Reverse foam sclerotherapy of the great saphenous vein with sapheno-femoral ligation compared to standard and invagination stripping: a prospective clinical series.

OBJECTIVES: Comparison of Reverse Foam Sclerotherapy of the great saphenous vein (GSV) combed with sapheno-femoral junction (SFJ) ligation to standard (Babcock) stripping and invagination (Pin) stripping in a prospective clinical series. DESIGN: Prospective clinical series. MATERIALS AND METHODS: 90 consecutive limbs of 82 patients with incompetence of the GSV resulting in varicose veins were prospectively randomised into 3 groups of 30, treated by SFJ ligation and either reverse foam sclerotherapy, standard stripping or invagination stripping of the GSV. Outcomes were assessed post-operatively and at 2-weeks follow-up. Peri-operative blood loss (24 hrs), analgesic requirement, bruising and residual varicosities were assessed. Bruising was assessed by both patients and independent assessors using questionnaires. RESULTS: SFJ ligation plus reverse foam sclerotherapy of the GSV was associated with significantly less blood loss, bruising and post-op discomfort than either of the stripping techniques. (p<0.001, Mann-Whitney) CONCLUSION: Standard stripping of the GSV and invagination stripping are not associated with major discomfort and problems in the early post-operative period. SFJ ligation and GSV reverse foam sclerotherapy yielded greater patient satisfaction with less post-op bruising and discomfort and reduced analgesic requirements.

Electroenzymatic reactions with oxygen on laccase-modified electrodes in anhydrous (pure) organic solvent.

The electroenzymatic reactions of Trametes hirsuta laccase in the pure organic solvent dimethyl sulfoxide (DMSO) have been investigated within the framework for potential use as a catalytic reaction scheme for oxygen reduction. The bioelectrochemical characteristics of laccase were investigated in two different ways: (i) by studying the electroreduction of oxygen in anhydrous DMSO via a direct electron transfer mechanism without proton donors and (ii) by doing the same experiments in the presence of laccase substrates, which display in pure organic solvents both the properties of electron donors as well as the properties of weak acids. The results obtained with laccase in anhydrous DMSO were compared with those obtained previously in aqueous buffer. It was shown that in the absence of proton donors under oxygenated conditions, formation of superoxide anion radicals is prevented at bare glassy carbon and graphite electrodes with adsorbed laccase. The influence of the time for drying the laccase solution at the electrode surface on the electroreduction of oxygen was studied. Investigating the electroenzymatic oxidation reaction of catechol and hydroquinone in DMSO reveals the formation of various intermediates of the substrates with different electrochemical activity under oxygenated conditions. The influence of the content of aqueous buffer in the organic solvent on the electrochemical behaviour of hydroquinone/1,4-benzoquinone couple was also studied.

Improved DET communication between cellobiose dehydrogenase and a gold electrode modified with a rigid self-assembled monolayer and green metal nanoparticles: The role of an ordered nanostructuration.

Efficient direct electron transfer (DET) between cellobiose dehydrogenase from Corynascus thermophilus (CtCDH) and a novel gold electrode platform, obtained by covalent linking of green AuNPs and AgNPs modified with a dithiol self-assembled monolayer, consisting of biphenyl-4,4'-dithiol (BPDT), was presented. The green AuNPs and AgNPs were synthesized using quercetin as reducing agent at room temperature. TEM experiments showed that the AuNPs and AgNPs were circular in shape with an average diameter of 5 and 8nm, respectively. Cyclic voltammetry of CtCDH immobilized onto the AuNPs/BPDT/AuE and the AgNPs/BPDT/AuE electrode platforms were carried out and compared with naked AuE, BPDT/AuE, AuNPs/AuE, and AgNPs/AuE. A pair of well-defined redox waves in neutral pH solution due to efficient DET of CtCDH was present with both MNPs/BPDT/AuE platforms. No DET communication was found with platforms without MNPs linked to BPDT. The apparent heterogeneous electron transfer rate constants (kS) of CtCDH were calculated to be 21.5±0.8s-1 and 10.3±0.7s-1, for the AuNPs/BPDT/AuE and the AgNPs/BPDT/AuE platforms, respectively. The modified electrodes were successively used to develop an eco-friendly biosensor for lactose detection. The CtCDH/AuNPs/BPDT/AuE based biosensor showed the best analytical performances with an excellent stability, a detection limit of 3µM, a linear range between 5 and 400µM and a sensitivity of 27.5±2.5µAcm-2mM-1. Such performances were favorably compared with other lactose biosensors reported in literature. The biosensor was successively tested to quantify lactose content in real milk and cream samples. No significant interference present in the sample matrices was observed.

