A second reversed-phase system using methanol-tetrahydrofuran-22 mM acetate buffer (15:6:11) was also developed.