Amperometric assays of total and free cholesterols in serum by the combined use of immobilized cholesterol esterase and cholesterol oxidase reactors and peroxidase electrode in a flow injection system

A flow injection system for assays of total cholesterol and free cholesterol was described. The total cholesterol assay system included an amperometric peroxidase electrode to measure hexacyanoferrate(III) converted from hydrogen peroxide, which was generated by injecting a 2-microliter sample into the packed-bed reactors of immobilized cholesterol esterase and cholesterol oxidase covalently bound to silica. The free cholesterol was assayed with the same system without the cholesterol esterase reactor. The peak current was linearly related to cholesterol in the range 2-160 mg/dl and to total cholesterol in the range 3-300 mg/dl; the assay speed was about 80 samples/h for free cholesterol and 40 samples/h for total cholesterol. Reliable results were obtained in the assays of free cholesterol and total cholesterol in human sera. Both the reactors and the peroxidase electrode retained over 90% of their original activities, even after repetitive use for 4 and 2 months, respectively.     

以芳香胺玻璃为载体的固定化的胆固醇脂酶和胆固醇氧化酶

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Quantitation of glycerophosphorylcholine by flow injection analysis using immobilized enzymes

A method for quantitating glycerophosphorylcholine by flow injection analysis is reported in the present paper. Glycerophosphorylcholine phosphodiesterase and choline oxidase, immobilized on controlled porosity glass beads, are packed in a small reactor inserted in a flow injection manifold. When samples containing glycerophosphorylcholine are injected, glycerophosphorylcholine is hydrolyzed into choline and sn-glycerol-3-phosphate. The free choline produced in this reaction is oxidized to betain and hydrogen peroxide. Hydrogen peroxide is detected amperometrically.Quantitation of glycerophosphorylcholine in samples containing choline and phosphorylcholine is obtained inserting ahead of the reactor a small column packed with a mixed bed ion exchange resin. The time needed for each determination does not exceed one minute.The present method, applied to quantitate glycerophosphorylcholine in samples of seminal plasma, gave results comparable with those obtained using the standard enzymatic- spectrophotometric procedure.An alternative procedure, making use of co-immobilized glycerophosphorylcholine phosphodiesterase and glycerol-3-phosphate oxidase for quantitating glycerophosphorylcholine, glycerophosphorylethanolamine and glycerophosphorylserine is also described.Abbreviations GPC sn-glycerol-3-phosphorylcholine - GPE sn-glycerol-3-phosphorylethanolamine - GPS sn-glycerol-3-phosphorylserine - GPA sn-glycerol-3-phosphoric acid - PDE glycerophosphorylcholine-phosphodiesterase - GPA-Ox glycerophosphate oxidase - Cho-Ox choline oxidase     

Effect of 5-thio-D-glucose on testosterone biosynthesis in vivo in mice testes

Testicular synthesis of (14C)cholesterol and (14C)testosterone from (14C)acetate were investigated in mice treated with 5-thio-D-glucose at a dose of 33 mg/kg body weight/day for 21 days. The testicular synthesis of free cholesterol as well as steroids were significantly decreased. The steroid synthesizing enzymes, cholesterol esterase, cholesterol side-chain cleaving enzyme, total alpha-hydroxysteroid dehydrogenase and total beta-hydroxysteroid dehydrogenase, were also analysed. Cholesterol esterase and total beta-hydroxysteroid dehydrogenase were significantly reduced whereas total alpha-hydroxysteroid dehydrogenase was unaffected. Hence, a decrease in free cholesterol for steroid synthesis and a decreased activity of the steroidogenic enzyme, beta-hydroxysteroid dehydrogenase, were responsible for the diminished synthesis of testosterone.     

Study of carbon nanotube modified biosensor for monitoring total cholesterol in blood

A carbon nanotube modified biosensor for monitoring total cholesterol in blood was studied. This sensor consists of a carbon working electrode and a reference electrode screen-printed on a polycarbonate substrate. Cholesterol esterase, cholesterol oxidase, peroxidase and potassium ferrocyanide were immobilized on the screen-printed carbon electrodes. Multi-walled carbon nanotubes (MWCN) were added to prompt electron transfer. Experimental results show that the carbon nanotube modified biosensor offers a reliable calibration profile and stable electrochemical properties.     

