Determination of zinc in vegetal tissue microsamples by platinum-wire loop in flame atomization atomic absorption spectrometry

The zinc content of 3 μL of vegetal samples (tree leaves, lichens and grape sap) atomized from a Pt-wire in the methane–air flame has been determined by atomic absorption spectrometry. The effect of gas flow rates and the atomization height in the flame on the absorption of zinc was evaluated at 213.9 nm. The best results were obtained at a height of 5 mm and gas flow rates of 200 L/h air and 26 L/h methane, respectively. The effect of Na, K, Ca, Mg, SO₄²⁻, and PO₄³⁻ on the absorption of zinc was studied too. The detection limit of 0.40 ± 0.21 ng was obtained at a significance level of 0.05, using the two-step Neyman–Pearson criterion. The zinc content of the samples has been determined with continuous nebulization and by atomization from the Pt-wire, using both the standard calibration curve and the standard addition method. The results of the two procedures agree within the determination errors.     

PAH-DNA adducts in environmentally exposed population in relation to metabolic and DNA repair gene polymorphisms

Epidemiologic studies indicate that prolonged exposure to particulate air pollution may be associated with increased risk of cardiovascular diseases and cancer in general population. These effects may be attributable to polycyclic aromatic hydrocarbons (PAHs) adsorbed to respirable air particles. It is expected that metabolic and DNA repair gene polymorphisms may modulate individual susceptibility to PAH exposure. This study investigates relationships between exposure to PAHs, polymorphisms of these genes and DNA adducts in group of occupationally exposed policemen (EXP, N = 53, males, aged 22–50 years) working outdoors in the downtown area of Prague and in matched “unexposed” controls (CON, N = 52). Personal exposure to eight carcinogenic PAHs (c-PAHs) was evaluated by personal samplers during working shift prior to collection of biological samples. Bulky-aromatic DNA adducts were analyzed in lymphocytes by ³²P-postlabeling assay. Polymorphisms of metabolizing (GSTM1, GSTP1, GSTT1, EPHX1, CYP1A1-MspI) and DNA repair (XRCC1, XPD) genes were determined by PCR-based RFLP assays. As potential modifiers and/or cofounders, urinary cotinine levels were analyzed by radioimmunoassay, plasma levels of vitamins A, C, E and folates by HPLC, cholesterol and triglycerides using commercial kits. During the sampling period ambient particulate air pollution was as follows: PM10 32–55 μg/m³, PM2.5 27–38 μg/m³, c-PAHs 18–22 ng/m³; personal exposure to c-PAHs: 9.7 ng/m³ versus 5.8 ng/m³ (P < 0.01) for EXP and CON groups, respectively. The total DNA adduct levels did not significantly differ between EXP and CON groups (0.92 ± 0.28 adducts/10⁸ nucleotides versus 0.82 ± 0.23 adducts/10⁸ nucleotides, P = 0.065), whereas the level of the B[a]P-“like” adduct was significantly higher in exposed group (0.122 ± 0.036 adducts/10⁸ nucleotides versus 0.099 ± 0.035 adducts/10⁸ nucleotides, P = 0.003). A significant difference in both the total (P < 0.05) and the B[a]P-“like” DNA adducts (P < 0.01) between smokers and nonsmokers within both groups was observed. A significant positive association between DNA adduct and cotinine levels (r = 0.368, P < 0.001) and negative association between DNA adduct and vitamin C levels (r = −0.290, P = 0.004) was found. The results of multivariate regression analysis showed smoking, vitamin C, polymorphisms of XPD repair gene in exon 23 and GSTM1 gene as significant predictors for total DNA adduct levels. Exposure to ambient air pollution, smoking, and polymorphisms of XPD repair gene in exon 6 were significant predictors for B[a]P-“like” DNA adduct. To sum up, this study suggests that polymorphisms of DNA repair genes involved in nucleotide excision repair may modify aromatic DNA adduct levels and may be useful biomarkers to identify individuals susceptible to DNA damage resulting from c-PAHs exposure.     

