Application of liquid chromatography–mass spectrometry to measure short chain fatty acids in blood

A new liquid chromatography–mass spectrometry method is described to determine concentrations of the short chain fatty acids acetic acid, propionic acid and butyric acid (SCFAs) in human blood plasma. The method is based on reversed phase chromatography followed by post-column neutralization of the mobile phase with ammonia and a consecutive measurement of the SCFAs ammonia adducts using negative electro spray ionization. Sample preparation involved simple organic acid deproteinization, resulting in 100% recovery. SCFAs eluted baseline separated within a 25 min run cycle. A linear response was obtained in the range between 0 and 250 μmol/l (R² ranged from 0.997 to 0.9999). The limit of detection ranged from 0.05 μmol/l for propionic and butyric acid and 0.1 μmol/l for acetic acid. The method was tested by analyzing plasma of arterial blood, from portal vein and hepatic vein blood from patients undergoing a pylorus-preserving pancreaticoduodenectomy. As expected, the highest SCFA concentrations were found in portal plasma, hepatic vein levels were in between, while arterial concentrations were lowest. This newly developed method is suitable to determine SCFA concentrations in human plasma samples.     

A method for determination of xanthine in meat by amperometric biosensor based on silver nanoparticles/cysteine modified Au electrode

A method is described for construction of an amperometric xanthine biosensor based on covalent Immobilization of xanthine oxidase (XOD) onto citrate capped silver nanoparticles deposited on Au electrode surface through cysteine self assembled monolayers (SAM). The biosensor showed optimum response within 5 s at pH 7.0 and 35 °C, when polarized at 0.5 V vs. Ag/AgCl. The linear working range of biosensor for xanthine was from 2 to 16 μM, with a detection limit of 0.15 μM and sensitivity of 0.17 mA/μM/cm². The mean analytical recovery of exogenously added xanthine in fish meat extract (5 g/l and 10 g/l) was 96.2 ± 2.3% and 95.2 ± 3.4%, respectively. Within and between batches coefficients of variation were <2.6% and <3.4%, respectively. The biosensor measured xanthine in fish, chicken, pork, and beef meat. The enzyme electrode lost 20% of its initial activity after its regular 180 uses over a period of 60 days, when stored at 4 °C in dry state.     

Quantitative determination of oxytocin receptor antagonist atosiban in rat plasma by liquid chromatography–tandem mass spectrometry

A kinetic study of atosiban was conducted following repeated intravenous administration in Wistar rats. Sample analysis was performed using liquid chromatography–tandem mass spectrometry (LC–MS/MS) following full validation of an in-house method. Eptifibatide, a cyclic peptide, was used as an internal standard (IS). The analyte and internal standard were extracted using solid phase extraction (SPE) method. Chromatographic separation was carried out using an ACE C18 5 μm 50 mm × 4.6 mm column with gradient elution. Mass spectrometric detection was performed using TSQ Quantum ultra AM. The lower limit of quantification was 0.01 μg/ml when 100 μl rat plasma was used. Plasma concentrations of atosiban were measured at 0 (pre-dose), 2, 15, 30, 45, 60, 120 min at the dosage levels of 0.125 mg/kg (low dose), 0.250 mg/kg (mid dose), and 0.500 mg/kg (high dose), respectively. Atosiban plasma concentration measured at Day 1 showed mean peak atosiban concentration (C_max) 0.40, 0.57, 1.95 μg/ml for low, mid and high dose treated animals and mean peak concentration on Day 28 was 0.41, 0.88, 1.31 μg/ml on Day 28 for low, mid and high dose treated animals.     

Identification and quantification of the glucose degradation product glucosone in peritoneal dialysis fluids by HPLC/DAD/MSMS