Enzymatic assay of cholesterol by reaction rate measurements

A dynamic method for free and total cholesterol assay based on the oxidation of cholesterol by cholesterol oxidase, and conversion of cholesteryl oleate to cholesterol by cholesterol esterase is discussed in this article. The reaction conditions for total cholesterol assay were a temperature of 310 K and pH of 7.4. For conversion of cholesteryl oleate to cholesterol, the samples were incubated with 0.6 unit/mL of cholesterol esterase and 0.02 g/mL of taurocholate. The determination of initial reaction rates in the oxidation of free cholesterol, which is directly related to the cholesterol concentration, was found to be rapid, reliable, and inexpensive. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 391-396, 1997.     

Combination of an enzymatic method and HPLC for the quantitation of cholesterol in cultured cells.

The study of the cellular events that lead to the foam cell formation requires the development of fast, accurate, and sensitive methods to quantify cholesterol in cultured cells. Here we describe a procedure that allows the rapid determination of free and total cholesterol in a reduced number of cells, which makes it very suitable for cholesterol determination in cell cultures. The method consists of the enzymatic conversion of cholesterol to cholest-4-ene-3-one by cholesterol oxidase followed by the analysis of the sample by high performance liquid chromatography (HPLC) to detect this oxidized product. Due to the relatively high wavelength at which cholest-4-ene-3-one has its maximum absorption (240 nm), other cellular components do not interfere with the chromatographic procedure and prior lipid extraction is not required. Moreover, the duration of each chromatogram is about 3 min, contributing to the celerity of the method. All the cholesteryl esters used (oleate, palmitate, stearate and linoleate) were quantitatively hydrolyzed by incubation with cholesterol esterase; this was observed to occur with both pure standards and in cell homogenates. Sensitivity is enough to allow the determination of free and total cholesterol in less than 5 x 10(3) cells. We have applied this method to human monocyte-derived macrophages and the values obtained for free and total cholesterol are in close agreement with published data.     

A chemiluminometric method for the determination of urea in serum using a three-enzyme bioreactor

A flow injection chemiluminometric assay for urea has been developed based on a minicolumn bioreactor packed with immobilized enzyme-bearing glass beads. The reactor contains immobilized urease, L -glutamate dehydrogenase and L -glutamate oxidase, aligned in this order (upstream to the downstream). When the sample is introduced into the bioreactor, urea is first hydrolysed by urease to produce ammonia, which is then converted into L -glutamate by L -glutamate dehydrogenase. L -Glutamate is finally oxidized by L -glutamate oxidase to produce hydrogen peroxide, which is quantified by measuring chemiluminescence emitted upon admixing with luminol and potassium ferricyanide. One assay cycle is completed within 1 minute. The method is sensitive (detection limit 0.5 nmol) and is linear in the range 0–30 mmol/l. It can be readily applied to the determination of urea in human serum, and requires no blank corrections for ammonia and/or L -glutamate present in serum samples.     

Development of disposable lipid biosensor for the determination of total cholesterol

A prototype chronoamperometric biosensor for the determination of total cholesterol was developed that consists of a homemade potentiostat and disposable strips immobilized with Fe(3)O(4), cholesterol oxidase (ChOx), and cholesterol esterase (ChE). The principle of sensing cholesterol is based on the detection of reduction signal of hydrogen peroxide generated in two enzymatic reactions. The co-immobilization of ChE and ChOx allows the sensor to detect both concentrations of esterified and free cholesterol. The effects of biosensor on catalyst, enzymes, applied potential, and buffer pH was investigated, and the operation conditions were optimized. The detection of cholesterol can be accomplished in one step, a 10 microL of sample was dropped onto the area of sensing strip and the reduction signal was obtained at an applied potential of -200 mV (vs. Ag/Ag(+)). The pre-reaction time was set at 15s before applying potential on the strip and the sampling time was 5s. The sensing device displays a linear response over the range of 100-400mg/dL (R(2)=0.999) for cholesteryl oleate. The coefficient variation was determined as 5.06% (N=20) for 100mg/dL cholesteryl oleate and the detection limit is 19.4 mg/dL (S/N=3). The probable interferences in bio-matrix were selected to test the selectivity and no significant response was observed in the biosensor.     

