Glucose biosensor based on immobilization of glucose oxidase in poly(o-aminophenol) film on polypyrrole-Pt nanocomposite modified glassy carbon electrode

Novel Pt nanoclusters embedded polypyrrole nanowires (PPy-Pt) composite was electrosynthesized on a glassy carbon electrode, denoted as PPy-Pt/GCE. A glucose biosensor was further fabricated based on immobilization of glucose oxidase (GOD) in an electropolymerized non-conducting poly(o-aminophenol) (POAP) film that was deposited on the PPy-Pt/GCE. The morphologies of the PPy nanowires and PPy-Pt nanocomposite were characterized by field emission scanning electron microscope (FE-SEM). Effect of experimental conditions involving the cycle numbers for POAP deposition and Pt nanoclusters deposition, applied potential used in glucose determination, temperature and pH value of the detection solution were investigated for optimization. The biosensor exhibited an excellent current response to glucose over a wide linear range from 1.5 × 10⁻⁶ to 1.3 × 10⁻² M (r = 0.9982) with a detection limit of 4.5 × 10⁻⁷ M (s/n = 3). Based on the combination of permselectivity of the POAP and the PPy films, the sensor had good anti-interference ability to ascorbic acid (AA), uric acid (UA) and acetaminophen. The apparent Michaelis–Menten constant (K_m) and the maximum current density (I_m) were estimated to be 23.9 mM and 378 μA/cm², respectively. In addition, the biosensor had also good sensitivity, stability and reproducibility.     

Direct glucose determination in blood using a reagentless optical biosensor

This paper demonstrates that glucose determination in blood can be done directly (without sample pretreatment) using a reagentless reversible biosensor based on the intrinsic spectroscopic properties of peroxidase (HRP). The biosensor, prepared by HRP and glucose oxidase entrapment in a polyacrylamide gel matrix, works in continuous mode, presents a linear response range from 1.5 × 10⁻⁶ up to 5.5 × 10⁻⁵ M and can be used for at least 750 measurements; in the best conditions (0.1 M pH 6 phosphate buffer, HRP and GOx amounts in the polymersation mixture for the sensor film preparation 0.0165 and 0.0010 g, respectively) the minimum samples rate is 30 h⁻¹. For glucose determination, blood is simply diluted in water (until haemolysis is completed) and fed into the sensor without a cleaning step between samples; the blood absorption is corrected in a simple way by working at a proper reference wavelength. The biosensor signals have been mathematically modeled in order to facilitate the design of sensors based on the same idea for other biochemical compounds.     

Bioelectrocatalytic application of titania nanotube array for molecule detection

A bioelectrocatalysis system based on titania nanotube electrode has been developed for the quantitative detection application. Highly ordered titania nanotube array with inner diameter of 60 nm and total length of 540 nm was formed by anodizing titanium foils. The functionalization modification was achieved by embedding glucose oxidases inside tubule channels and electropolymerizing pyrrole for interfacial immobilization. Morphology and microstructure characterization, electrochemical properties and bioelectrocatalytic reactivities of this composite were fully investigated. The direct detection of hydrogen peroxide by electrocatalytic reduction reaction was fulfilled on pure titania nanotube array with a detection limit up to 2.0 × 10⁻⁴ mM. A biosensor based on the glucose oxidase–titania/titanium electrode was constructed for amperometric detection and quantitative determination of glucose in a phosphate buffer solution (pH 6.8) under a potentiostatic condition (−0.4 V versus SCE). The resulting glucose biosensor showed an excellent performance with a response time below 5.6 s and a detection limit of 2.0 × 10⁻³ mM. The corresponding detection sensitivity was 45.5 μA mM⁻¹ cm⁻². A good operational reliability was also achieved with relative standard deviations below 3.0%. This novel biosensor exhibited quite high response sensitivity and low detection limit for potential applications.     

