Function Of Cholesterol Concentration Research Articles

Cholesterol stabilization of phospholipid vesicles against bile-induced solubilization.

Sphingomyelin (SM) and cholesterol complex to form functional liquid-ordered (Lo) domains. It has been suggested that the detergent resistance of these domains plays a key role during gastrointestinal digestion of the milk fat globule membrane (MFGM), which is rich in both SM and cholesterol. Small-angle X-ray scattering was employed to determine the structural alterations that occur when milk sphingomyelin (MSM)/cholesterol, egg sphingomyelin (ESM)/cholesterol, soy phosphatidylcholine (SPC)/cholesterol, and milk fat globule membrane (MFGM) phospholipid/cholesterol model bilayer systems were incubated with bovine bile under physiological conditions. The persistence of diffraction peaks was indicative of multilamellar vesicles of MSM with cholesterol concentrations >20 % mol, and also for ESM with or without cholesterol. The complexation of ESM with cholesterol is therefore capable of inhibiting the resulting vesicles from disruption by bile at lower cholesterol concentrations than MSM/cholesterol. After subtraction of background scattering by large aggregates in the bile, a Guinier fitting was used to determine changes in the radii of gyration (Rgs) over time for the biliary mixed micelles after mixing the vesicle dispersions with bile. Swelling of the micelles by phospholipid solubilization from vesicles was a function of cholesterol concentration, with less swelling of the micelles occurring as the cholesterol concentration was increased. With 40% mol cholesterol, the Rgs of the bile micelles mixed with MSM/cholesterol, ESM/cholesterol, and MFGM phospholipid/cholesterol were equal to the control (PIPES buffer + bovine bile), indicating negligible swelling of the biliary mixed micelles.

Effects of cholesterol and temperature on structural properties and dynamic behavior of niosome bilayers with melatonin Inclusion: A Coarse-Grained simulation study

The effects of cholesterol and temperature on the physical properties of niosome bilayers with and without melatonin inclusion have been investigated using coarse-grained (CG) simulations. CG models of Span 60 and melatonin have been newly developed and then validated by comparing them with all atomistic (AA) simulation results. The CG simulations of niosome bilayers with melatonin inclusion at various cholesterol concentrations (0–70 mol% Chol) were carried out at room temperature, 298 K. A niosome bilayer with melatonin inclusion at a 50 mol% Chol level was simulated for various temperatures (300 K–340 K). The bilayer structure and dynamic properties of these systems were examined as a function of cholesterol concentration and temperature. Melatonin molecules in the niosome bilayer preferentially diffuse into the water phase and the niosome bilayer develops a more disordered structure as temperature increases. Melatonin showed two possible orientations. The first is parallel to the bilayer surface and the second is tilted at an angle to the bilayer normal. Maximal efficiency of melatonin entrapment of a niosome bilayer is found at a 50 mol% Chol cholesterol concentration when T = 298 K and decreases as temperature increases. A niosome bilayer with an equal ratio of Span 60 to cholesterol shows high physical stability and greater efficiency of melatonin entrapment over a wide temperature range (298 K–310 K). This excellent property can be suggested for designing and developing niosomes to improve drug delivery into cells for therapeutic purposes.

Mechanism by Which Cholesterol Induces Sphingomyelin Conformational Changes at an Air/Water Interface.

This work investigates the interactions in cholesterol and sphingomyelin monolayers at the molecular level by high-resolution broadband sum frequency generation vibrational spectroscopy (HR-BB-SFG-VS). The SFG spectra of natural egg sphingomyelin (ESM) as a function of cholesterol concentration are obtained at an air/water interface under different polarization combinations. The analysis of the spectra shows that cholesterol can induce sphingomyelin conformational changes at an air/water interface. The mechanism is proposed. When cholesterol is inserted into the ESM monolayer, the inherent intramolecular hydrogen bonds between the phosphate moiety and 3OH in the sphingosine backbones are destroyed. During this process, the sphingosine backbones become more ordered, while the conformation of the N-linked long acid chain remains unaltered. The OH of the cholesterol head group can bind to the -PO-2 of the ESM molecule, and the orientation of the -PO-2 in the head groups changes to be more parallel to the interface.

