A rapid gel electrophoretic chip for serum cholesterol determination

We present a rapid gel electrophoretic chip, composed of 2.5% (w/v) acrylamide and 1% (w/v) agarose gel, for serum cholesterol determination using a photo lithography technique. After optimizations, we determined the lipoprotein concentration of standard serum using a conventional enzyme method. The serum was diluted, stained and loaded for 15 min onto the chip. After loading, the intensities of low density lipoprotein cholesterol (LDL-C) and high density lipoprotein cholesterol (HDL-C) bands separated at the chip were estimated using an image analyzer. The intensities of these bands corresponded to concentrations obtained from a standard enzyme-based method. The detected LDL-C and HDL-C concentrations were linear up to 146 mg dL(-1) and 53 mg dL(-1) respectively. Finally, we carried out the cholesterol analysis using real biological samples obtained from nine volunteers using our electrophoretic chip. The LDL-C and HDL-C levels detected using our chip correlated well with the results obtained using the conventional enzyme-based method r(2) = 0.98 and r(2) = 0.86 for LDL-C and HDL-C, respectively. Although our sample size is small and confined only to health volunteers, we have demonstrated that this proof-of-concept gel electrophoretic chip can determine lipoproteins, simultaneously.     

Inhibition of cholesterol synthesis from mevalonate by aminotriazole treatment in vivo

3-Amino-1,2,4-triazole (aminotriazole) is an irreversible inhibitor of catalase which is a marker enzyme of peroxisomes. We studied the effect of aminotriazole treatment on biosyntheses of cholesterol and bile acid in vivo. When catalase activity of peroxisomes of rat liver was inhibited by aminotriazole treatment, bile acid content in the bile was significantly decreased to about 70% of the control, but that in the liver was not changed. Cholesterol content in the bile was significantly decreased to about 80% of the control, while in liver and serum the content was not significantly changed. When [2-14C]mevalonate was administered to rats, radioactivities of cholesterol in the liver, serum and bile were all drastically decreased by aminotriazole treatment, and an unidentified radioactive product was detected. Radioactivity of bile acid in the bile was also greatly decreased. In a similar experiment with [4-14C]cholesterol, aminotriazole treatment had no effect on the radioactivity of either cholesterol or bile acid in the liver, serum and bile. In this case, the unidentified product could not be detected. These results indicate that when catalase activity of liver peroxisomes is suppressed by aminotriazole treatment, biosynthesis of bile acid from exogenous cholesterol is not inhibited, but a step in the pathway of biosynthesis of endogenous cholesterol from mevalonate is inhibited.     

Kinetics of cholesterol oxidation by cholesterol oxidase

Kinetic studies of cholesterol oxidation catalyzed by soluble cholesterol oxidase fromBrevibacterium were conducted. The optimum temperature and pH were found to be 40–45°C and 7.0, respectively. A plot of initial reaction rate versus cholesterol concentration is sigmoidal in shape. Analysis of the data suggests that the reaction follows a concerted model and not a stepwise model.     

The role of cholesterol in UV light B-induced apoptosis

Modification of major lipid raft components, such as cholesterol and ceramide, plays a role in regulation of programmed cell death under various stimuli. However, the relationship between cholesterol level modification and the activation of apoptotic signaling cascades upon UVB light has not been established. In this report, we demonstrate that upon UVB irradiation cholesterol levels in membrane rafts of skin cells increase, which leads to Fas-receptor (Fas) aggregation in the rafts. Utilizing a continuous velocity floatation technique, we show that Fas accumulated in the lipid rafts of human melanoma M624 cells after UVB irradiation. The subsequent events of death-inducing signaling complex formation were also detected in the lipid raft fractions. Depletion of cholesterol by methyl-β-cyclodextrin reduces Fas aggregation, while overloading increases. Disruption of lipid rafts also prevents Fas death domain-associated protein (Daxx) from dissociating from Fas in the lipid rafts, which is accompanied with a reduced apoptotic, but increased nonapoptotic death of UVB-irradiated human keratinocytes, HaCaT cells. Results indicate that cholesterol located in the plasma membrane of skin cells is required for lipid raft domain formation and activation of UVB-induced apoptosis.     