Immobilization of horseradish peroxidase on chitosan/silica sol-gel hybrid membranes for the preparation of hydrogen peroxide biosensor

A simple and effective strategy for fabrication of hydrogen peroxide (H₂O₂) biosensor has been developed by entrapping horseradish peroxidase (HRP) in chitosan/silica sol–gel hybrid membranes (CSHMs) doped with potassium ferricyanide (K₃Fe(CN)₆) and gold nanoparticles (GNPs) on platinum electrode surface. The hybrid membranes are prepared by cross-linking chitosan (CS) with 3-aminopropyltriethoxysilane (APTES), while the presence of GNPs improved the conductivity of CSHMs, and the Fe(CN)₆^3−/4− was used as a mediator to transfer electrons between the electrode and HRP due to its excellent electrochemistry activity. UV–Vis absorption spectroscopy was employed to characterize the different components in the CSHMs and their interaction. The parameters influencing the performance of the resulting biosensor were optimized and the characteristic of the resulting biosensor was characterized by cyclic voltammetry and chronoamperometry. Linear calibration for hydrogen peroxide was obtained in the range of 3.5 × 10^− 6 to 1.4 × 10^− 3 M under the optimized conditions with the detection limit (S/N = 3) of 8.0 × 10^− 7 M. The apparent Michaelis–Menten constant of the enzyme electrode was 0.93 mM. The enzyme electrode retained about 78% of its response sensitivity after 30 days. The system was applied for the determination of the samples, and the results obtained were satisfactory.     

Fabrication of sucrose biosensor based on single mode planar optical waveguide using co-immobilized plant invertase and GOD

In present studies, the new optical sensing platform based on optical planar waveguide (OPWG) for sucrose estimation was reported. An evanescent-wave biosensor was designed by using novel agarose–guar gum (AG) biopolymer composite sol–gel with entrapped enzymes (acid invertase (INV) and glucose oxidase (GOD)). Partially purified watermelon invertase isolated from Citrullus vulgaris fruit (specific activity 832 units mg⁻¹) in combination with GOD was physically entrapped in AG sol–gel and cladded on the surface of optical planar waveguide. Na⁺–K⁺ ion-exchanged glass optical waveguides were prepared and employed for the fabrication of sucrose biosensor. By addressing the enzyme modified waveguide structure with, the optogeometric properties of adsorbed enzyme layer (12 μm) at the sensor solid–liquid interface were studied. The OPWG sensor with short response time (110 s) was characterized using the 0.2 M acetate buffer, pH 5.5. The fabricated sucrose sensor showed concentration dependent linear response in the range 1 × 10⁻¹⁰ to 1 × 10⁻⁶ M of sucrose. Lower limit of detection of this novel AG–INV–GOD cladded OPWG sensor was found to be 2.5 × 10⁻¹¹ M sucrose, which indicates that the developed biosensor has higher sensitivity towards sucrose as compared to earlier reported sensors using various transducer systems. Biochips when stored at room temperature, showed high stability for 81 days with 80% retention of original sensitivity. These sucrose sensing biochips showed good operational efficiency for 10 cycles. The proper confinement of acid invertase and glucose oxidase in hydrogel composite was confirmed by scanning electron microscopy (SEM) images. The constructed OPWG sensor is versatile, easy to fabricate and can be used for sucrose measurements with very high sensitivity.     

The development of an amperometric microbial biosensor using Acetobacter pasteurianus for lactic acid

A microbial biosensor, using Acetobacter pasteurianus cells and an oxygen electrode, was developed for the determination of lactic acid. The bacterial cells were retained on a nylon membrane and attached to the surface of the oxygen electrode. In view of response time, stability and sensitivity, the biosensor performed best at 26°C and in pH 6 phthalate buffer containing magnesium sulfate. The activity of the retained cells was stable for approximately 170 h and was regenerable. The biosensor exhibited a hyperbolic response to both D- and L-lactic acid in the range of 10⁻⁴ M to 25 × 10⁻³ M. However, in the range 10⁻⁴ M to 15 × 10⁻⁴ M the response was linear. The microbial biosensor was applicable for detecting lactate concentration in yogurt and milk, since it was not sensitive to lactose, sucrose and glucose — three major components of such dairy products.     