Glucose degradation products (GDPs) formed during heat sterilization of peritoneal dialysis (PD) fluids exert cytotoxic effects and promote the formation of advanced glycation end-products in the peritoneal cavity. As a result, long-term application of continuous ambulatory peritoneal dialysis is limited. The composition and concentration of GDPs in PD fluids must be known to evaluate their biological effects. The present study describes a targeted screening for novel GDPs in PD fluids. For this purpose, dicarbonyl compounds were converted with o-phenylenediamine to give the respective quinoxaline derivatives, which were selectively monitored by HPLC/diode array detector. Glucosone was thereby identified as a novel major GDP in PD fluids. Product identity was confirmed by LC/MSMS analysis using independently synthesized glucosone as a reference compound. Furthermore, a method was developed to quantify glucosone in PD fluids by HPLC/UV after derivatization with o-phenylenediamine. The method's limit of detection was 0.6 μM and the limit of quantitation 1.1 μM. A linear calibration curve was obtained between 1.1 and 113.9 μM (R² = 0.9999). Analyzed at three different concentration levels, recovery varied between 95.6% and 102.0%. The coefficient of variation ranged between 0.4% and 4.7%. The method was then applied to the measurement of glucosone in typical PD fluids. Glucosone levels in double chamber bag PD fluids varied between not detectable and 6.7 μM. In single chamber bag fluids, glucosone levels ranged between 28.7 and 40.7 μM.     

A novel electrochemical biosensor based on horseradish peroxidase immobilized on Ag-nanoparticles/poly(l-arginine) modified carbon paste electrode toward the determination of pyrogallol/hydroquinone

A novel electrochemical biosensor for the determination of pyrogallol (PG) and hydroquinone (HQ) has been constructed based on the poly l-arginine (poly(l-Arg))/carbon paste electrode (CPE) immobilized with horseradish peroxidase (HRP) and silver nanoparticles (AgNPs) through the silica sol–gel (SiSG) entrapment. The electrochemical properties of the biosensor were characterized by employing the electrochemical techniques. The proposed biosensor showed a high sensitivity and fast response toward the determination of PG and HQ around 0.18 V. Under the optimized conditions, the anodic peak current of PG and HQ was linear with the concentration range of 8 μM to 30 × 10^?5 M and 1–150 μM. The limit of detection (LOD) and limit of quantification (LOQ) were found to be 6.2 μM, 20 μM for PG and 0.57 μM, 1.92 μM for HQ respectively. The electrochemical impedance spectroscopy (EIS) studies have confirmed that the occurrence of electron transfer at HRP-SiSG/AgNPs/poly(l-Arg)/CPE was faster. Moreover the stability, reproducibility and repeatability of the biosensor were also studied. The proposed biosensor was successfully applied for the determination of PG and HQ in real samples and the results were found to be satisfactory.     

Novel liquid chromatographic determination of cystatin C in human urine

A rapid and sensitive method for quantitative determination of cystatin C (CC) protein in human urine via HPLC was developed and validated. Acetone has been used as a precipitating agent of CC protein from the urine biological matrix. Separation and detection were accomplished by ion pair liquid chromatography and UV detection. Gradient elution mode was utilized to elute CC with a UV detection of 224 nm. The analysis time was 14 min per sample using Ace C8 (150 × 4.6 mm i.d., 5 μm) as a chromatographic column with a flow rate of 1.0 mL/min. Calibration curve with good linearity (r² = 0.99) within the range from 0.390 to 0.001 mg/mL was obtained. Limits of detection and quantification were 0.001 and 0.002 mg/mL, respectively. Inter-assay and intra-assay variabilities were <15% for all levels and <20% at the limit of quantification level. Major advantages of the method: specific where no false positive results might be obtained and fast where sample pretreatment needs only 1 h.     

Nucleic acid changes during photodynamic inactivation of bacteria by cationic porphyrins

Light activation of photosensitizing dyes in presence of molecular oxygen generates highly cytotoxic reactive oxygen species leading to cell inactivation. Nucleic acids are molecular targets of this photodynamic action but not considered the main cause of cell death. The in vivo effect of the photodynamic process on the intracellular nucleic acid content of Escherichia coli and Staphylococcus warneri was evaluated herein.Two cationic porphyrins (Tetra-Py⁺-Me and Tri-Py⁺-Me-PF) were used to photoinactivate E. coli (5.0 μM; 10⁸ cells mL^?1) and S. warneri (0.5 μM; 10⁸ cells mL^?1) upon white light irradiation at 4.0 mW cm^?2 for 270 min and 40 min, respectively. Total nucleic acids were extracted from photosensitized bacteria after different times of irradiation and analyzed by agarose gel electrophoresis. The double-stranded DNA was quantified by fluorimetry and the porphyrin binding to bacteria was determined by spectrofluorimetry.E. coli was completely photoinactivated with both porphyrins (5.0 μM), whereas S. warneri was only completely inactivated by Tri-Py⁺-Me-PF (0.5 μM). The hierarchy of nucleic acid changes in E. coli was in the order: 23S rRNA > 16S rRNA > genomic DNA. The nucleic acids of S. warneri were extensively reduced after 5 min with Tri-Py⁺-Me-PF but almost unchanged with Tetra-Py⁺-Me after 40 min of irradiation. The amount of Tri-Py⁺-Me-PF bound to E. coli after washing the cells is higher than Tetra-Py⁺-Me and the opposite was observed for S. warneri. The binding capacity of the photosensitizers is not directly related to the PDI efficiency or nucleic acid reduction and this reduction occurs in parallel with the decrease of surviving cells.     