Effect of liposome-encapsulation on immunomodulating and antiviral activities of interferon-gamma 1.

The effect of liposome-encapsulation on the immunomodulating and antiviral activities of interferon-gamma (IFN-gamma) was evaluated in this study. The immunomodulating activity was measured by increases in phagocytic activity and in nitric oxide production by peritoneal macrophages from mice treated with both free and LIP-IFN-gamma (4000 U/mouse, intraperitoneal injection). Resident peritoneal macrophages harvested from mice treated with free unencapsulated IFN-gamma or muramyl dipeptide showed significant increases in macrophage yield, and enhanced ability to phagocytize zymosan particles. In mice treated with liposome-encapsulated IFN-gamma (LIP-IFN-gamma), both macrophage yield and phagocytic activity further increased by 2-fold over unencapsulated IFN-Y. In addition, the activation of peritoneal macrophages with LIP-IFN-gamma showed enhanced production of NO when the cells were cultured ex vivo. Using a murine respiratory influenza infection model, intranasally administered LIP-IFN-gamma conferred protection to 70% in mice challenged intranasally with 10 LD50 doses of influenza A/PR/8 virus compared with a 20% survival rate using free IFN-gamma. Together these results suggest that liposome-encapsulation increases the immunomodulating and antiviral activities of IFN-gamma. Liposome-encapsulation of IFN-gamma may provide additional therapeutic advantages by reducing IFN-gamma toxicity while prolonging its body retention.

Mediatorless biosensor for H(2)O(2) based on recombinant forms of horseradish peroxidase directly adsorbed on polycrystalline gold.

Four forms of horseradish peroxidase (HRP) have been used to prepare peroxidase-modified gold electrodes for mediatorless detection of peroxide: native HRP, wild type recombinant HRP, and two recombinant forms containing six-His tag at the C-terminus and at the N-terminus, respectively. The adsorption of the enzyme molecules on gold was studied by direct mass measurements with electrochemical quartz crystal microbalance. All the forms of HRP formed a monolayer coverage of the enzyme on the gold surface. However, only gold electrodes with adsorbed recombinant HRP forms exhibited high and stable current response to H(2)O(2) due to its bioelectrocatalytic reduction based on direct electron transfer between gold and HRP. The sensitivity of the gold electrodes modified with recombinant HRPs was in the range of 1.4-1.5 A M(-1) cm(-2) at -50 mV versus Agmid R:AgCl. The response to H(2)O(2) in the concentration range 0.1-40 microM was not dependent on the presence of a mediator (i.e. catechol) giving strong evidence that the electrode currents are diffusion limited. Lower detection limit for H(2)O(2) detection was 10 nM at the electrodes modified with recombinant HRPs.

Characterisation of a thermophilic L-glutamate dehydrogenase biosensor for amperometric determination of L-glutamate by flow injection analysis.