Axonal plasma membrane-mediated toxicity of cholesterol in Alzheimer's disease: A microsecond molecular dynamics study

Alzheimer's disease is increasingly being recognized as an immune-mediated disease of brain. Since physiological brain health and brain immune function is dependent upon homeostatic neuronal membrane structure and function, alterations in membrane lipid biochemistry may predispose to disease. Brain is rich in cholesterol, and cholesterol metabolism dysfunction is a known risk factor for AD. Employing extensive microsecond all-atom molecular dynamics simulations, we investigated the properties of model neuronal membranes as a function of cholesterol concentration; phospholipid and phospholipid/cholesterol bilayers were also simulated to compare against available experimental data. Increased cholesterol concentrations compact and stiffen the lipid membrane, reducing permeability while modulating local water densities in the peri-membranous environment. Conversely, lower cholesterol mole fraction yields membranes with increased molecular disorder, enhanced fluidity, higher molecular tilting, and augmented interdigitation between bilayer leaflet lipids. Our findings provide a molecular insight on effect of cholesterol composition on various biochemical processes occurring at neuronal axon plasma membrane. These calculations also endeavor to establish a membrane-based link between cholesterol as an AD risk factor and possible AD pathology.

Fabrication of Au/ZnO/MWCNTs electrode and its characterization for electrochemical cholesterol biosensor

In this work, a new sensitive enzyme-based electrode for electrochemical cholesterol biosensor was fabricated based on a nanocomposite of Au nanoparticles, ZnO nanoparticles and multi-wall carbon nanotubes (Au/ZnO/MWCNTs). The nanocomposite was prepared by sol–gel method and deposited on FTO substrate by dip coating, followed by cholesterol oxidase (ChOx) enzyme immobilized (ChOx/Au/ZnO/MWCNTs). Structural properties and morphology of the nanocomposite have been studied using X-ray diffraction (XRD) and Field emission scanning electron microscopy (FESEM). The sample was subjected to Fourier transform infrared spectroscopy (FTIR) to determine functional groups. Electrochemical behavior of the electrode was studied by cyclic voltammetry (CV) and differential pulse voltammetry (DPV) techniques as a function of cholesterol concentration. Electrochemical impedance spectroscopy (EIS) was also considered to study of surface modified electrodes. The ChOx/Au/ZnO/MWCNTs electrode has been found to have enhanced electron transfer and display excellent analytical linear performances. The fabricated electrode exhibited low detection limit (0.1 μM), high sensitivity (25.89 μA/μM) evaluated from DPV data in the detection range of 0.1–100 µM and high selectivity in the determination of cholesterol over glucose and uric acid. The application of the ChOx/Au/ZnO/MWCNTs electrode in detection of cholesterol in human serum was also confirmed.

Colorimetric point-of-care detection of cholesterol using chitosan nanofibers

Recent developments in biosensor-related research have provided economic and highly sensitive biosensors for numerous biological analytes. Several strategies have evolved for developing a sensitive biosensor for cholesterol detection. In this study, we have developed a point-of-care, chitosan nanofiber-based cholesterol biosensor, involving colorimetric detection of the analyte. Chitosan nanofibers, fabricated using electrospinning, were utilized for immobilizing cholesterol oxidase and peroxidase enzymes. A uniform and bead-free chitosan nanofibers were obtained using chitosan and polyvinyl alcohol (Cs: PVA), at the ratio of 0.7:1 w/w, in a solvent system containing 10%v/v of methanol in 63% acetic acid. The nanofibers were characterized using scanning electron microscopy (SEM), which revealed the formation of smooth fibers, with an average diameter of 60–90 nm. The fabricated nanofibers offered a greater surface area for immobilizing high quantities of enzymes and demonstrated the potential to be developed into a strip-based intervention, based on a simple, visual detection system. A colorimetric detection method was developed using the chromogenic substrate, 3,3́,5,5́-tetramethylbenzidine hydrochloride. Analysis by UV–vis spectrophotometry demonstrated a linear increase in the absorbance, with increasing concentrations of cholesterol. The intensity of color change, as a function of cholesterol concentration was used for developing a color gradient scale for the investigated biosensor.