Copper toxicity and sulfur metabolism in Chinese cabbage are affected by UV radiation

Biomass production, dry matter content, specific leaf area and pigment content of Chinese cabbage were all quite similar, when plants were grown in the absence or presence of UV-A + B (2.2 mW cm⁻²). Elevated Cu²⁺ concentrations (2–10 μM) in the root environment and UV radiation had negative synergistic effects for Chinese cabbage and resulted in a more rapid and stronger decrease in plant biomass production and pigment content. The quantum yield of photosystem II photochemistry (F_v/F_m) was only decreased at ≥5 μM Cu²⁺ in the presence of UV radiation, when leaf tissue started to become necrotic. The enhanced Cu toxicity in the presence of UV was largely due to a UV-induced enhanced accumulation of Cu in both roots and shoots. An enhanced Cu content strongly affected the uptake and assimilation of sulfur in plants. The total sulfur content of the root increased at ≥2 μM Cu²⁺ in presence of UV and at 10 μM Cu²⁺ in absence of UV and that of the shoot increased at ≥2 μM Cu²⁺ in presence of UV and at ≥5 μM Cu²⁺ in absence of UV. In the shoot it could be attributed mainly to an increase in sulfate content. Moreover, there was a strong increase in the water-soluble non-protein thiol content upon Cu²⁺ exposure in the root and, to a lesser extent in the shoot, both in the presence and absence of UV. The regulation of the uptake of sulfate responded to the occurrence of Cu toxicity directly, since it was more rapidly affected in the presence than in the absence of UV radiation. For instance, the expression and activity of the high affinity sulfate transporter, Sultr1;2, were enhanced at ≥2 μM in the presence of UV, and at ≥5 μM Cu²⁺ in the absence of UV. In the shoot, the expression of the vacuolar sulfate transporter, Sultr4;1, was upregulated at ≥5 μM Cu²⁺ in the presence and absence of UV whilst the expression of a second vacuolar sulfate transporter, Sultr4;2, was upregulated at 10 μM Cu²⁺ in the presence of UV. It is suggested that high Cu tissue levels may interfere/react with the signal compounds involved in the regulation of expression and activity of sulfate transporters. The expression of adenosine 5′-phosphosulfate reductase in the root was hardly affected and was slightly down-regulated at 2 μM in the presence of UV and at 10 μM in the absence of UV. The expression and activity of sulfate transporters were enhanced upon exposure at elevated Cu²⁺ concentrations; this may not be simply due to a greater sulfur demand at higher Cu levels, but more likely is the consequence of Cu toxicity, since it occurred more rapidly in the presence compared to the absence of UV.     

A new use of β-Ala-Lys (AMCA) as a transport reporter for PEPT1 and PEPT2 in renal brush border membrane vesicles from the outer cortex and outer medulla