Epinephrine quantification in pharmaceutical formulations utilizing plant tissue biosensors

A plant tissue biosensor associated with flow injection analysis is proposed to determine epinephrine in pharmaceutical samples. The polyphenol oxidase enzymes present in the fibers of a palm tree fruits (Livistona chinensis), catalyses the oxidation of epinephrine to epinephrinequinone as a primary product. This product is then electrochemically reduced (at −0.10 V versus Ag/AgCl_sat) on the biosensor surface and the resulting current is used for the quantification of epinephrine. The biosensor provides a linear response for epinephrine in the concentration range from 5.0 × 10⁻⁵ to 3.5 × 10⁻⁴ mol l⁻¹. The limit of detection estimated for this interval was 1.5 × 10⁻⁵ mol l⁻¹ and the correlation coefficient of 0.998, working under a flow rate of 2.0 ml min⁻¹ and using a sample loop of 100 μl. The repeatability (R.S.D. for 10 consecutive determinations of a 3.0 × 10⁻⁴ mol l⁻¹ epinephrine solution) was 3.1%. The results obtained by the method here proposed were compared with the official UV spectrophotometric procedure and also using a plant tissue reactor. The responses obtained with the proposed strategies were in good agreement with both ways of analyses, whereas the values obtained by the official spectrophotometric method was strongly affected by benzoic acid, present in the formulation of pharmaceutical product utilized for inhalation. Such favorable results obtained with the carbon paste biosensor or utilizing the bioreactor, joined with the simplicity of its preparation turns these procedures very attractive for epinephrine quantification in pharmaceutical products.     

A performance comparison of choline biosensors: anodic or cathodic detections of H2O2 generated by enzyme immobilized on a conducting polymer

Amperometric choline biosensors were fabricated by the covalent immobilization of an enzyme of choline oxidase (ChO) and a bi-enzyme of ChO/horseradish peroxidase (ChO/HRP) onto poly-5,2′:5′,2″-terthiophene-3′-carboxylic acid (poly-TTCA) modified electrodes (CPMEs). A sensor modified with ChO utilized the oxidation process of enzymatically generated H₂O₂ in a choline solution at +0.6 V. The other one modified with ChO/HRP utilized the reduction process of H₂O₂ in a choline solution at −0.2 V. Experimental parameters affecting the sensitivity of sensors, such as pH, applied potential, and temperature were optimized. A performance comparison of two sensors showed that one based on ChO/HRP/CPME had a linear range from 1.0×10⁻⁶ to 8.0×10⁻⁵ M and the other based on ChO/CPME from 1.0×10⁻⁶ to 5.0×10⁻⁵ M. The detection limits for choline employing ChO/HRP/CPME and ChO/CPME were determined to be about 1.0×10⁻⁷ and 4.0×10⁻⁷ M, respectively. The response time of sensors was less than 5 s. Sensors showed good selectivity to interfering species. The long-term storage stability of the sensor based on ChO/HRP/CPME was longer than that based on ChO/CPME.     

Direct electrochemistry of cytochrome c at ordered macroporous active carbon electrode

Three-dimensionally (3D) ordered macroporous active carbon has been fabricated and used as electrode substrate for the direct electrochemistry of horse heart cytochrome c (Cyt c). The Cyt c immobilized on the surface of the ordered macroporous active carbon shows a pair of well-defined and nearly reversible redox waves at the formal potential of −0.033 V in pH 6.8 phosphate buffer solution. The interaction between Cyt c and the 3D macroporous active carbon makes the formal potential shift negatively compared to that of Cyt c in solution. Spectrophotometric and electrochemical methods have been used to investigate the interaction between Cyt c and the porous active carbon. The immobilized Cyt c maintains its biological activity, and shows a surface controlled electrode process with the electron-transfer rate constant (k_s) of 17.6 s⁻¹ and the charge-transfer coefficient (a) of 0.52, and displays the features of a peroxidase in the electrocatalytic reduction of hydrogen peroxide (H₂O₂). A potential application of the Cyt c-immobilized porous carbon electrode as a biosensor to monitor H₂O₂ has been investigated. The steady-state current response increases linearly with H₂O₂ concentration from 2.0 × 10⁻⁵ to 2.4 × 10⁻⁴ mol l⁻¹. The detection limit (3σ) for determination of H₂O₂ has been found to be 1.46 × 10⁻⁵ mol l⁻¹.     