Cholesterol concentration effect on the bilayer properties and phase formation of niosome bilayers: A molecular dynamics simulation study

Niosome bilayers formed by the mixture of sorbitan monostearate (Span60) and cholesterol molecules with different cholesterol concentrations (0 to 70mol% Chol) were studied by molecular dynamics simulations at the temperature of 298K and the pressure of 1bar. Structural properties and dynamics of niosome bilayers were characterized as a function of cholesterol concentration. We found that addition of cholesterol in the niosome bilayer significantly changes the molecular structure and stability. Increasing of cholesterol concentrations causes the area per molecule to increase and bilayer thickness to decrease, suggesting that the niosome bilayer is expanded from the gel to the liquid ordered phase. This contrasts with DOPC or DMPC lipid bilayers which show a condensation effect with increasing cholesterol content. The calculated isothermal area compressibility of the niosome bilayer shows a remarkable increase at moderate concentration (40–50mol% Chol) and slight change at the higher concentrations. This suggests that the preparation of niosome with the higher cholesterol concentrations does not significantly improve the bilayer compressibility. With increasing cholesterol concentration the Span60 tails gradually adopt a more conformation while the orientation and dynamics of the Span60 groups are rather unaffected. The hydrogen bond interactions of the Span60/chol/water and Span60/water system increased with increasing cholesterol concentration, leading to improved bilayer stability.

Cross-Linking-Mass Spectrometry Studies of Cholesterol Interactions with Human α1 Glycine Receptor

The glycine receptor (GlyR) belongs to a superfamily of pentameric ligand-gated ion channels (pLGICs) that mediate fast neurotransmission. GlyR typically modulates inhibitory transmission by antagonizing membrane depolarization through anion influx. Allosteric interactions between the receptor and its lipid surroundings affect receptor function, and cholesterol is essential for pLGIC activity. Cholesterol at compositions below ∼33 mol percent has been shown to have negligible chemical activity, suggesting that specific interactions between membrane proteins and cholesterol become significant only at concentrations above this stoichiometric threshold. Human α1 GlyR was purified from baculovirus infected insect cells and reconstituted in unilamellar vesicles at cholesterol/lipid ratios above and below the cholesterol activity threshold with equivalent aliquots of azi-cholesterol, a photoactivatable nonspecific cross-linker. After photoactivation, cross-linked cholesterol-GlyR was trypsinized and mass fingerprinted. Mass shifted peptides containing cholesterol were identified by electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-Q-TOF MS), and sites of direct covalent attachment to peptides were refined by targeted MS/MS. Differential patterns of dozens of cholesterol-GlyR cross-links were identified in these comparative studies, with sites of cross-linking found primarily in the fourth transmembrane helix and extramembranous connecting loops and mapping the lipid-accessible surface of the receptor. Unique cross-linking observed in both reduced and elevated cholesterol composition suggests different apo-state structural conformations of GlyR as a function of cholesterol concentration and, in the latter studies, identified potential specific binding sites for cholesterol in the receptor.