Effects of food nitrogen content and concentration on the forms of nitrogen excreted by the calanoid copepod, Acartia tonsa

Nitrogen excreted as ammonium, urea, and dissolved primary amines (DPA), and nitrogen ingested by the planktonic calanoid copepod, Acartia tonsa, were measured while fed 4 foods with different N/C ratios in high (500 μg C l^− 1) and low (50 μg C l^− 1) concentrations. Adult copepods were fed the ciliate, Uronema marinum (N/C = 0.26), the diatom, Thalassiosira weissflogii, in log-phase growth (N/C = 0.20), and in senescent-phase growth (N/C = 0.12), and detritus derived from the saltmarsh grass, Spartina alterniflora, (N/C = 0.04). Total nitrogen excreted ranged from 0.06 to 0.18 μg N copepod^− 1 d^− 1 whereas nitrogen ingested exhibited considerably more variation (0.01 to 0.39 μg N copepod ^− 1d ^− 1). Ammonium was the dominant form of nitrogen excreted and was influenced by both food concentration and N/C ratio. Copepods fed foods with N/C ratios resembling their own body composition (log-phase diatoms and ciliates) excreted more ammonium when fed higher concentrations of food. In contrast, copepods fed foods with lower N/C ratios than their own body composition excreted more ammonium when fed lower concentrations of food, suggesting that they were catabolizing body protein for survival. Excretion of urea varied with food N/C ratio, with more urea excreted when the copepods were fed higher N/C foods. The excretion of DPA did not vary with either food concentration or food N/C ratio. Homeostasis serves to conserve the N/C ratio of copepods. Thus nitrogen excretion by healthy copepods should be expected to increase with ingestion only when copepods have high quantities of nitrogen-rich foods relative to the body composition of the copepods.     

A substitutive substrate for measurements of beta-ketoacyl reductases in two fatty acid synthase systems

Bacterial β-ketoacyl-ACP reductase (FabG) and the β-ketoacyl reductase domain in mammalian fatty acid synthase (FAS) have the same function and both are rendered as the novel targets for drugs. Herein we developed a convenient method, using an available compound ethyl acetoacetate (EAA) as the substitutive substrate, to measure their activities by monitoring decrease of NADPH absorbance at 340 nm. In addition to the result, ethyl 3-hydroxybutyrate (EHB) was detected by HPLC analysis in the reaction system, indicating that EAA worked effectively as the substrate of FabG and FAS since its β-keto group was reduced. Then, the detailed kinetic characteristics, such as optimal ionic strength, pH value and temperature, and kinetic parameters, for FabG and FAS with this substitutive substrate were determined. The K_m and k_cat values of FabG obtained for EAA were 127 mM and 0.30 s^− 1, while those of this enzyme for NADPH were 10.0 μM and 0.59 s^− 1, respectively. The corresponding K_m and k_cat values of FAS were 126 mM and 4.63 s^− 1 for EAA; 8.7 μM and 4.09 s^− 1 for NADPH. Additionally, the inhibitory kinetics of FabG and FAS, by a known inhibitor EGCG, was also studied.     

A novel spectrophotometric method for determination of kinetic constants of aldehyde oxidase using multivariate calibration method

Although phenanthridine has been frequently used as a specific substrate for the assessment of aldehyde oxidase activity, the use of this method is questionable due to a lower limit of detection and its validity for kinetic studies. In the present study, a novel sensitive multivariate calibration method based on partial least squares (PLS) has been developed for the measurement of aldehyde oxidase activity using phenanthridine as a substrate. Phenanthridine and phenanthridinone binary mixtures were prepared in a dynamic linear range of 0.1–30.0 μM and the absorption spectra of the solutions were recorded in the range of 210–280 nm in Sorenson's phosphate buffer (pH 7.0) containing EDTA (0.1 mM). The optimized PLS calibration model was used to calculate the concentration of each chemical in the prediction set. Hepatic rat aldehyde oxidase was partially purified and the initial oxidation rates of different concentrations of phenanthridine were calculated using the PLS method. The values were used for calculating Michaelis–Menten constants from a Lineweaver–Burk double reciprocal plot of initial velocity against the substrate concentration. The limits of detection for phenanthridine and phenanthridinone were found to be 0.04 ± 0.01 and 0.03 ± 0.01 μM (mean ± SD, n = 5), respectively. Using this method, the K_m value for the oxidation of phenanthridine was calculated as 1.72 ± 0.09 μM (mean ± SD, n = 3). Thus, this study describes a novel spectrophotometric method that provides a suitable, sensitive and easily applicable means of measuring the kinetics of phenanthridine oxidation by aldehyde oxidase without the need for expensive instrumentation.     