Integral membrane proteins PEPT1 and PEPT2 are essential for reabsorbing almost all hydrolysed or filtered di- and tripeptides alongside a wide range of peptidomimetic drugs in the kidney. The aim of this study was to investigate the potential use of the fluorophore-conjugated dipeptide β-Ala-Lys (AMCA) as a biosensor for measuring peptide transport activity in brush border membrane vesicles isolated from the outer cortex (BBMV-OC) and outer medulla (BBMV-OM) (representing PEPT1 and PEPT2 respectively). The vesicles were isolated using a dual magnesium precipitation and centrifugation technique. Intravesicular fluorescence accumulation was measured after incubating extra-vesicular media at pH 6.6 and different concentrations of β-Ala-Lys (AMCA) with vesicles pre-equilibrated at pH 7.4. Both BBMV-OC and BMMV-OM showed accumulation of an intravesicular fluorescence signal after 20 min incubation. Changing the extra-vesicular pH to 7.4 caused a significant reduction in the β-Ala-Lys (AMCA) uptake into BBMV-OC at concentrations > 100 μM. When different concentrations of dipeptide, Gly-Gln was added, there was a significant inhibition of 100 μM β-Ala-Lys (AMCA) uptake into BBMV-OC and BMMV-OM, reaching 69% and 80%, respectively. Kinetic analysis of β-Ala-Lys (AMCA) at 20 min showed that the K_m and V_max were 783.7 ± 115.7 μM and 2191.2 ± 133.9 ΔF/min/mg for BBMV-OC, while BMMV-OM showed significantly higher affinity, but lower capacity at K_m = 93.6 ± 21.9 μM and V_max = 935.8 ± 50.2 ΔF/min/mg. These findings demonstrate the applicability of β-Ala-Lys (AMCA) as a biosensor to measure the transport activity of the renal-type PEPT1 and PEPT2 in BBMV-OC and BMMV-OM respectively.     

Quantification of nortriptyline in plasma by HPLC and fluorescence detection

A simple, sensitive and specific high-performance liquid chromatography method has been developed for the determination of nortriptyline (NT) in plasma samples. The assay involved derivatization with 9H-fluoren-9-ylmethyl chloroformate (Fmoc-Cl) and isocratic reversed-phase (C₁₈) chromatography with fluorescence detection. The developed method required only 100 μl of plasma sample, deproteinized and derivatized in one step. Calibration curves were lineal over the concentration range of 5–5000 ng/ml. The derivatization reaction was performed at room temperature in 20 min and the obtained NT derivative was stable for at least 48 h at room temperature. The within-day and between-day relative standard deviation was below 8%. The limit of detection (LOD) was 2 ng/ml, and the lower limit of quantification (LLOQ) was established at 10 ng/ml. The method was applied on plasma collected from rats, at different time intervals, after intravenous administration of 0.5 mg of NT.     

l-NAME prevents GM1 ganglioside-induced vasodilation in the rat brain

Monosialoganglioside (GM1) is a glycosphingolipid present in most cell membranes that displays antioxidant and neuroprotective properties. It has been recently described that GM1 induces vasodilation. However, the mechanisms underlying GM1-induced vasodilation were not evaluated to date. Therefore, in this study we investigated whether the nonspecific NOS inhibitor l-NAME prevents GM1-induced vasodilation in rats. The systemic injection of GM1 (50 mg/kg, i.p.) increased the outer diameter of pial vessels by 50% in anesthetized animals at 30 min, and this effect was fully prevented by the administration of the nitric oxide synthase inhibitor N^G-nitro-l-arginine methyl ester (l-NAME, 60 mg/kg, i.p. 15 min before GM1 injection).A 30 min exposure of cerebral cortex slices to GM1 (100 μM) increased the content of nitrite plus nitrate (NOx) by 50%. Addition of l-NAME (100 μM) to the incubation medium fully prevented GM1-induced NOx increase. Conversely, a 60 min exposure of slices to GM1 (100 μM) decreased NOx content, revealing a biphasic effect of GM1. Our results suggest that NO plays an important role in the vasodilation induced by GM1.     

Lead,zinc, cadmium hyperaccumulation and growth stimulation in Arabis paniculata Franch