Graft copolymerization of acrylic acid onto guar gum initiated by vanadium (V)–mercaptosuccinic acid redox pair

Guar gum has been modified by graft copolymerization with acrylic acid in aqueous medium using vanadium (V)–mercaptosuccinic acid redox system. The optimum reaction conditions affording maximum grafting ratio, efficiency, add on and conversion have been determined. The grafting parameters have been found to increase with increase in vanadium (V) concentration upto 1.0 × 10⁻² mol dm⁻³, but these parameters decrease on further increasing the vanadium (V) concentration. On increasing the mercaptosuccinic acid concentration from 1.0 × 10⁻² to 4.0 × 10⁻² mol dm⁻³ grafting ratio, efficiency and add on increase up to 2.0 × 10⁻² mol dm⁻³ but decrease with further increase in mercaptosuccinic acid concentration. On varying the acrylic acid concentration from 5.0 × 10⁻² to 30.0 × 10⁻² mol dm⁻³, maximum grafting ratio, efficiency and add on have been obtained at 20.0 × 10⁻² mol dm⁻³. The grafting ratio, add on and conversion increase, on increasing the H⁺ ion concentration from 1.5 × 10⁻¹ to 6.0 × 10⁻¹ mol dm⁻³. On increasing the guar gum concentration the grafting parameters increase. The grafting ratio, add on and conversion have been found to increase with time period while efficiency started decreasing after 120 min. It has been observed that %G increases on increasing the temperature up to 35 °C. The graft copolymer has been characterized by IR spectroscopy and thermogravimetric analysis.     

Synthesis of graft copolymer (k-carrageenan-g-N,N-dimethylacrylamide) and studies of metal ion uptake, swelling capacity and flocculation properties

Graft copolymer of k-carrageenan and N,N-dimethylacrylamide has been synthesized by free radical polymerization using peroxymonosulphate/glycolic acid redox pair in an inert atmosphere. The grafting parameters i.e. grafting ratio, add on and efficiency decrease with increase in concentration of k-carrageenan from 0.6 to 1.4 g dm⁻³ and hydrogen ion from 3 × 10⁻³ to 7 × 10⁻³ mol dm⁻³, but these grafting parameters increase with increase in concentration of N,N-dimethylacrylamide from 16 × 10⁻² to 32 × 10⁻² mol dm⁻³, and peroxymonosulphate from 0.8 × 10⁻² to 2.4 × 10⁻² mol dm⁻³. The metal ion sorption, swelling behaviour and flocculation properties have been studied. The intrinsic viscosity of pure and grafted samples has been measured by using Ubbelohde capillary viscometer. Flocculation capability of k-carrageenan and k-carrageenan-g-N,N-dimethylacrylamide for both coking and non-coking coals has been studied for the treatment of coal mine waste water. The graft copolymer has been characterized by Infrared (IR) spectroscopy and thermogravimetric analysis.     

Glutathione-proline activation of feeding behavior in the zoanthid Palythoa psammophilia Walsh & Bowers

Palythoa psammophilia Walsh & Bowers has a well coordinated, stereotyped feeding response, the culminating step of which is ingestion; this may be elicited by the synergistic effect of the tripeptide glutathione and the -imino acid, proline. Either activator acting separately causes responses only at high concentrations (above 10⁻⁵ M for glutathione; above 10⁻⁴ M for proline) in a reduced number of animals and at a low rate (5.00 ± 1.73 min in 5 × 10⁻³ M solutions of glutathione; 11.10±3.74 min in 5 × 10⁻³ M solutions of proline). Highest percentages of response were obtained in combinations where glutathione was at a concentration of 5 × 10⁻³ M and proline at 5 × 10⁻⁴ M or in combinations of glutathione at concentrations 5 × 10⁻⁶ M and proline at 5 × 10⁻⁵ M. The speed of ingestion is considerably enhanced when these activators are combined (1.17±1.18 min).     

Optimizing the formulation of a myoglobin molecularly imprinted thin-film polymer--formed using a micro-contact imprinting method

Thin-film myoglobin molecularly imprinted polymers have been fabricated using a micro-contact approach. By initially selecting the cross-linker on the basis of it having a minimal recognition for the template and using this as a starting point for functional monomer selection, we have produced myoglobin imprinted polymers with exceptionally high selectivities.