Performance of anaerobic baffled reactor (ABR) treating synthetic wastewater containing p-nitrophenol

In this study, the effect of increasing p-nitrophenol (PNP) concentrations on the performance of anaerobic baffled reactor (ABR) (chemical oxygen demand (COD), removals, volatile fatty acid (VFA), p-aminophenol (PAP) and methane gas productions) was investigated through 240 days. The PNP concentrations were raised to 700 from 10 mg/L corresponding to PNP loading rates of 0.97 and 67.9 g/m³ day. The PNP and COD removal efficiencies were 99 and 90% at PNP loading rates as high as 33.9 g/m³ day, respectively, through the acclimation of anaerobic granular sludge. After this loading rate, the removal efficiencies decreased to 79%. The COD removal efficiencies were high in compartment 1 (E = 78–93%) while a small amount of COD removal was achieved in compartments 2 and 3. The PNP removal efficiencies were approximately 90% in all PNP loading rates except for loading rate of 0.97 g/m³ day. The maximum PNP removal efficiency was measured as 99% at a loading rate of 8.32 g/m³ day. The optimum PNP loading rate for maximum COD, PNP removals and methane yield was 8.32 g/m³ day. The total, methane gas productions and methane percentages were approximately 2160–2400 mL/day and 950–1250 mL/day and 44–52% for the PNP loading rates varying between 4.36 and 33.9 g/m³ day, respectively. For PNP loading rates varying between 33.9 and 67.9 g/m³ day, the total, methane gas productions and methane percentages were approximately 2160 and 960 mL/day and 44%, respectively. The highest total volatile fatty acid (TVFA) concentrations were found in the first compartment with fluctuated values varied between 50 and 200 mg/L indicating the acidogenesis. p-Aminophenol was found as the main intermediate through anaerobic degradation of PNP which later was broken down to phenol and ammonia.     

Behavioral effects of low dissolved oxygen on the bivalve Macoma balthica

Hypoxia, a dissolved oxygen concentration (DO) below 2 mg l^– 1, is a significant stressor in many estuarine ecosystems. Many sedentary organisms, unable to move to avoid hypoxic areas, have metabolic and behavioral adaptations to hypoxic stress. We tested the effects of hypoxia on the behavior and mortality of the clam Macoma balthica, using four levels of dissolved oxygen in flow-through tanks. We used five replicates of each of four treatments: (1) Hypoxic (DO mean ± SE = 1.1 ± 0.06 mg O₂ l^– 1), (2) Moderately hypoxic (DO 2.6 ± 0.05 mg O₂ l^– 1), (3) Nearly normoxic (DO 3.2 ± 0.04 mg O₂ l^– 1), (4) Normoxic (DO = 4.9 ± 0.13 mg O₂ l^– 1). We lowered the dissolved oxygen with a novel fluidized mud-bed, designed to mimic field conditions more closely than the common practice of solely bubbling nitrogen or other gasses. This method for lowering the DO concentrations for a laboratory setup was effective, producing 1.4 l min^–1 of water with a DO of 0.8 mg O₂ l^– 1 throughout the experiment. The setup greatly reduced the use of compressed nitrogen and could easily be scaled up to produce more low-DO water if necessary. The lethal concentration for 50% of the M. balthica population (LC₅₀) was 1.7 mg O₂ l^– 1 for the 28-day experimental period. M. balthica decreased its burial depth under hypoxic and moderately hypoxic (~2.5 mg O₂ l^– 1) conditions within 72 hours of the onset of hypoxia. By the sixth day of hypoxia the burial depth had been reduced by 26 mm in the hypoxic tanks and 10 mm in the moderately hypoxic tanks. Because reduced burial depth makes the clams more vulnerable to predators, these results indicate that the sub-lethal effects of hypoxia could change the rate of predation on M. balthica in the field.     