Carbon paste wax electrodes incorporating thermophilic L-glutamate dehydrogenase, NADP and a polymeric toluidine blue O (poly-TBO) mediator have been characterised for the amperometric determination of L-glutamate at 313-318 K in a flow injection analysis (FIA) system. The biosensors exhibit good sensitivity, mechanical stability and reproducibilty, unlike carbon paste- or carbon wax-based electrodes under the same conditions. The carbon paste wax electrode responds linearly to L-glutamate up to 40 mM, the detection limit is 0.3 mM and the RSD (n = 10) for 5 mM L-glutamate was 7.6%. The response to some potential interferents has been quantified. Addition of finely ground hexaammineruthenium (III) trichloride ([Ru(NH3)6]Cl3) to the carbon paste wax electrodes decreases the FIA peak width and increases the peak current. The metal complex appears to accelerate the rate of oxidation of NAD(P)H by poly-TBO.

Biosensors based on novel peroxidases with improved properties in direct and mediated electron transfer.

Native horseradish peroxidase (HRP) on graphite has revealed approximately 50% of the active enzyme molecules to be in direct electron transfer (ET) contact with the electrode surface. Some novel plant peroxidases from tobacco, peanut and sweet potato were kinetically characterised on graphite in order to find promising candidates for biosensor applications and to understand the nature of the direct ET in the case of plant peroxidases. From measurements of the mediated and mediatorless currents of hydrogen peroxide reduction at the peroxidase-modified rotating disk electrodes (RDE), it was concluded that the fraction of enzyme molecules in direct ET varies substantially for the different plant peroxidases. It was observed that the anionic peroxidases (from sweet potato and tobacco) demonstrated a higher percentage of molecules in direct ET than the cationic ones (HRP and peanut peroxidase). The peroxidases with a high degree of glycosylation demonstrated a lower percentage of molecules in direct ET. It could, thus, be concluded that glycosylation of the peroxidases hinders direct ET and that a net negative charge on the peroxidase (low pI value) is beneficial for direct ET. Especially noticeable are the values obtained for sweet potato peroxidase (SPP), revealing both a high percentage in direct ET and a high rate constant of direct ET. The peroxidase electrodes were used for determination of hydrogen peroxide in RDE mode (mediatorless). SPP gave the lowest detection limit (40 nM) followed by HRP and peanut peroxidase.

Amperometric thin film biosensors based on glucose dehydrogenase and Toluidine Blue O as catalyst for NADH electrooxidation.

Amperometric glucose sensors were constructed based on solid graphite electrodes, surface-modified with NAD+ dependent glucose dehydrogenase (GDH), Toluidine Blue O (TBO), and protective ionic polymers. The electrocatalytic oxidation of NADH was evaluated from cyclic voltammetry with TBO dissolved, adsorbed, and electrostatically or covalently bound to polymers. The NADH and glucose sensors constructed were investigated and operated at 0 mV vs. Ag/AgCl using single potential step chronoamperometry. The operational stability of the glucose sensors was limited by leakage of NAD+. A glucose sensitivity much higher than carbon paste electrode was found. A sensitivity as high as 25 microA cm-2 mM-1 was achieved.

Amperometric biosensor for the determination of phenolic compounds using a tyrosinase graphite electrode in a flow injection system.

Selective and sensitive devices for the monitoring of phenol and phenolic compounds are required in clinical and environmental analysis. This paper describes a biosensor for the analysis of phenolic compounds in a flow injection system. The enzyme electrode is based on the use of immobilized tyrosinase and the amperometric detection of the enzymatic product at -50 mV vs. SCE. The enzyme is covalently immobilized on the surface of a carbodiimide-activated graphite electrode. The biosensor responds to a variety of phenolic substrates with different conversion efficiencies. The detection limit for phenol is 0.003 microM (S/N = 3), a quantification limit of 0.01 microM (rsd 3.7%), and an extended dynamic range up to 5 microM is achieved with a sample frequency of 110 samples per hour.

Enzymatic specificity and hydrolysis pattern of the catalytic domain of the xylanase Xynl from Rhodothermus marinus.