Metal hyperaccumulation is of great interest in recent years because of its potential application for phytoremediation of heavy metal contaminated soils. In this study, a field survey and a hydroponic experiment were conducted to study the accumulation characteristics of lead (Pb), zinc (Zn) and cadmium (Cd) in Arabis paniculata Franch., which was found in Yunnan Province, China. The field survey showed that the wild population of A. paniculata was hyper-tolerant to extremely high concentrations of Pb, Zn and Cd, and could accumulate in shoots an average level of 2300 mg kg^?1 dry weight (DW) Pb, 20,800 mg kg^?1 Zn and 434 mg kg^?1 Cd, with their translocation factors (TFs) all above one. Under the hydroponic culture, stimulatory effects of Pb, Zn and Cd on shoot dry biomass were noted from 24 to 193 μM Pb, 9 to 178 μM Cd and all Zn supply levels in nutrient solution, while the effects were not obvious in the roots. Chlorophyll concentrations in Pb, Zn and Cd treatments showed an inverted U-shaped pattern, consistent with the change of plant biomass. Pb, Zn and Cd concentrations in the shoots and roots increased sharply with increasing Pb, Zn and Cd supply levels. They reached > 1000 mg kg^?1 Pb, 10,000 mg kg^?1 Zn and 100 mg kg^?1 Cd DW in the 24 μM Pb, 1223 μM Zn and 9 μM Cd treatment, respectively, in which the plants grew healthy and did not show any symptoms of phytotoxicity. The TFs of Zn were basically higher than one and the amount of Zn taken by shoots ranged from 78.7 to 90.4% of the total Zn. However, the TFs of Pb and Cd were well below one, and 55.0–67.5% of total Pb and 57.8–83.5% of total Cd was accumulated in the shoots. These results indicate that A. paniculata has a strong ability to tolerate and hyperaccumulate Pb, Zn and Cd. Meanwhile, suitable levels of Pb, Zn and Cd could stimulate the biomass production and chlorophyll concentrations of A. paniculata. Thus, it provides a new plant material for understanding the mechanisms of stimulatory effect and co-hyperaccumulation of multiple heavy metals.     

Nitric oxide interacts with mitochondrial complex III producing antimycin-like effects

The effect of NO between cytochromes b and c of the mitochondrial respiratory chain were studied using submitochondrial particles (SMP) from bovine heart and GSNO and SPER-NO as NO sources. Succinate-cytochrome c reductase (complex II-III) activity (222±4 nmol/min. mg protein) was inhibited by 51% in the presence of 500 μM GSNO and by 48% in the presence of 30 μM SPER-NO, in both cases at ~1.25 μM NO. Neither GSNO nor SPER-NO were able to inhibit succinate-Q reductase activity (complex II; 220±9 nmol/min. mg protein), showing that NO affects complex III. Complex II-III activity was decreased (36%) when SMP were incubated with l-arginine and mtNOS cofactors, indicating that this effect is also produced by endogenous NO. GSNO (500 μM) reduced cytochrome b₅₆₂ by 71%, in an [O₂] independent manner. Hyperbolic increases in O₂^•- (up to 1.3±0.1 nmol/min. mg protein) and H₂O₂ (up to 0.64±0.05 nmol/min. mg protein) productions were observed with a maximal effect at 500 μM GSNO. The O₂^•-/H₂O₂ ratio was 1.98 in accordance with the stoichiometry of the O₂^•- disproportionation. Moreover, H₂O₂ production was increased by 72–74% when heart coupled mitochondria were exposed to 500 μM GSNO or 30 μM SPER-NO. SMP incubated in the presence of succinate showed an EPR signal (g=1.99) compatible with a stable semiquinone. This EPR signal was increased not only by antimycin but also by GSNO and SPER-NO. These signals were not modified under N₂ atmosphere, indicating that they are not a consequence to the effect of NOx species on complex III area. These results show that NO interacts with ubiquinone-cytochrome b area producing antimycin-like effects. This behaviour comprises the inhibition of electron transfer, the interruption of the oxidation of cytochromes b, and the enhancement of [UQH^•]_ss which, in turn, leads to an increase in O₂^•- and H₂O₂ mitochondrial production rates.     

Amperometric biosensor based on a site-specific immobilization of acetylcholinesterase via affinity bonds on a nanostructured polymer membrane with integrated multiwall carbon nanotubes