The alternating current polarographic behavior and determination of lansoprazole and omeprazole in dosage forms and biological fluids

The alternating current (AC_t) polarographic behavior of lansoprazole (LNS) and omeprazole (OMP) was studied in Britton Robinson buffers (BRb) over the pH range 4.1–11.5. In BRb of pH 9.6 and 10.5, well-defined AC_t peaks were obtained for both LNS and OMP, respectively. The current–concentration plots were rectilinear over the ranges of 0.4–20 µg mL^− 1 and 0.2–10 µg mL^− 1 for LNS and OMP respectively. The minimum detection limits (S/N = 2) were 0.02 µg mL^− 1 (5.4 × 10^− 8 M) and 0.01 µg mL^− 1 (2.9 × 10^− 8 M) for LNS and OMP, respectively. The proposed method was successfully applied to the analysis of the two drugs in their commercial capsules. The average percent recoveries were favorably compared to those obtained by reference methods. Co-administered drugs such as naproxen and methotrexate did not interfere with the proposed method. The proposed method was further extended to the in-vitro determination of the lansoprazole in spiked plasma, the percentage recoveries was 98.47 ± 1.29 (n = 4). The pathway for the electrode reaction for both drugs involved reduction of the sulphonyl group into the corresponding thiol group at the Dropping Mercury Electrode. The advantages of the method were time saving and more sensitive than the other published voltammetric method. Yet The present study is the first report on the use of alterating current polarography (AC_t) in this respect.     

Immobilization of hemoglobin on electrodeposited cobalt-oxide nanoparticles: direct voltammetry and electrocatalytic activity

Cyclic voltammetry at potential range − 1.1 to 0.5 V from aqueous buffer solution (pH 7) containing CoCl₂ produced a well defined cobalt oxide (CoOx) nanoparticles deposited on the surface of glassy carbon electrode. The morphology of the modified surface and cobalt oxide formation was examined with SEM and cyclic voltammetry techniques. Hemoglobin (Hb) was successfully immobilized in cobalt-oxide nanoparticles modified glassy carbon electrode. Immobilization of hemoglobin onto cobalt oxide nanoparticles have been investigated by cyclic voltammetry and UV–visible spectroscopy. The entrapped protein can take direct electron transfer in cobalt-oxide film. A pair of well defined, quasi-reversible cyclic voltammetric peaks at about − 0.08 V vs. SCE (pH 7), characteristic of heme redox couple (Fe(III)/Fe(II)) of hemoglobin, and the response showed surface controlled electrode process. The dependence of formal potential (E^0′) on the solution pH (56 mV pH^− 1) indicated that the direct electron transfer reaction of hemoglobin was a one-electron transfer coupled with a one proton transfer reaction process. The average surface coverage of Hb immobilized on the cobalt oxide nanoparticles was about 5.2536 × 10^− 11 mol cm^− 2_, indicating high loading ability of nanoparticles for hemoglobin entrapment. The heterogeneous electron transfer rate constant (k_s) was 1.43 s^− 1, indicating great of facilitation of the electron transfer between Hb and electrodeposited cobalt oxide nanoparticles. Modified electrode exhibits a remarkable electrocatalytic activity for the reduction of hydrogen peroxide and oxygen. The Michaels–Menten constant K_m of 0.38 mM, indicating that the Hb immobilized onto cobalt oxide film retained its peroxidases activity. The biosensor exhibited a fast amperometric response < 5 s, a linear response over a wide concentration range 5 μM to 700 μM and a low detection limit 0.5 μM. According to the direct electron transfer property and enhanced activity of Hb in cobalt oxide film, a third generation reagentless biosensor without using any electron transfer mediator or specific reagent can be constructed for determination of hydrogen peroxide in anaerobic solutions.     