The catalytic domain of a xylanase from Rhodothermus marinus was produced in Escherichia coli. The catalytic domain belongs to glycosyl hydrolase family 10. The produced protein has a 22-amino acid leader peptide followed by a 411-amino acid truncated xylanase. The molecular mass was 48 kDa and the recombinant xylanase had a pI of 4.9. The pH and temperature optima for activity were determined to be 7.5 and 80 degrees C, respectively. At that temperature the enzyme had a half-life of 1 h 40 min. An addition of 1 mM calcium stabilized the activity of the enzyme at 80 degrees C. The xylanase had its highest specific activity on oat spelt xylan but was active also on other xylans and to a limited extent on some other polysaccharides (soluble glucans). No exo- or endo-cellulase activity was observed. Hydrolysis of xylo-oligomers and oat spelt xylan was studied and the predominant products of hydrolysis were xylobiose and xylotriose. The enzyme was inactive on xylobiose, xylotriose and on the soluble fraction from oat spelt xylan. The R. marinus xylanase is shown to have a strong preference for internal linkages and is therefore classified as an endo-xylanase.

A reagentless amperometric biosensor for alcohol detection in column liquid chromatography based on co-immobilized peroxidase and alcohol oxidase in carbon paste.

A reagentless carbon paste electrode chemically modified with covalently bound alcohol oxidase and horse-radish peroxidase was examined as a selective sensor in flow injection and column liquid chromatography. A combination of carbodiimide, glutaraldehyde, and polyethyleneimine was used for immobilizing the enzymes in the paste. The surface of the electrodes was protected by first forming a layer of electropolymerized ortho-phenylenediamine followed by deposition of a cation exchange membrane (Eastman AQ 29D). The electrodes were used for detection of hydrogen peroxide, methanol, ethanol, propanol, isopropanol, and butanol. Preliminary investigations of the use of this sensor for bioprocess control are reported.

High-performance anion-exchange chromatography-electrospray mass spectrometry for investigation of the substituent distribution in hydroxypropylated potato amylopectin starch.

The use of high-performance anion-exchange chromatography (HPAEC) with pulsed amperometric detection (PAD) coupled on-line with electrospray mass spectrometry (ESI-MS) for analysis of the substitution pattern in chemically modified starch, has been investigated. In order to characterise the distribution of substitution groups along the polymer chain, hydroxypropylated potato amylopectin starch (HPPAP) was subjected to enzymic hydrolysis, followed by analysis of the degradation products by HPAEC-PAD-MS. When using conventional chromatographic techniques for characterisation of enzymic hydrolysates, standard compounds are required for identification of the hydrolysis products. However, the on-line coupling with ESI-MS allowed identification of all products obtained, substituted as well as unsubstituted, and also of those compounds that co-eluted, without the need for standards. Further, HPAEC-PAD-MS was shown to be useful for analysis of the substitution pattern in modified starch; from results obtained it was suggested that the hydroxypropyl groups were homogeneously distributed in the amylopectin molecule. It was also shown that the starch hydrolysing enzymes were hindered by the hydroxypropyl groups and preferentially cleaved glucosidic linkages between unsubstituted glucose units.

Enzyme-based biosensor as a selective detection unit in column liquid chromatography.

A reagentless enzyme electrode based on co-immobilized alcohol oxidase and horseradish peroxidase was used as the working electrode in an amperometric flow-through cell connected to a column liquid chromatographic (CLC) system for the selective detection of methanol and ethanol. The enzymes were covalently immobilized in carbon paste (graphite-phenylmethylsilicone oil) in the presence of polyethylenimine. Electrodes prepared from the enzyme-modified carbon paste were optimized with respect to their sensitivity and selectivity. Different membranes were cast or electropolymerized directly on the surface of the electrode to increase the long-term stability of the biosensor. The compatibility with the reversed-phase chromatographic system was established. A PLRP-S polymer-based separation column was used with phosphate buffer as the mobile phase. The selectivity of the enzyme electrode was also determined by injecting some easily oxidizable and possibly interfering species normally present in biological samples. The enzyme electrode was also used in an on-line system, consisting of a microdialysis probe as the sampling unit, the CLC system and the biosensor detection device, for the selective following of the ethanol produced when a paper pulp industrial waste water was fermented with Saccharomyces cerevisiae.