Amperometric cholesterol biosensor based on immobilized cholesterol esterase and cholesterol oxidase on conducting polypyrrole films

Fabrication of an amperometric cholesterol biosensor by co-immobilization of cholesterol esterase (ChEt) and cholesterol oxidase (ChOx) onto conducting polypyrrole (PPY) films using electrochemical entrapment technique is described. Electrochemical polymerization was carried out using a two-electrode cell configuration at 0.8 V. Characterization of resulting amperometric biosensor for the estimation of cholesterol has been experimentally determined in terms of linear response range, optimum pH, applied potential, temperature, and shelf-life. These PPY/ChEt/ChOx electrodes can be used for cholesterol ester estimation from 1 to 8 mM and have shelf-life of about 4 weeks at 4 °C during which about 15 estimations of cholesterol ester could be made. The sensitivity of PPY/ChEt/ChOx electrode has been found to be 0.15 μA/mM and the apparent K_m value for this electrode is 9.8 mM. Conductivity of the polymer films found to be about 3×10⁻³ S/cm.     

Development of cholesterol biosensor based on immobilized cholesterol esterase and cholesterol oxidase on oxygen electrode for the determination of total cholesterol in food samples

The development of a cholesterol biosensor by co-immobilization of cholesterol esterase (ChEt) and cholesterol oxidase (ChOX) on oxygen electrode is described. The electrode consists of gold cathode and Ag/AgCl anode. The enzymes were immobilized by cross-linking with glutaraldehyde and Bovine Serum Albumin (BSA). The immobilized enzymatic membrane was attached to the tip of the electrode by a push cap system. The optimum pH and temperature of the sensor was determined, these are 6 and 25 degrees C respectively. The developed sensor was calibrated from 1-75 mg/dl of cholesterol palmiate and found linear in the range of 2-50 mg/dL. The calibration curve was drawn with V(i) (ppm/min)(initial velocity) vs different concentrations of cholesterol palmiate (mg/dL). The application of the sensor to determine the total cholesterol in different real food samples such as egg, meat was investigated. The immobilized enzymatic layer can be reused over 30 times and the stability of the enzymatic layer was studied up to 9 weeks.     

TOF‐SIMS study of cystine and cholesterol stones

Two different human stones, cystine and cholesterol from the kidney and gall bladder, were examined by time‐of‐flight secondary ion mass spectrometry using Ga⁺ primary ions as bombarding particles. The mass spectra of kidney stone were compared with those measured for the standard compounds, cystine and cysteine. Similar spectra were obtained for the stone and cystine. The most important identification was based on the existence of the protonated molecules [M + H]⁺ and deprotonated molecules [M‐H]^‐. The presence of cystine salt was also revealed in the stone through the sodiated cystine [M + Na]⁺ and the associated fragments, which might be due to the patient treatment history. In the gallstone, the deprotonated molecules [M‐H]⁺ of cholesterol along with relatively intense characteristic fragments [M‐OH]⁺ were detected. Copyright © 2012 John Wiley & Sons, Ltd.     

Immobilization of cholesterol esterase and cholesterol oxidase onto sol-gel films for application to cholesterol biosensor

Cholesterol oxidase (ChOx) and cholesterol esterase (ChEt) have been covalently immobilized onto tetraethylorthosilicate (TEOS) sol-gel films. The tetraethylorthosilicate sol-gel/ChEt/ChOx enzyme films thus prepared have been characterized using scanning electron microscopic (SEM), UV-vis spectroscopic, Fourier-transform-infrared (FTIR) spectroscopic and amperometric techniques, respectively. The results of photometric measurements carried out on tetraethylorthosilicate sol-gel/ChEt/ChOx reveal thermal stability up to 55 °C, response time as 180 s, linearity up to 780 mg dL⁻¹ (12 mM), shelf life of 1 month, detection limit of 12 mg dL⁻¹ and sensitivity as 5.4 × 10⁻⁵ Abs. mg⁻¹ dL⁻¹.     

Determination of cholesterol in sub-nanomolar quantities in biological fluids by high-performance liquid chromatography.