Vinyl imidazole carrying metal-chelated beads for reversible use in yeast invertase adsorption

Poly(ethylene glycol dimethacrylate-n-vinyl imidazole) [poly(EGDMA–VIM)] hydrogel (average diameter 150–200 μm) was prepared copolymerizing ethylene glycol dimethacrylate (EGDMA) with n-vinyl imidazole (VIM). Poly(EGDMA–VIM) beads had a specific surface area of 59.8 m²/g. Poly(EGDMA–VIM) beads were characterized by swelling studies and scanning electron microscope (SEM). Cu²⁺ ions were chelated on the poly(EGDMA–VIM) beads (452 μmol Cu²⁺/g), then the metal-chelated beads were used in the adsorption of yeast invertase in a batch system. The maximum invertase adsorption capacity of the poly(EGDMA–VIM)–Cu²⁺ beads was observed as 35.2 mg/g at pH 4.5. The adsorption isotherm of the poly(EGDMA–VIM)–Cu²⁺ beads can be well fitted to the Langmuir model. Adsorption kinetics data were tested using pseudo-first- and -second-order models. Kinetic studies showed that the adsorption followed a pseudo-second-order reaction. The value of the Michaelis constant K_m of invertase was significantly larger upon adsorption, indicating decreased affinity by the enzyme for its substrate, whereas V_max was smaller for the adsorbed invertase. The optimum temperature for the adsorbed preparation of poly(EGDMA–VIM)–Cu²⁺-invertase at 50 °C, 10 °C higher than that of the free enzyme at 40 °C. Storage stability was found to increase with adsorption. Adsorbed invertase retains an activity of 82% after 10 batch successive reactions, demonstrating the usefulness of the enzyme-loaded beads in biocatalytic applications.     

Determination of bacterial cell number and cell volume by means of flow cytometry, transmission electron microscopy, and epifluorescence microscopy

Comparative measurements of bacterial total counts and volumes of flow cytometry (FCM), transmission electron (TEM), and epifluorescence microscopy (EFM), were undertaken during a four week mesocosm experiment. Total counts of bacteria measured by TEM, EFM, and FCM were in the range of 1 · 10⁶−6 cells ml⁻¹, 1 · 10⁶−3 · 10¹⁶ cells ml⁻¹, and 5 · 10⁵ cells ml⁻¹ respectively. The mean volume of the bacterial community, measured by means of EFM and TEM, increased from 0.12–0.15 μm³ at the start of the experiment to 0.39–0.53 μm³ at the end. Generally, there was good agreement between the two methods and regression analyses gave r = 0.87 (p < < 0.01) for cell volume and r = 0.97 (p < < 0.01) for cell number. DAPI stained bacteria with volumes less than 0.2 μm³ were not detected by flow cytometry and these were generally an order of magnitude lower than counts made by TEM and EFM. For samples where the mean bacterial cell volume was longer than 0.3 μm³, all three methods were in agreement both with respect to counts and volume estimates.     

Conceptual model of seagrass die-off in Florida Bay: Links to biogeochemical processes