Liquid chromatographic determination of total and beta-N-oxalyl-L-alpha,beta-diaminopropionic acid in lathyrus sativus seeds using both refractive index and bioelectrochemical detection.

A further improved chromatographic method for the simultaneous determination of the total amount of ODAP, selectively the amount of its neurotoxic form, beta-ODAP, and free L-glutamate in raw Lathyrus sativus (grass pea) seed samples is described using post-column refractive index in combination with bioelectrochemical detection. The biosensor is based on crosslinking horseradish peroxidase (HRP) and an Os-containing mediating polymer with poly(ethyleneglycol)(400) diglycidyl ether (PEGDGE), forming an inner hydrogel layer and then immobilising L-glutamate oxidase (GlOx) as an outer layer on top of a graphite electrode. Addition of polyethylenimine (PEI) to the hydrogel is believed to have sensitivity and stability enhancing effect on the biosensor. The double-layer approach in the biosensor construction avoided direct electrical wiring of GlOx and resulted in a higher sensitivity of 4.6 mA/M cm2 with respect to beta-ODAP and a wider linear range (1-250 microM) for both L-glutamate and beta-ODAP when compared with a single-layer approach where GlOx, HRP, and Os-polymer are crosslinked together. The limit of detection for the chromatographic-biosensor system was found to be 2 microM with respect to beta-ODAP and 0.7 microM with respect to L-glutamate. The refractive index detection on-line with the biosensor enabled full control of the chromatographic system for the determination of the total amount of ODAP, selectively the amount of beta-ODAP and L-glutamate. Ten grass pea samples have been collected from Lathyrism prone areas of Ethiopia to test the applicability of the presently developed analytical system for real sample analysis. The toxin levels of grass pea collections were determined in an aqueous extracts and ranged from 0.52 to 0.76%, dry mass basis. Comparison of results of an established spectrophotometric assay and that of the present system has shown an extraordinary degree of agreement as revealed by parallel "t" test (90% confidence limit). The present system has operational stability of more than 50 h. Analysis time per sample is 10 min after extraction for 90 min.

Optimal membrane choice for microdialysis sampling of oligosaccharides.

An analytical methodology based on microdialysis sampling, high-performance anion-exchange chromatography and integrated pulsed electrochemical detection for the monitoring of oligosaccharides in bioprocesses is presented. Amylopectin and model maltooligosaccharide standards; glucose, maltose, maltotriose, maltotetraose, maltopentaose, maltohexaose and maltoheptaose were used to demonstrate its versatility in view to sampling in enzymatic bioprocesses. The performance and characteristics of membranes with the same cut-off ranging between 3 and 100 kDa, were evaluated with respect to their extraction fraction (EF), permeability factors, temperature stability and protein (enzyme) interaction. All investigated membranes showed some non-specific interaction with enzymes. The EF and non-specific membrane-enzyme interactions were higher for the polysulfone membranes compared with the polyamide and polyethersulfone membranes. For all saccharides, the EF was independent of the concentration even for a 250-fold change in concentration. The EF and morphology of the membranes in their dehydrated state, as observed using scanning electron microscopy did not show any significant difference between membranes exposed to a 90 degrees C temperature for 3 and 24 h indicating their applicability to the study of high temperature bioprocesses.

Investigation of electrochemical properties of FMN and FAD adsorbed on titanium electrode.