Acetylcholinesterase (AChE) was immobilized on chemically modified poly-(acrylonitrile-methyl-methacrylate-sodium vinylsulfonate) membranes in accordance with three different methods, the first of which involved random enzyme immobilization via glutaraldehyde, the second one—site-specific enzyme immobilization via glutaraldehyde and Concanavalin A (Con A) and the third method—modified site-specific enzyme immobilization via glutaraldehyde in the presence of a mixture of multiwall carbon nanotubes and albumin (MWCNs + BSA), glutaraldehyde and Con A. Preliminary tests for the activity of immobilized AChE were carried out using these three methods. The third method was selected as the most efficient one for the immobilization of AChE and the prepared enzyme carriers were used for the construction of amperometric biosensors for the detection of acetylthiocholine (ATCh).A five level three factorial central composite design was chosen to determine the optimal conditions for the enzyme immobilization with three critical variables: concentration of enzyme, Concanavalin A and MWCNs. The design illustrated that the optimum values of the factors influencing the amperometric current were C_E: 70 U mL⁻¹; C_{Con A}: 1.5 mg mL⁻¹ and C_MWCN: 11 mg mL⁻¹, with an amperometric current 0.418 μA. The basic amperometric characteristics of the constructed biosensor were investigated. A calibration plot was obtained for a series of ATCh concentrations ranging from 5 to 400 μM. A linear interval was detected along the calibration curve from 5 to 200 μM. The correlation coefficient for this concentration range was 0.995. The biosensor sensitivity was calculated to be 0.065 μA μM⁻¹ cm⁻². The detection limit with regard to ATCh was calculated to be 0.34 μM. The potential application of the biosensor for detection and quantification of organophosphate pesticides was investigated as well. It was tested against sample solutions of Paraoxon. The biosensor detection limit was determined to be 1.39 × 10⁻¹² g L⁻¹ of Paraoxon, as well as the interval (10⁻¹¹ to 10⁻⁸ g L⁻¹) within which the biosensor response was linearly dependant on the Paraoxon concentration. Finally the storage stability of the enzyme carrier was traced for a period of 120 days. After 30-day storage the sensor retained 76% of its initial current response, after 60 days—68% and after 120 days—61%.     

Construction of a bacterial biosensor for zinc and copper and its application to the development of multifunctional heavy metal adsorption bacteria

In this study, we designed and applied molecular biosensors for heavy metals, zinc and copper, for use in bioremediation strategies. Bacteria utilize two component systems to sense changes in the environment by multiple signal components including heavy metals and control gene expression in response to changes in signal molecules. zraP and cusC promoters were selected from a genetic circuit of the ZraSR and CusSR two-component system and were fused to a dual-labeling reporter protein as an interactive biological component for zinc and copper to generate a signal from the constructed biosensor. The biosensor efficiently senses zinc and copper with a calculated detection limit of 16 μM and 26 μM, respectively, and was shown to be a sensitive and effective heavy metal monitoring bacterial system. To extend the application of the bacterial biosensor, we assembled a bioadsorption system that can trigger bacteria to sense and adsorb 13 ± 0.3 mg/L of zinc and 11.4 ± 0.42 mg/L of copper per gram of dry cell weight with induction at a concentration of 100 mg/L of the respective metal ion.     

Determination of puerarin by capillary electrophoresis with chemiluminescence detection

A new analytical method for puerarin using capillary electrophoretic (CE) separation and chemiluminescence (CL) detection has been developed. The detection was based on the enhanced CL intensity of the reaction between luminol and potassium ferricyanide by puerarin in alkaline solution. A laboratory-built CE–CL apparatus was deployed for the puerarin detection. Under the optimal conditions, a linear range from 5.0 × 10^?8 to 2.5 × 10^?6 M and a detection limit of 1.0 × 10^?8 M (S/N = 3) for puerarin were achieved. The determination of puerarin was achieved in less than 5 min, and the proposed method was applied to the determination of puerarin in pharmaceutical, human urine and human plasma samples.     

Development of electrochemical sulfite biosensor based on SOX/PBNPs/PPY modified Au electrode

A sulfite oxidase (SO_X) (EC 1.8.3.1) purified from Syzygium cumini leaves was immobilized onto Prussian blue nanoparticles/polypyrrole (PBNPs/PPY) nanocomposite film electrodeposited onto the surface of gold (Au) electrode. An electrochemical sulfite biosensor was fabricated using SO_X/PBNPs/PPY/Au electrode as working electrode, Ag/AgCl as standard electrode and Pt wire as auxiliary electrode connected through a potentiostat. The working electrode was characterized by Fourier Transform infrared (FTIR) spectroscopy, cyclic voltammetry (CV), scanning electron microscopy (SEM) and electrochemical impedance spectroscopy (EIS) at different stages of its construction. The biosensor showed optimum response within 2 s, when operated at 20 mV s⁻¹ in 0.1 M Tris–HCl buffer, pH 8.0 and at 30 °C. Linear range and minimum detection limit were 0.5–1000 μM and 0.1 μM (S/N = 3) respectively. The sensor was evaluated with 95.0% recovery of added sulfite in red wine samples and 1.9% and 3.3% within and between batch coefficients of variation respectively. There was a good correlation (r = 0.96) between red wine samples sulfite value by standard DTNB method and the present method. The sensor was employed for determination of sulfite level in red, white and rose wine samples. The enzyme electrode was used 300 times over a period of 4 months, when stored at 4 °C.     