Concentration-Dependent Differential State Crosslinking of azi-Cholesterol with Human Alpha 1 Glycine Receptor using Mass Spectrometry

The glycine receptor (GlyR) belongs to a superfamily of pentameric ligand-gated ion channels (pLGICs) that mediate fast neurotransmission. GlyR typically modulates inhibitory transmission by antagonizing membrane depolarization through anion influx. Allosteric interactions between the receptor and its lipid surroundings affect receptor function, and cholesterol is essential for pLGIC activity. Cholesterol at compositions below ∼33 mol% has negligible chemical activity. Taken together, the findings suggest that specific interactions between membrane proteins and cholesterol become significant only at concentrations above this stoichiometric threshold, while interactions at lower cholesterol levels will be primarily indicative of non-specific accessibility to the surrounding bilayer. Human α1 GlyR was purified from baculovirus infected insect cells and reconstituted in unilamellar vesicles at cholesterol:lipid ratios above and below the cholesterol activity threshold with equivalent aliquots of azi-cholesterol. State-dependent crosslinking of the resting and desensitized states was achieved by the absence and inclusion of saturating concentrations of glycine (10 mM), respectively. After photoactivation, covalently crosslinked cholesterol-GlyR were trypsinized and mass fingerprinted. Mass shifted peptides containing cholesterol were identified by ESI-Q-TOF MS, and sites of cholesterol crosslinks in peptides were refined by targeted MS/MS. Differential sites of crosslinking as a function of cholesterol concentration in both states were identified, with crosslinks found primarily in transmembrane helices and extramembranous loops of GlyR. Specific concentration-dependent sites of interaction between cholesterol and GlyR in both states were identified, highlighting sites of non-specific interactions of the receptor with its surrounding lipids, as well as potential specific cholesterol binding sites in GlyR. Differential cholesterol crosslinking patterns between resting and desensitized states were observed, highlighting state-dependent differences in GlyR lipid accessibility.

Cholesterol Decreases the Size and the Mechanical Resistance to Rupture of Sphingomyelin Rich Domains, in Lipid Bilayers Studied as a Model of the Milk Fat Globule Membrane

Sphingomyelin-rich microdomains have been observed in the biological membrane surrounding milk fat globules (MFGM). The role played by cholesterol in these domains and in the physical properties and functions of the MFGM remains poorly understood. The objective of this work was therefore to investigate the phase state, topography, and mechanical properties of MFGM polar lipid bilayers as a function of cholesterol concentration, by combining X-ray diffraction, atomic force microscopy imaging, and force spectroscopy. At room temperature, i.e. below the phase transition temperature of the MFGM polar lipids, the bilayers showed the formation of sphingomyelin-rich domains in the solid ordered (so) phase that protruded about 1 nm above the liquid disordered (ld) phase. These so phase domains have a higher mechanical resistance to rupture than the ld phase (30 nN versus 15 nN). Addition of cholesterol in the MFGM polar lipid bilayers (i) induced the formation of liquid ordered (lo) phase for up to 27 mol % in the bilayers, (ii) decreased the height difference between the thicker ordered domains and the surrounding ld phase, (iii) promoted the formation of small sized domains, and (iv) decreased the mechanical resistance to rupture of the sphingomyelin-rich domains down to ∼5 nN. The biological and functional relevance of the lo phase cholesterol/sphingomyelin-rich domains in the membrane surrounding fat globules in milk remains to be elucidated. This study brought new insight about the functional role of cholesterol in milk polar lipid ingredients, which can be used in the preparation of food emulsions, e.g. infant milk formulas.

Crosslinking/MS Studies of Cholesterol Interactions with Human α1 Glycine Receptor