The affinity of the polymers, for myoglobin, when prepared with a variety of different cross-linkers and no functional monomer was evaluated. Of these, tetraethylene glycol dimethacrylate (TEGDMA) exhibited the lowest affinity for the template species. Methyl methacrylate (MMA) was chosen as the functional monomer as when it was used in conjunction with TEGDMA, it exhibited maximum selectivity for the template compared to polymers made with other functional monomers.

With a MMA to TEGDMA ratio of 1 to 3, the myoglobin molecularly imprinted polymer adsorbed 15.03 ± 0.89 × 10⁻¹¹ mole/cm² of template from a 5.68 × 10⁻⁷ M myoglobin solution, compared to 2.58 ± 0.02 × 10⁻¹¹ mole/cm² for a polymer of similar composition, but formed in the absence of a template. Various washing conditions, using alkaline media to remove the template, were investigated. An extraction solvent comprising 2 wt.% SDS and 0.6 wt.% NaOH used at 80 °C for 30 min was shown to give the highest imprinting factor i.e. 5.83 with 72.82% myoglobin removal.

The saturation kinetics of template binding to the thin-film MIP were examined and found to display a simple two-phase profile typical of non-cooperative binding. A Scatchard binding plot showed the dissociation constant (K_d) for the specific binding phase to be 3.4 × 10⁻⁷ M and the binding site capacity to be 7.24 × 10⁻¹¹ mole/cm². For the non-specific binding phase, K_d was found to be 1.355 × 10⁻⁵ M and the binding site capacity was determined as 9.62 × 10⁻¹⁰ mole/cm².

Selectivity experiments were carried out in both single protein and binary protein systems all using a total protein concentration of 5.68 × 10⁻⁷ M. The molar ratio of adsorbed myoglobin to IgG, HSA and hemoglobin was found to 115.5, 230.9 and 2.5, respectively. While, in binary competition systems, myoglobin selectivity to IgG, HSA and hemoglobin was, respectively, 94.18, 98.21 and 61.09%. Rebinding in natural biological matrices, i.e. human serum or urine, showed the imprinted films to have significantly greater uptake than non-imprinted films. Re-binding in undiluted urine was found to be a facile process, with the imprinting factor, i.e. the ratio of MIP to NIP binding, being determined as 37.4.     

Enzyme-entrapping membranes for biosensors obtained by radiation-induced polymerization

A new method of physically immobilizing enzymes in poly(2-hydroxyethyl methacrylate) membranes was developed in order to obtain suitable biosensors. It was possible to prepare an enzyme sensor based on an oxygen Clark electrode and on glucose oxidase immobilized by low-temperature gamma radiation-induced polymerization. Temperature and pH effects on the activity of immobilized enzyme are described and the response characteristics of the resulting biosensor are summarized. The determination of glucose in standard solutions was carried out and a linear calibration curve, with an R² value of 0·9993, from the detection limit 5 × 10⁻⁵ to 1·2 × 10⁻³ was obtained. The biosensor was employed to analysis of control sera and the results were compared to those obtained by enzymatic-spectrophotometric detection.     

Amperometric glucose biosensor based on self-assembly hydrophobin with high efficiency of enzyme utilization

Hydrophobins are a family of natural self-assembling proteins with high biocompability, which are apt to form strong and ordered assembly onto many kinds of surfaces. These physical-chemical and biological properties make hydrophobins suitable for surface modification and biomolecule immobilization purposes. A class II hydrophobin HFBI was used as enzyme immobilization matrix on platinum electrode to construct amperometric glucose biosensor. Permeability of HFBI self-assembling film was optimized by selecting the proper HFBI concentration for electrode modification, in order to allow H₂O₂ permeating while prevent interfering compounds accessing. HFBI self-assembly and glucose oxidase (GOx) immobilization was monitored by quartz crystal microbalance (QCM), and characterization of the modified electrode surface was obtained by scanning electron microscope (SEM). The resulting glucose biosensors showed rapid response time within 6 s, limits of detection of 0.09 mM glucose (signal-to-noise ratio = 3), wide linear range from 0.5 to 20 mM, high sensitivity of 4.214 × 10⁻³ A M⁻¹ cm⁻², also well selectivity, reproducibility and lifetime. The all-protein modified biosensor exhibited especially high efficiency of enzyme utilization, producing at most 712 μA responsive current for per unit activity of GOx. This work provided a promising new immobilization matrix with high biocompatibility and adequate electroactivity for further research in biosensing and other surface functionalizing.     