Seagrasses are recognized as important plant communities in coastal estuaries and lagoons across both tropical and temperate climes; thus, large-scale seagrass die-off events worldwide are of general concern. In Florida Bay, at the southern terminus of the Florida peninsula, seagrass die-off events up to 4000 ha have been reported and smaller scale mortality events are noted annually. In the present study, we examined several hypothesized causative factors (high temperature, hypersalinity, sulfide toxicity) of seagrass (Thalassia testudinum) mortality in Florida Bay. To test sulfide effects, in situ sulfide production was stimulated by applying a labile carbon source (glucose) to sulfate reducers in the sediment at five sites across the bay (northeastern, northcentral, and southwestern basins). During the one year study, high temperature (32–36 °C) and salinity (> 50 psu) were recorded in the bay associated with a regional drought. We also experienced major seagrass die-off events at two of our southwestern bay sites. These field conditions provided an excellent opportunity to closely examine cause–effect relationships among stressors and die-off events in the field, and verify results of our previous mesocosm experiments. Even though glucose amendments stimulated porewater sulfides in bay sediments (4–8 mmol L^− 1), no significant differences in biomass, short shoot density or final growth rates were found between control and glucose plots. In addition, the highest growth rates and shoot densities were concomitant with maximum water column salinity (> 50 psu) and temperature (32–36 °C), when porewater sulfides were also in the millimolar range. Large-scale seagrass mortality events, encompassing  50% of the entire meadow at one site, occurred at southwestern bay sites when plants were down regulating (slower growth and shoot density), probably in response to shorter day length and lower temperature (30–34 to 23–26 °C) from October, 2004 to January, 2005. Sulfate reduction rates (SRR) were also 2-fold higher in the southwestern (214–488 nmol cm^− 3 d^− 1) versus northcentral and northeastern (97–240 nmol cm^− 3 d^− 1) bay sites, possibly limited by labile carbon, which we found to stimulate SRR 3-fold in northeastern and northcentral bay sites (461–708 nmol cm^− 3 d^− 1) and 4-fold at southwestern bay sites (1211–2036 nmol cm^− 3 d^− 1). Based on a synthesis of the field data reported herein, our mesocosm experiments to date, and contributions by others, we present a conceptual model of seagrass die-off in Florida Bay outlining a cascade of stressors, stimulated by P enrichment, which leads to high O₂ consumption in the system triggering a seagrass die-off event.     

Participation of the inducible nitric oxide synthase on atrial natriuretic peptide plasma concentration during endotoxemic shock

Atrial natriuretic peptide (ANP) is a hormone secreted in response to atrial or ventricular volume expansion and pressure overload, respectively. However, it has been found in studies with animals and patients an increase in ANP plasma concentration, during advanced septic shock, despite the fall in mean arterial pressure (MAP).

Several studies support the hypothesis that NO may be involved in the regulation of ANP release. Since NO may have an effect on ANP release, we hypothesized that NO pathway may participate in the control of the ANP release induced by the endotoxemic shock. Thus, the purpose of the present study was to assess the effect of the intravenous (i.v.) and intracereboventricular (i.c.v.) administration of aminoguanidine, an iNOS blocker, on plasma ANP levels and MAP during experimental endotoxemic shock.

Experiments were performed on adult male Wistar rats weighing 180–240 g. Rats were injected i.v. by bolus injection with 1.5 mg/kg of Lipopolysaccharide (LPS) or saline (0.5 mL) and were decapitated 2, 4 and 6 h after LPS injection for ANP determination by radioimmunoassay. In a separate set of experiments, rats received intravenous (i.v.) (100 mg/kg) or intracerebroventricular (i.c.v.) (250 μg in a final volume of 2 μL) injection of aminoguanidine (AG). Thirty minutes after the i.c.v. or i.v. injections, animals received LPS and were decapitated 2, 4 and 6 h later to determine plasma ANP concentration. In the two set of experiments MAP and heart rate (HR) were measured each 15 min for a period of 6 h using a polygraph.

When animals were injected with LPS, a reduction (p < 0.01) in MPA and an increase in HR occurred. A significant increase in plasma ANP concentration occurred, coinciding with the period of drop in blood pressure.

We found a significant increase in plasma ANP concentration after AG plus LPS injection, when compared to the rats treated with LPS plus saline. Further, the administration of AG plus LPS attenuated the decrease in the MAP after LPS and attenuated the increase in the HR when compared to the rats treated with LPS plus saline.

Our study suggests that inducible NOS pathway may activate an inhibitory control mechanism that attenuates ANP secretion, which is not regulated by the changes in blood pressure.     