A highly sensitive method for the determination of cholesterol in biological fluids is described. Unsaponifiable lipids from rat serum and thoracic duct lymph chylomicron samples were treated with cholesterol oxidase. The product of the enzymatic reaction, delta 4-cholestenone, was analysed by normal-phase high-performance liquid chromatography (HPLC) using hexane-isopropanol (95:5, v/v) as a mobile phase and detected with a UV spectrophotometer at 240 nm. When the standard samples containing varying amounts of cholesterol (0.15-3 nmol) were treated with cholesterol oxidase and analysed by HPLC (injected amounts 0.09-1.8 nmol of cholesterol), the peak areas increased proportionally with the amounts of authentic cholesterol with a correlation coefficient of 0.996. The values in these biological fluids determined by the HPLC method were identical to those obtained by enzymatic-colorimetric or gas chromatographic methods. Moreover, the detection limit (0.09 nmol) of the present method (0.15 nmol are required for the sample preparation) is lower than those of conventional methods (approximately 30 nmol). Because of the excellent sensitivity and reproducibility, this method is well suited for the determination of cholesterol in biological fluids where cholesterol concentration is low.     

Amperometric determination of total cholesterol in serum with use of immobilized cholesterol esterase and cholesterol oxidase

Cholesterol esterase and cholesterol oxidase were immobilized on octyl-agarose gel, activated with cyanogen bromide and placed in a reactor. The sensor system for total cholesterol was assembled with the immobilized enzyme reactor, a hydrogen peroxide electrode and a peristaltic pump. Characteristics of the sensor system were investigated by using cholesterol palmitate as a standard substrate. A linear relationship was obtained between peak current and cholesterol palmitate concentration below 1000 mg dl^-1 (10.3 mM). A 10-μl sample could be assayed in 5 min. Total cholesterol in human serum was determined in the range 100–400 mg dl^-1. The standard deviation for the determination of 50 samples of 300 mg dl^-1 was 6 mg dl^-1 (2%). The system was used for 300 assays without loss of enzymatic activity. The correlation coefficient was 0.94 for 27 samples of human sera analyzed by the system proposed and by the conventional chemical method.     

Comparison of cholesterol oxidation product preparation methods for subsequent gas chromatographic analysis

An evaluation was made of the stability of cholesterol hydroperoxides (CHPs) under the analytical conditions and preparation methods commonly used for determination and quantification of cholesterol oxidation products (COPs). CHPs were prepared by photoxidation and separated by silica thin-layer chromatography. CHPs were individually collected by normal-phase liquid chromatography and then subjected either to reduction or to cold saponification. The corresponding hydroxyl derivatives were generated by reduction, whereas cold saponification gave rise predominantly to 7-ketocholesterol. In another test, silylated and non-silylated CHPs were separately injected into a gas chromatograph at 310 degrees C, collected, and re-injected into a gas chromatography-mass spectrometry system. The silylated CHPs were more stable than the non-silylated ones, giving 7-ketocholesterol, 7alpha- and 7beta-hydroxycholesterol as main degradation products. Two unknown degradation peaks were detected in both silylated and nonsilylated CHPs, having 384 as main m/z fragment. The study of their mass spectra led to the conclusion that peaks A and B correspond to 6alpha- and 6beta-hydroxycholesterol, respectively.     

Determination of cholesterol with a microporous membrane chemiluminescence cell with cholesterol oxidase in solution

A reagent solution, containing cholesterol oxidase buffered at pH 7, is contained in a pressurized reservoir and forced through a microporous membrane at 2–5 μl min^?1 into a stream flowing at 2–10 ml min^?1 which contains injected slugs of cholesterol as the analyte. The hydrogen peroxide produced then reacts with luminol in pH 9.0 Tris buffer, catalyzed by horseradish peroxidase, to produce chemiluminescence, the intensity of which is related to the cholesterol concentration. The working range is 0.4–40 mg dl^?1; precision is 1–4% over this range. The detection limit is 0.2 mg dl^?1 or 5 μM. Sample throughput is 60 h^?1, and only 0.01 unit of enzyme is consumed per sample. Blood serum samples may be analyzed for either free or total cholesterol by using standard addition and pre-treatment with Somogyi reagents for removal of reducing species.     