The glycine receptor (GlyR) belongs to a superfamily of pentameric ligand-gated ion channels (pLGICs) known to mediate fast neurotransmission throughout the brain and spinal cord. GlyR typically modulates inhibitory transmission by antagonizing membrane depolarization through anion influx. Allosteric interactions between the receptor and its lipid surroundings affect receptor function, and cholesterol is essential for pLGIC activity. However, cholesterol at compositions below approximately 33 mol percent is reported to have negligible chemical activity. Taken together, the findings suggest that specific interactions between membrane proteins and cholesterol may become significant only at concentrations above this stoichiometric threshold, while interactions at lower cholesterol levels are primarily indicative of non-specific accessibility to the surrounding bilayer. Human α1 GlyR was purified from baculovirus infected insect cells and reconstituted in unilamellar vesicles at cholesterol:lipid ratios above and below the cholesterol activity threshold. Equivalent aliquots of azi-cholesterol, a photoactivatable cholesterol analog, were incorporated into reconstituted vesicles of varying cholesterol/lipid ratios. After photoactivation, covalently crosslinked cholesterol-GlyR were trypsin digested and mass fingerprinted. Mass shifted peptides containing cholesterol were identified by ESI-Q-TOF MS, and sites of cholesterol crosslinks in peptides were identified by targeted MS/MS. Differential sites of crosslinking as a function of cholesterol concentration were identified, with crosslinks found primarily in TM4 and extramembranous loops of GlyR. Concentration-dependent sites of interaction between cholesterol and GlyR were identified, highlighting sites of non-specific interactions of the receptor with its surrounding lipids, as well as potential specific cholesterol binding sites in GlyR.

Molecularly "wired" cholesterol oxidase for biosensing.

The influence of several factors on the activity of cholesterol oxidase (ChOx) transiently exposed to a room temperature ionic liquid (RTIL) was studied. Presence of flavin adenine dinucleotide (FAD, prosthetic group of ChOx) during exposure to RTIL makes the procedure enzyme-friendly, while the use of RTIL (green reagent) makes it environmentally-friendly. Following exposure to RTIL and its subsequent removal, FAD becomes part of the molecular structure of the refolded protein (a molecular "wire"). This makes the procedure used here a molecular one. The factors studied were: FAD presence in RTIL during modification, water presence during exposure to RTIL, and ratio FAD:RTIL during "wiring". Performance parameters monitored were: enzyme activity before and after "wiring" (expressed as (dA/dt)/mg enzyme, and measured spectrophotometrically), peak current in an amperometric biosensor for cholesterol detection, and linearity of the biosensor response depending on cholesterol concentration. After RTIL removal, the modified enzyme (ME) retained a high percentage of the added FAD, which supplemented that of the native enzyme (functioning as a "wire" and enhancing electron transfer kinetics), and a fraction of the initial activity. Used in an amperometric biosensor, ME showed catalytic activity, linear behavior as a function of cholesterol concentration, and stability.

Examining the Effects of Cholesterol: Laurdan and Patman see it Differently

The liquid-ordered phase induced by cholesterol in phosphatidylcholine bilayers can be detected with Laurdan fluorescence by an increase in the associated Generalized Polarization (GP) value. This increase in GP is usually interpreted as a reduction in the access of water molecules to the bilayer at depths approaching the phospholipid glycerol backbones. Comparisons of Laurdan fluorescence over a broad range of temperatures with various saturated and unsaturated phosphatidylcholines demonstrated that cholesterol has little effect on GP at temperatures below the melting point of the pure lipid (tm). However, above tm, increasing cholesterol concentrations monotonically raised the value of GP. The resulting GP increments generally did not vary with temperature above tm. In contrast, the observations were more complex with Patman, a charged derivative of Laurdan. First, cholesterol raised the value of Patman GP at temperatures below tm, and its effect as a function of concentration was bimodal. Second, although cholesterol increased Patman GP above tm (similar to Laurdan), the effect size was smaller and was bimodal as a function of cholesterol concentration. Finally, the GP values at high versus low cholesterol concentration converged as temperature was raised well beyond tm. In some cases, such as with unsaturated lipids ≥54°C above tm, this convergence of Patman GP values reached a cross-over point; Patman GP was reduced by the presence of cholesterol beyond this point. These results suggest that the charge associated with Patman, or possibly the slightly deeper location of the probe in the bilayer, provides a means for identifying additional effects of cholesterol on the membrane that are not visible with Laurdan.

The Influence of Cholesterol on Fast Dynamics Inside of Vesicle and Planar Phospholipid Bilayers Measured with 2D IR Spectroscopy.