Characterization of orange peel pectin and effect of sugars, -ascorbic acid, ammonium persulfate, salts on viscosity of orange peel pectin solutions

The effects of temperature and concentration on the viscosity of orange peel pectin solutions were examined at five different temperatures between 20 and 60°C and five concentration levels between 2.5–20 kg/m³. The effects of temperature was described by an Arrhenius-type equation. The activation energy for viscous flow was in the range 19.53–27.16 kJ/mol, depending on the concentration. The effect of concentration was described by two types of equation, power-law and exponential. Equations were derived which describes the combined effects of temperature and concentration on the viscosity for two different models in the range of temperatures and concentrations studied. Orange peel pectin was extracted by using HCl (pH 2.5, 90°C, 90 min) ammonium oxalate (0.25%, pH 3.5, 75°C, 90 min) and EDTA (0.5%, 90°C, 90 min) extraction procedures. The best result was obtained with ammonium oxalate extraction in which the pectin content of the final product was 30.12%, although the efficiency among the procedures varied.The average molecular weight was measured by light scattering technique. Magnitudes of intrinsic viscosity and molecular weight of pectins obtained by extraction with HCl, ammonium oxalate and EDTA were 0.262, 0.281, 0.309 m³/kg and 84 500, 91 400, 102 800 kg/kgmol, respectively. The molecular weight dependence of the intrinsic viscosity of the orange peel pectin solutions was expressed by Mark–Houwink–Sakurada equation. The data were fitted to equation as η_i=2.34×10⁻⁵(M_w,ave)^0.8224 which helps to evaluate the average molecular weight of pectin solutions from orange peel with a knowledge of their intrinsic viscosity.     

Simultaneous determination of 5-fluorouracil and uracil by high-performance liquid chromatography using four serial columns

A sensitive assay was developed for the quantitation of 5-fluorouracil (5-FU) and uracil using liquid–liquid extraction (LLE) and HPLC with UV detection. Analyses were performed with four μBondapak C₁₈ columns connected in series using 20 mM acetic acid with 1% ACN as mobile phase. The calibration curves were linear across the range of 26–1000 ng ml⁻¹ (0.21–7.8 μM) for 5-FU and 1.0–14.0 μg ml⁻¹ (0.01–110 μM) for uracil. This assay has been implemented to determine the plasma concentrations for pharmacokinetic studies for 5-FU and uracil in conjunction with clinical trials.     

Salivary hydrogen peroxide produced by holding or chewing green tea in the oral cavity

Tea (Camellia sinensis) catechins have been studied for disease prevention. These compounds undergo oxidation and produce H₂O₂. We have previously shown that holding tea solution or chewing tea leaves generates high salivary catechin levels. Herein, we examined the generation of H₂O₂ in the oral cavity by green tea solution or leaves. Human volunteers holding green tea solution (0.1-0.6%) developed salivary H₂O₂ with C_max = 2.9-9.6 μM and AUC_0 → ∞ = 8.5-285.3 μM min. Chewing 2 g green tea leaves produced higher levels of H₂O₂ (C_max = 31.2 μM, AUC_0 → ∞ = 1290.9 μM min). Salivary H₂O₂ correlated with catechin levels and with predicted levels of H₂O₂ (C_{max(expected)} = 36 μM vs C_{max(determined)} = 31.2 μM). Salivary H₂O₂ and catechin concentrations were similar to those that are biologically active in vitro. Catechin-generated H₂O₂ may, therefore, have a role in disease prevention by green tea.     

Effect of tropisetron versus placebo on cold-induced oxygen consumption and shivering in male volunteers

In a prospective, randomized, cross-over, placebo-controlled study in healthy male volunteers, we tested the effect of the 5-HT₃ antagonist tropisetron on cold-induced oxygen consumption and shivering.

Cooling was performed by intravenous infusion of isotonic salt solution at 4 °C. Whole-body oxygen consumption (VO₂) was measured with a metabolic monitor. Shivering was qualitatively assessed. When the shivering score evaluated “2” (intense shivering), 5 mg tropisetron or NaCl 0.9% was injected and repeated if necessary, to completely stop shivering.