Effect of mercury and gold on growth,nutrient uptake,and anatomical changes in Chilopsis linearis

Plants of Chilopsis linearis were grown with 0, 50, 100, and 200 μM Hg [as Hg(CH₃COO)₂] and 0 and 50 μM Au (as KAuCl₄) in hydroponics. The results showed that seedling grown with 50 μM Au + 50 μM Hg and 50 μM Au + 100 μM Hg had roots 25 and 55% shorter than control roots, respectively. The element uptake determination using ICP/OES demonstrated that Hg at 50 and 100 μM (with and without Au) significantly increased (p < 0.05) the S concentration in leaves. On the other hand, the concentration of Fe significantly increased in roots of plants treated with Au–Hg. In addition, the stems of plants treated with Hg at 100 μM, with and without Au, had 239 and 876 mg Hg/kg dry biomass (d wt), respectively. Also, at 50 μM Hg, with and without Au, stems accumulated 375 and 475 mg Hg/kg d wt. The Hg concentration in leaves (287 mg Hg/kg d wt) was higher (p < 0.05) for the treatment containing 50 μM Au + 100 μM Hg. Without Au, the Hg concentration in leaves decreased to 75 mg Hg/kg d wt. Toxicity symptoms induced by Hg in cortex cells and the vascular system were lower in plants exposed to 50 μM Au + 50 μM Hg compared to plants exposed to 50 μM Hg only. Further, the SEM micrographs revealed deposition of Au–Hg particles inside the root. Although the concentrations of Hg used in this study showed different degree of toxicity, the plants displayed good agronomic value.     

Ground level environmental protein concentrations in various ecuadorian environments: Potential uses of aerosolized protein for ecological research

Large quantities of free protein in the environment and other bioaerosols are ubiquitous throughout terrestrial ground level environments and may be integrative indicators of ecosystem status. Samples of ground level bioaerosols were collected from various ecosystems throughout Ecuador, including pristine humid tropical forest (pristine), highly altered secondary humid tropical forest (highly altered), secondary transitional very humid forest (regrowth transitional), and suburban dry montane deforested (suburban deforested). The results explored the sensitivity of localized aerosol protein concentrations to spatial and temporal variations within ecosystems, and their value for assessing environmental change. Ecosystem specific variations in environmental protein concentrations were observed: pristine 0.32 ± 0.09 μg/m³, highly altered 0.07 ± 0.05 μg/m³, regrowth transitional 0.17 ± 0.06 μg/m³, and suburban deforested 0.09 ± 0.04 μg/m³. Additionally, comparisons of intra-environmental differences in seasonal/daily weather (dry season 0.08 ± 0.03 μg/m³ and wet season 0.10 ± 0.04 μg/m³), environmental fragmentation (buffered 0.19 ± 0.06 μg/m³ and edge 0.15 ± 0.06 μg/m³), and sampling height (ground level 0.32 ± 0.09 μg/m³ and 10 m 0.24 ± 0.04 μg/m³) demonstrated the sensitivity of protein concentrations to environmental conditions. Local protein concentrations in altered environments correlated well with satellite-based spectral indices describing vegetation productivity: normalized difference vegetation index (NDVI) (r² = 0.801), net primary production (NPP) (r² = 0.827), leaf area index (LAI) (r² = 0.410). Moreover, protein concentrations distinguished the pristine site, which was not differentiated in spectral indices, potentially due to spectral saturation typical of highly vegetated environments. Bioaerosol concentrations represent an inexpensive method to increase understanding of environmental changes, especially in densely vegetated ecosystems with high canopies or in areas needing high spatial and temporal resolution. Further research to expand understanding of the applicability of bioaerosol concentrations for environmental monitoring is supported by this pilot study.