Phospholipid bilayers are frequently used as models for cell membranes. Here the influence of cholesterol on the structural dynamics in the interior of 1,2-dilauroyl-sn-glycero-3-phosphocholine (dilauroylphosphatidylcholine, DLPC) vesicles and DLPC planar bilayers are investigated as a function of cholesterol concentration. 2D IR vibrational echo spectroscopy was performed on the antisymmetric CO stretch of the vibrational probe molecule tungsten hexacarbonyl, which is located in the interior alkyl regions of the bilayers. The 2D IR experiments measure spectral diffusion, which is caused by the structural fluctuations of the bilayers. The 2D IR measurements show that the bilayer interior alkyl region dynamics occur on time scales ranging from a few picoseconds to many tens of picoseconds. These are the time scales of the bilayers' structural dynamics, which act as the dynamic solvent bath for chemical processes of membrane biomolecules. The results suggest that at least a significant fraction of the dynamics arise from density fluctuations. Samples are studied in which the cholesterol concentration is varied from 0% to 40% in both the vesicles (72 nm diameter) and fully hydrated planar bilayers in the form of aligned multibilayers. At all cholesterol concentrations, the structural dynamics are faster in the curved vesicle bilayers than in the planar bilayers. As the cholesterol concentration is increased, at a certain concentration there is a sudden change in the dynamics, that is, the dynamics abruptly slow down. However, this change occurs at a lower concentration in the vesicles (between 10% and 15% cholesterol) than in the planar bilayers (between 25% and 30% cholesterol). The sudden change in the dynamics, in addition to other IR observables, indicates a structural transition. However, the results show that the cholesterol concentration at which the transition occurs is influenced by the curvature of the bilayers.

Investigating the impact of cholesterol on magnetically aligned sphingomyelin/cholesterol multilamellar vesicles using static 31P NMR

The effect of cholesterol (5-40mol%) on the magnetic induced orientation of sphingomyelin/cholesterol multilamellar vesicles (MLVs) was examined using static solid state (31)P NMR spectroscopy. The orientation was modeled assuming an ellipsoidal deformation of the vesicles and was monitored as a function of cholesterol concentration and temperature. In addition, the static (31)P chemical shift anisotropy (CSA) was used to assess the motional and dynamical changes occurring in the bilayer are reported. An exploration of the factors determining the magnetic orientation in sphingomyelin/cholesterol bilayers from the gel (s(o)) to liquid crystalline (or liquid-ordered, l(o)) phases is presented and discussed.

Probing Cholesterol Dependent Lipid Properties Using Molecular Dynamics Simulations of BODIPY-PC in Explicit DMPC and DPPC Membranes

Dunn and co-workers have used polarized total internal reflection fluorescence microscopy to measure the tilt angle of a single molecule (BODIPY-PC) relative to the membrane normal to characterize the membrane properties. Recently, they found that increasing quantities of cholesterol in monolayer and bilayer membrane systems leads to a much smaller tilt angle of the BODIPY-PC molecule. To better understand the influence of cholesterol on membrane properties and its relation to BODIPY-PC tilting, we have performed molecular dynamics (MD) simulations of BODIPY-PC in explicit dipalmitoylphosphatidylcholine (DPPC) and dimyristoylphosphocholine (DMPC) monolayer and bilayer membrane systems with systematically increasing concentrations of cholesterol (0, 5, 10, 15, 20, 33, 50%). After 4 ns of equilibration, each system has been simulated for another 16 ns. Preliminary results indicate that the BODIPY-PC tilt angle decreases as a function of increasing cholesterol concentration until 10% cholesterol after which there appears to be cholesterol saturation and the rigidifying affect ceases. Other points of interest include confirming the applicability of monolayer data for bilayer systems and explaining any quantifiable differences between the DPPC and DMPC membrane systems. We will present the orientation (azimuthal and rotational angle distribution) of BODIPY-PC, the BODIPY-PC tilting energetics, various membrane properties, and the spatial distribution of the cholesterol as a function of cholesterol concentration.