The VO₂ before shivering (178±9 ml/min/m²) rose significantly during shivering (291±21 ml/min/m²). 5 mg of tropisetron in 2 volunteers and 10 mg in 3 volunteers stopped shivering but it returned (grade 0–1). The VO₂ decreased significantly (209±17 ml/min/m²). Placebo had no effect.

Tropisetron reduced cold-induced VO₂ and intensity of hypothermic shivering. That an additional dose of tropisetron could not stop the shivering totally may indicate that the effect of tropisetron is not dose dependent.     

Enantiorecognition of triiodothyronine and thyroxine enantiomers using different chiral selectors by HPLC and micro-HPLC

This paper deals with the chiral separation of triiodothyronine (T₃) and thyroxine (T₄) by HPLC and micro-HPLC. The separation of T₃ and T₄ is of great pharmaceutical and clinical interest, since the enantiomers exhibit different pharmacological activities. The HPLC measurements were performed on a chiral stationary ligand-exchange phase using l-4-hydroxyproline bonded via 3-glycidoxypropyltrimethoxysilane to silica gel as a selector. Also a chiral teicoplanin (Chirobiotic ™®) phase was used.

In micro-HPLC the chiral separation behaviour of l-4-hydroxyproline, and of the macrocyclic antibiotics teicoplanin and teicoplanin aglycone was investigated for the enantioseparation of T₃ and T₄. l-4-Hydroxyproline was bonded to 3 μm and the glycopeptide antibiotics were bonded to 3.5 μm silica gel and separations were accomplished by microbore HPLC columns (10 cm × 1 mm I.D.). With both techniques and all chiral selectors investigated T₃ and T₄ were baseline resolved. micro-HPLC was found to be superior to analytical HPLC with respect to low consumption of packing material, mobile phase and analyte.     

HPLC method for determination of verapamil in human plasma after solid-phase extraction

A simple, rapid and precise HPLC method has been developed for the assay of verapamil in human plasma. The clean up of the plasma samples was tested using several adsorbents for solid-phase extraction and best recovery was obtained using mixed-mode cartridges (HLB - hydrophilic-lipophilic balance) ranging between 94.70 and 103.71%. HPLC separation was performed with isocratic elution on Lichrospher 60 RP-select B column (250 mm × 4 mm I.D., 5 μm particle size). The mobile phase was 40% acetonitrile and 0.025 mol/L KH₂PO₄ with pH 2.5 at flow rate of 1 mL/min. Diltiazem was used as internal standard and the detection wavelength was 200 nm. The calibration curves were linear in the range of 10–500 ng/mL. The developed method is convenient for routine analysis of verapamil in human plasma.     

Glucocorticoids suppress vasopressin gene expression in human suprachiasmatic nucleus

Sleep impairment is one of the major side effects of glucocorticoid therapy. The mechanism responsible for this circadian disorder is unknown, but alterations in the suprachiasmatic nucleus (SCN), the biological clock of the human brain, are presumed to play a major role. In the present study, the amount of vasopressin mRNA (AVP mRNA) expression in the SCN was investigated in 10 glucocorticoid-exposed patients and 10 glucocorticoid free, age- and clock time of death-matched controls. The total amount of AVP mRNA, expressed as masked silver grains in the SCN, was two times lower in glucocorticoid-exposed patients (n = 10; 5115 ± 1314 μm²) than that in controls (n = 10; 11,021 ± 1408 μm²) (P = 0.006). There was also a 53% decrease in the total number of profiles in the SCN that expressed AVP mRNA in glucocorticoid-exposed patients (16,759 ± 3110) compared with those in controls (31,490 ± 3816) (P = 0.01). In conclusion, glucocorticoids have an inhibitory effect on AVP mRNA expression in the human SCN, which may be the biological basis of the circadian rhythm disturbances during glucocorticoid therapy.