Electrochemical impedance spectroscopy for study of aptamer-thrombin interfacial interactions

A simple and highly sensitive electrochemical impedance spectroscopy (EIS) biosensor based on a thrombin-binding aptamer as molecular recognition element was developed for the determination of thrombin. The signal enhancement was achieved by using gold nanoparticles (GNPs), which was electrodeposited onto a glassy carbon electrode (GCE), as a platform for the immobilization of the thiolated aptamer. In the measurement of thrombin, the change in interfacial electron transfer resistance of the biosensor using a redox couple of [Fe(CN)₆]^3−/4− as the probe was monitored. The increase of the electron transfer resistance of the biosensor is linear with the concentration of thrombin in the range from 0.12 nM to 30 nM. The association and dissociation rate constants of the immobilized aptamer–thrombin complex were 6.7 × 10³ M⁻¹ s⁻¹ and 1.0 × 10⁻⁴ s⁻¹, respectively. The association and dissociation constants of three different immobilized aptamers binding with thrombin were measured and the difference of the dissociation constants obtained was discussed. This work demonstrates that GNPs electrodeposited on GCE used as a platform for the immobilization of the thiolated aptamer can improve the sensitivity of an EIS biosensor for the determination of protein. This work also demonstrates that EIS method is an efficient method for the determination of association and dissociation constants on GNPs modified GCE.     

Estradiol inhibits the estrone sulfatase activity in normal and cancerous human breast tissues

It is well accepted that estradiol (E₂) plays an important role in the genesis and evolution of breast cancer. Quantitative evaluation indicates that in human breast tumor, estrone sulfate (E₁S) ‘via sulfatase’ is a much more likely precursor for E₂ than is androstenedione ‘via aromatase’. In previous studies, it was demonstrated that in isolated MCF-7 and T-47D breast cancer cell lines, estradiol can block estrone sulfatase activity. In the present study, the effect of E₂ was explored using total normal and cancerous breast tissues. This study was carried out with post-menopausal patients with breast cancer. None of the patients had a history of endocrine, metabolic or hepatic diseases or had received treatment in the previous 2 months. Each patient received local anaesthetic (lidocaine 1%) and two regions of the mammary tissue were selected: (A) the tumoral tissue and (B) the distant zone (glandular tissue) which was considered as normal. Samples were placed in liquid nitrogen and stored at –80 °C until enzyme activity analysis. Breast cancer histotypes were ductal and post-menopausal stages were T2. Homogenates of tumoral or normal breast tissues (45–75 mg) were incubated in 20 mM Tris–HCl, pH 7.2 with physiological concentrations of [³H]-E₁S (5 × 10⁻⁹ M) alone or in the presence of E₂ (5 × 10⁻⁵ to 5 × 10⁻⁷ M) during 30 min or 3 h. E₁S, E₁ and E₂ were characterized by thin layer chromatography and quantified using the corresponding standard. The sulfatase activity is significantly more intense with the breast cancer tissue than normal tissue, since the concentration of E₁ was 3.20 ± 0.15 and 0.42 ± 0.07 pmol/mg protein, respectively after 30 min incubation. The values were 27.8 ± 1.8 and 3.5 ± 0.21 pmol/mg protein, respectively after 3 h incubation. Estradiol at the concentration of 5 × 10⁻⁷ M inhibits this conversion by 33% and 31% in cancerous and normal breast tissues, respectively and by 53% and 88% at the concentration of 5 × 10⁻⁵ M after 30 min incubation. The values were 24% and 18% for 5 × 10⁻⁷ M and 49% and 42% for 5 × 10⁻⁵ M, respectively after 3 h incubation. It was observed that [³H]-E₁S is only converted to [³H]-E₁ and not to [³H]-E₂ in normal or cancerous breast tissues, which suggests a low or no 17β-hydroxysteroid dehydrogenase (17β-HSD) Type 1 reductive activity in these experimental conditions. In conclusion, estradiol is a strong anti-sulfatase agent in cancerous and normal breast tissues. This data can open attractive perspectives in clinical trials using this hormone.     