Nanostructured zinc oxide platform for cholesterol sensor

Nanostructured zinc oxide (nano-ZnO) film has been fabricated onto indium tin oxide (ITO) containing preferred (002) plane and 10 nm crystallite size using sol-gel technique for immobilization of cholesterol oxidase (ChOx). Electrochemical response of ChOx/nano-ZnO/ITO bioelectrode determined as a function of cholesterol concentration using cyclic voltammetry technique reveals improved detection range (5–400 mg/dl), low detection limit (0.5 mg/dl), fast response time (10 s), sensitivity (0.059 μA/mg dl−1 cm−2), and low value (0.98 mg/dl) of Michaelis–Menten constant (Km). It is shown that nano-ZnO film provides better environment and enhanced electron transfer between ChOx and electrode.

Solution synchrotron x-ray diffraction reveals structural details of lipid domains in ternary mixtures

The influence of cholesterol on lipid bilayer structure is significant and the effect of cholesterol on lipid sorting and phase separation in lipid-raft-forming model membrane systems has been well investigated by microscopy methods on giant vesicles. An important consideration however is the influence of fluorescence illumination on the phase state of these lipids and this effect must be carefully minimized. In this paper, we show that synchrotron x-ray scattering on solution lipid mixtures is an effective alternative technique for the identification and characterization of the l_{o} (liquid ordered) and l_{d} (liquid disordered) phases. The high intensity of synchrotron x rays allows the observation of up to 5 orders of diffraction from the l_{o} phase, whereas only two are clearly visible when the l_{d} phase alone is present. This data can be collected in approximately 1 min/sample , allowing rapid generation of phase data. In this paper, we measure the lamellar spacing in both the liquid-ordered and liquid-disordered phases simultaneously, as a function of cholesterol concentration in two different ternary mixtures. We also observe evidence of a third gel-phaselike population at 10-12 mol % cholesterol and determine the thickness of the bilayer for this phase. Importantly we are able to look at phase coexistence in the membrane independent of photoeffects.

Poly (pyrrole-co-N-methyl pyrrole) for application to cholesterol sensor

Cholesterol oxidase (ChOx) has been electrochemically entrapped onto p-toluene sulphonate (PTS) doped poly (pyrrole-co-N-methyl pyrrole) (1:1) on indium-tin-oxide (ITO) glass plates. This ChOx-copolymer-PTS/ITO bioelectrode has been characterized using cyclic voltammetry (CV), Fourier transform infrared (FTIR) spectroscopy, UV-visible spectroscopy, scanning electron microscopy (SEM) techniques. The cholesterol bioelectrode shows response time of 19 s, linearity from 50 to 400 mg/dL as a function of cholesterol concentration. It exhibits optimum pH range between 6.5 and 7.5, shelf-life of up to 6 weeks at 4 °C and shows almost undisturbed response in presence of interferents like ascorbic acid, uric acid and glucose.

Amplification of Diacylglycerol Activation of Protein Kinase C by Cholesterol

The combined effects of cholesterol, a major cell membrane component, and the lipid second messenger diacylglycerol on the activity of protein kinase C (PK-C) and the structure of phosphatidylcholine/phosphatidylserine bilayers were investigated using specific PK-C assays and 2H NMR. Whereas the classical activation of PK-C was observed as an effect of diacylglycerol, in the absence of this second messenger, cholesterol did not affect PK-C activity. A novel effect of amplified PK-C activation was observed in the presence of both cholesterol and diacylglycerol concentrations within the physiological range of each of these components. 2H NMR results suggest that this phenomenon is due to cholesterol- and diacylglycerol-induced increased propensity of the lipids to adopt nonbilayer phases, effectively destabilizing the bilayer structure. The magnitude of the effect was a function of cholesterol concentration, implying that laterally separated cell membrane domains with distinct cholesterol concentrations have the capacity to differ in their sensitivity to extracellular stimuli.