Bionanoparticles of amphiphilic copolymers polyacrylate bearing cholesterol and ascorbate for drug delivery

In this study, a series of amphiphilic polymers with poly(ascorbyl acrylate) (PAAA) as hydrophilic blocks and polyacrylate bearing side-chain cholesteryl mesogens (PCholDEGA) as hydrophobic blocks were prepared using a combination of four-step reactions consisting of two consecutive reversible addition-fragmentation chain transfer (RAFT), desulfurization, and hydrogenolysis under normal pressure. The thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) as well as wide-angle X-ray diffraction (WAXD) studies showed that the copolymers with PCholDEGA as major block had relatively high stability and clear isotropization temperature (T(i)). Small-angle X-ray diffraction (SAXD) investigation exhibited that the copolymers had bilayer smectic A structure. Their self-assembly behavior was monitored by turbidity change using UV-vis spectrometer, and the morphology and size of the nanoparticles via self-assembly were detected using transmission electron microscopy (TEM) and dynamic light scattering (DLS). The entrapment efficiency and loading capacity of these amphiphilic copolymers were investigated using nile red and drug molecule Ibuprofen. These polymeric micelles with PAAA shell extending into the aqueous solution and strong hydrophobic PCholDEGA core have potential abilities to act as promising nanovehicles with high loading and targeting delivery.     

Covalent immobilization of cholesterol esterase and cholesterol oxidase on polyaniline films for application to cholesterol biosensor

Cholesterol esterase (ChEt) and cholesterol oxidase (ChOx) have been covalently immobilized on electrochemically prepared polyaniline (PANI) films. These PANI/ChEt/ChOx enzyme films have been characterized using UV-visible, Fourier transform infrared (FTIR) spectroscopy and scanning electron microscopy (SEM). Electrochemical behavior of these films has been studied using cyclic voltammetry (CV) and amperometric techniques, respectively. The PANI/ChEt/ChOx enzyme films show broad oxidation peak from 0.2 to 0.5 V. These PANI/ChEt/ChOx biosensing electrodes have a response time of about 40s, linearity from 50 to 500 mg/dl of cholesterol oleate concentration. These PANI/ChEt/ChOx films are thermally stable up to 46 degrees C. This polyaniline based cholesterol biosensor has optimum pH in the range of 6.5-7.5, sensitivity as 7.5x10(-4) nA/mg dl and a lifetime of about 6 weeks.     

Nonsteroidal benzophenone-containing analogues of cholesterol

The four benzophenones, 10-13, containing the natural side chain of cholesterol (1) have been synthesized to explore whether the tetracyclic nucleus of 1 is essential for its biochemical properties. The syntheses of analogues 10, 11, and 13 feature efficient introduction of the alkyl side chain by Suzuki coupling. Preliminary biochemical evaluation of 10 and 12 suggests that the sterol tetracyclic nucleus is not required for biological compatibility with 1.     

Steady-state oxidation of cholesterol catalyzed by cholesterol oxidase in lipid bilayer membranes on platinum electrodes

Cholesterol oxidase is immobilized in electrode-supported lipid bilayer membranes. Platinum electrodes are initially modified with a self-assembled monolayer of thiolipid. A vesicle fusion method is used to deposit an outer leaflet of phospholipids onto the thiolipid monolayer forming a thiolipid/lipid bilayer membrane on the electrode surface. Cholesterol oxidase spontaneously inserts into the electrode-supported lipid bilayer membrane from solution and is consequently immobilized to the electrode surface. Cholesterol partitions into the membrane from buffer solutions containing cyclodextrin. Cholesterol oxidase catalyzes the oxidation of cholesterol by molecular oxygen, forming hydrogen peroxide as a product. Amperometric detection of hydrogen peroxide for continuous solution flow experiments are presented, where flow was alternated between cholesterol solution and buffer containing no cholesterol. Steady-state anodic currents were observed during exposures of cholesterol solutions ranging in concentration from 10 to 1000 μM. These data are consistent with the Michaelis-Menten kinetic model for oxidation of cholesterol as catalyzed by cholesterol oxidase immobilized in the lipid bilayer membrane. The cholesterol detection limit is below 1 μM for cholesterol solution prepared in buffered cyclodextrin. The response of the electrodes to low density lipoprotein solutions is increased upon addition of cyclodextrin. Evidence for adsorption of low density lipoprotein to the electrode surface is presented.     