Nitric oxide stimulates prostaglandin synthesis in cultured rabbit gastric cells

Both prostaglandins (PGs) and nitric oxide (NO) have cytoprotective and hyperemic effects in the stomach. However, the effect of NO on PG synthesis in gastric mucosal cells is unclear. We examined whether sodium nitroprusside (SNP), a releaser of NO, stimulates PG synthesis in cultured rabbit gastric mucus-producing cells. These cells did not release NO themselves. Co-incubation with SNP (2 × 10⁻⁴, 5 × 10⁻⁴, 10⁻³ M) increased PGE₂ synthesis, and SNP (10⁻³ M) increased PGI₂ synthesis in these cells. Hemoglobin, a scavenger of NO, (10⁻⁵ M) eliminated the increase in PGE₂ synthesis by SNP, but methylene blue, an inhibitor of soluble guanylate cyclase, (5 × 10⁻⁵ M) did not affect the increase in PGE₂ synthesis by SNP. 8-bromo guanosine 3′ : 5′-cyclic monophosphate (8-bromo cGMP), a cGMP analogue, (10⁻⁶, 10⁻⁵, 10⁻⁴, 10⁻³ M) did not affect PGE₂ synthesis. These findings suggest that NO increased PGE₂ and PGI₂ synthesis via a cGMP-independent pathway in cultured rabbit gastric cells.     

Cortisol and testosterone: Inhibitory agents of the mineralocorticoid pathway. Is there a physiopathological meaning in adrenal tumors?

The authors incubated adrenal mitochondria to study the in vitro action of cortisol and testosterone on the transformation of corticosterone and 18-hydroxycorticosterone into aldosterone. The results show that cortisol at concentrations of 5 × 10⁻⁶ and 10⁻⁴ M inhibit the conversion of corticosterone into aldosterone by 23.6 to 90%; testosterone 5 × 10⁻⁵ and 10⁻⁴ M inhibit the reaction by 78.4 and 87.2%, respectively. The inhibition of the conversion of 18-hydroxycorticosterone into aldosterone is 12.5 to 91% by cortisol with concentrations ranging from 5 × 10⁻⁷ to 5 × 10⁻⁵ M and testosterone 5 × 10⁻⁵ and 10⁻⁴ M inhibits the reaction by 87.3 and 91%, respectively. Aldosterone (10⁻⁸ and 10⁻⁶ M) does not inhibit aldosterone biosynthesis from corticosterone or 18-hydroxycorticosterone. It thus appears that cortisol and testosterone have an effect on the aldosterone biosynthesis pathways in mitochondria. This action may be located at the binding site of the cytochrome P450 11β, which catalyzes all hydroxylation steps in the mineralocorticoid biosynthesis pathway. Because cortisol and testosterone may interfere with aldosterone biosynthesis, and since functional zonation is expected in adrenal carcinomas, the presence of these steroids in substantial amounts could explain the very low plasma aldosterone level usually observed, in adrenal carcinomas studies in our laboratory.     

SEC of mono-carboxymethyl cellulose (CMC) in a wide range of pH; Mark–Houwink constants

A method for determination of carboxymethyl cellulose (CMC) molecular weight (M_W) and chemical heterogeneity (degree of oxidation (DO)) using a bi-detector HPSEC (UV-detector online with refractometer) has been developed. It has been found that the use of 0.5 N NaOH or 0.4 M acetate buffer as the eluent ensures CMC separation according to M_W. It has been revealed that the universal calibration for the polyelectrolyte CMC and the neutral polymer dextran is valid under the conditions applied. The Mark–Houwink equations for CMC in 0.5 N NaOH and 0.4 M acetate buffer have been estimated to be [η]=5.37×10⁻⁴ M_W^0.73 and [η] =2.24×10⁻⁴ M_W^0.83 dl g⁻¹, respectively. The equation log K=1.64−4.00 ml g⁻¹ for CMC has been estimated. An approach for determining DO from adsorption at 290 or 313 nm has been developed.