The electrochemical properties (such as the values of the formal potentials, the dependence of the formal potentials on solution pH, the reversibility of the electrochemical process) of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) adsorbed on a titanium electrode were dependent on the electrolyte. The formal potentials of adsorbed FMN and FAD in phosphate, HEPES and PIPES buffers at pH 7 were similar to those for dissolved flavins (-460 to -480 mV vs. SCE) and changed linearly with a slope of about 52 mV per pH unit in the pH region 3 to 8. In TRIS buffer, the formal potentials of adsorbed FMN and FAD were also pH-dependent, however, with invariance in the pH range 4.5 to 5.5. In non-buffered solutions (KCl, LiCl, NaCl, CsCl, CaCl(2), Na(2)SO(4) at different concentrations), the electrochemical behavior of adsorbed FMN and FAD differed from that of dissolved flavins and was dependent on the electrolyte (especially at pH 4.5 and pH 5). Under certain conditions (electrolyte, concentration, pH), a two-step oxidation of FMN could be observed.

Mediator-modified electrodes for catalytic NADH oxidation: high rate constants at interesting overpotentials.

Carbon paste electrodes were modified with a nitrofluorenone derivative, 2,4,7-trinitro-9-fluorenone, adsorbed on zirconium phosphate (ZP). After electrochemical reduction of the fluorenone derivative, it turns into a very efficient mediator for electrocatalytic NADH oxidation, with a formal potential of about +250 mV vs. Ag/AgCl. The electrochemistry and the electrocatalytic properties of the mediator were investigated with cyclic voltammetry and rotating disk electrode methodology. The second order rate constant with NADH was evaluated and found to be higher than 10(6) M(-1) s(-1), thus approaching true diffusion controlled currents for NADH oxidation.

Characterisation of the substituent distribution in hydroxypropylated potato amylopectin starch.

The distribution of substituents in hydroxypropylated potato amylopectin starch (amylose deficient) modified in a slurry of granular starch (HPPAPg) or in a polymer 'solution' of dissolved starch (HPPAPs), was investigated. The molar substitution (MS) was determined by three different methods: proton nuclear magnetic resonance (1H NMR) spectroscopy, gas-liquid chromatography (GLC) with mass spectrometry, and a colourimetric method. The MS values obtained by 1H NMR spectroscopy were higher than those obtained by GLC-mass spectrometry analysis and colourimetry. The relative ratio of 2-, 3-, and 6-substitution, as well as un-, mono-, and disubstitution in the anhydroglucose unit (AGU) were determined by GLC-mass spectrometry analysis. Results obtained showed no significant difference in molar distribution of hydroxypropyl groups in the AGU between the two derivatives. For analysis of the distribution pattern along the polymer chain, the starch derivatives were hydrolysed by enzymes with different selectivities. Debranching of the polymers indicated that more substituents were located in close vicinity to branching points in HPPAPg than in HPPAPs. Simultaneous alpha-amylase and amyloglucosidase hydrolysis of HPPAPg liberated more unsubstituted glucose units than the hydrolysis of HPPAPs, indicating a more heterogeneous distribution of substituents in HPPAPg.

Controlled potential iodometric determination of iron after extraction with di-isopropyl ether.

A selective method for the determination of small amounts of iron in different kinds of samples with complex matrices has been developed. The iron in a sample is converted into the ferric state and after extraction with di-isopropyl ether the ferric iron is iodometrically determined with a controlled-potential apparatus previously described. Iron concentrations lower than 1 ppm can be determined.

Prussian-Blue-based amperometric biosensors in flow-injection analysis.

Optimisation of the electrodeposition of Prussian Blue onto mirrored glassy carbon electrodes yielded a modified electrode practically insensitive to oxygen reduction. At the same time the electrode activity towards hydrogen peroxide reduction was extremely high. This allowed the detection of hydrogen peroxide by electroreduction over a wide potential range. Flow-injection investigations of this electrode inserted into a flowthrough electrochemical cell of the confined wall-jet type showed that the response for hydrogen peroxide is limited by diffusion. Glucose and alcohol biosensors were made by immobilisation of glucose oxidase and alcohol oxidase respectively, within a Nafion layer, onto the top of the Prussian-Blue-modified electrodes. By increasing the density of Nafion and decreasing the measuring potential the glucose biosensor was made completely insensitive to both ascorbate and acetominophes.