Competitive solubilization of cholesterol and phytosterols in nonionic microemulsions studied by pulse gradient spin-echo NMR

The actual mechanism of cholesterol reduction by phytosterols is yet to be explored. One hypothesis states that cholesterol and phytosterols compete on the solubilization locus within gastric bile salt micelles. In this study competitive solubilization within microemulsions as vehicles for dietary intake of cholesterol and phytosterols was studied by pulse gradient spin-echo nuclear magnetic resonance. The loaded microemulsions undergo phase transitions as a function of dilution, the type of solubilized sterol, and the weight ratio of the cosolubilized sterols. Microemulsions containing 10-20 wt% of aqueous phase, show similar diffusivity of the oil and aqueous phases in all examined systems (excluding PS-loaded one) reflecting the minor influence of these solubilizates on the structure of the inner and the outer phases. The closeness of these structures enables the mobility of water molecules between them. Upon further dilution (>20 wt% aqueous phase), significant differences in decrease rate of the oil and increase of the water phases mobilities (occurring upon inversion), were detected within the studied systems. It was concluded that the solubilized sterols influence the structural transitions based on their location within the structures and their competitive solubilization. The phytosterols solubilized mostly in the continuous oil phase and between the surfactant tails. Cholesterol is solubilized in the vicinity of the surfactant headgroups and affects the surface curvature. In mixtures of cholesterol and phytosterols, structural changes are dictated mostly by the presence of the cholesterol.     

Significance of cholesterol methyl groups

Cholesterol is an indispensable molecule in mammalian cell membranes. To truly understand its role in the functions of membranes, it is essential to unravel cholesterol's structure-function relationship determined by underlying molecular interactions. For this purpose, we elaborate on this issue by considering the previously proposed idea that cholesterol's effects on a number of physical properties of membranes have been optimized during the evolution by removal of its excess methyl groups from the alpha-face of cholesterol, thus "smoothening" the structure. Consequently, the methyl groups still attached to cholesterol are one of the most intriguing structural features of the molecule. An obvious question arises: Why do these methyl groups still exist, and could cholesterol properties be further optimized by their removal? Because of the nature of the biosynthetic pathways of cholesterol, and the evidence of decreased interactions between sterols and lipid acyl chains when methyl groups are present, it seems plausible that removal of the methyl groups might indeed lead to stronger ordering and condensing effects of the cholesterol molecule. Atomic-scale molecular dynamics simulations of numerous modified sterols embedded in saturated lipid bilayers demonstrate, however, that the issue is more subtle. The analysis reveals a complex interplay between the lipid acyl chains and the structural details of cholesterol. Changes in cholesterol structure typically do not improve its performance in terms of promoting membrane order. This view is substantiated by a detailed analysis of the simulation data. In particular, it highlights the importance of the methyl group C18 for cholesterol properties. The C18 group resides between the third and fourth ring of cholesterol on its "rough" beta-side, and the results provide compelling evidence that C18 is crucial for the proper orientation of the sterol. More generally, the data provide insight into the role of the methyl groups of cholesterol.     

Determination of total cholesterol in serum by high-performance liquid chromatography with electrochemical detection

A simple and sensitive HPLC method that does not require derivatization for determining cholesterol has been developed. Investigation of voltammetric behavior of cholesterol showed that cholesterol could be oxidized on a glassy carbon electrode in non-aqueous solvents. This was applied to the development of a method by HPLC with electrochemical detection (HPLC-ED). The HPLC-ED was optimized using the separation of cholesterol and oxysterols including 26-hydroxycholesterol and 24S-hydroxycholesterol. The separation was carried out with a Develosil C30-UG-3 column; acetonitrile-2-propanol (9:1, v/v) containing 50mM LiClO(4) as a mobile phase; and an applied potential at 1.9V versus Ag/AgCl. The current peak height was linearly related to the amount of cholesterol injected from 0.5-100 microM (r>0.999). The detection limit (S/N=3) of cholesterol was 0.36 microM (1.8 pmol). Cholesterol at 100 microM was directly detected with a relative standard deviation (RSD) of less than 1.0% (n=8). Total cholesterol and free cholesterol in control human serum were determined by the present method with the recovery of more than 90% and the RSD (n=6) of less than 3.0%.     

Sensitive high-density lipoprotein cholesterol assay

A procedure has been described in which a currently acceptable simple procedure for the precipitation of low-density and very-low-density lipoproteins is paired with a modified sensitized enzyme reagent system containing sodium 2-hydroxy-3,5-dichlorobenzenesulfonate for the determination of cholesterol by equilibrium reaction with the high density lipoprotein fraction of serum. The resulting increase in absorbances by using the auxochromic derivative of phenol is some four to five times that of the phenol system and therefore makes it possible to discriminate with more assurance within the critical ±50 mg/liter concentration difference. Factors such as comparative calibration slopes for the phenol reaction and its auxochrome substitute, the water displacement error, the presence of bilirubin, and the contamination by LDL-VLDL precipitating reagents are considered and discussed.