Degradation of cholesterol by Bacillus subtilis SFF34 isolated from Korean traditional fermented flatfish

AIMS: To examine cholesterol degradation by Bacillus subtilis SFF34. METHODS AND RESULTS: Cholesterol degradation and cholesterol oxidase production by B. subtilis SFF34 were investigated in a medium containing 0.2% cholesterol. In addition, the oxidized product of cholesterol by the purified cholesterol oxidase was detected using a gas chromatograph. Cholesterol oxidase production reached its maximal level (3.14 U ml(-1) after 24 h of incubation in the cholesterol medium. The residual cholesterol content reduced to 0.98 mg g(-1) after 60 h of cultivation in the cholesterol medium. Two cholesterol oxidases were purified from the culture supernatant fluid and their reaction product against cholesterol was identified as 4-cholesten-3-one. CONCLUSIONS: B. subtilis SFF34 degraded cholesterol and produced a high level of extracellular cholesterol oxidase. SIGNIFICANCE AND IMPACT OF THE STUDY: Bacillus subtilis will be very useful for the reduction of cholesterol in many fermented foods and as a source of cholesterol oxidase.     

Cholesterol Reporter Molecules

Cholesterol is a major constituent of the membranes in most eukaryotic cells where it fulfills multiple functions. Cholesterol regulates the physical state of the phospholipid bilayer, affects the activity of several membrane proteins, and is the precursor for steroid hormones and bile acids. Cholesterol plays a crucial role in the formation of membrane microdomains such as "lipid rafts" and caveolae. However, our current understanding on the membrane organization, intracellular distribution and trafficking of cholesterol is rather poor. This is mainly due to inherent difficulties to label and track this small lipid. In this review, we describe different approaches to detect cholesterol in vitro and in vivo. Cholesterol reporter molecules can be classified in two groups: cholesterol binding molecules and cholesterol analogues. The enzyme cholesterol oxidase is used for the determination of cholesterol in serum and food. Susceptibility to cholesterol oxidase can provide information about localization, transfer kinetics, or transbilayer distribution of cholesterol in membranes and cells. The polyene filipin forms a fluorescent complex with cholesterol and is commonly used to visualize the cellular distribution of free cholesterol. Perfringolysin O, a cholesterol binding cytolysin, selectively recognizes cholesterol-rich structures. Photoreactive cholesterol probes are appropriate tools to analyze or to identify cholesterol binding proteins. Among the fluorescent cholesterol analogues one can distinguish probes with intrinsic fluorescence (e.g., dehydroergosterol) from those possessing an attached fluorophore group. We summarize and critically discuss the features of the different cholesterol reporter molecules with a special focus on recent imaging approaches.     

The kinetics of cholesterol oxidase synthesis by Nocardia rhodocrous.

The production of cholesterol oxidase by 3 liter batch cultures of Nocardia rhodocrous growing on a glycerol/yeast extract medium was investigated. Cholesterol was shown to be a good inducer of the enzyme. The optimum time for cholesterol addition and the quantity to be added were determined, resulting in a 15-fold yield increase. Cholesterol oxidase synthesis was influenced by the dissolved oxygen tension. Maximum cholesterol oxidase production was obtained at 30-40% air saturation. The effect of growth conditions on the extraction of cholesterol oxidase by Triton X-100 was investigated. The scale-up of the fermentation to 800 liters in a pilot-plant fermenter is described.     

Cholesterol oxidase in microemulsion: Enzymatic activity on a substrate of low water solubility and inactivation by hydrogen peroxide

Cholesterol oxidase from Nocardia erythropolis, Pseudomonas, and Streptomyces species was active in microemulsion in which cholesterol is well solubilized. The activity was stable in nonionic microemulsions whereas in cationic and anionic microemulsions the activity decreased with time. The coupled activity test using horseradish peroxidase which is very stable in microemulsion, was modified. The activity at very low water concentration in nonionic microemulsions increased with the water content. The kinetic constants were determined: the Michaelis constant is in the range 10 to 28 mm in the microemulsions, compared to 10 to 28 μm in buffer. The maximum velocity was reduced by a factor of 3 to 5 compared to that in buffer. Neither substrate excess nor product inhibition was detected. The preparative oxidation of cholesterol revealed the inactivation of the cholesterol oxidase by hydrogen peroxide. In contrast to glucose oxidase, hydrogen peroxide inactivated cholesterol oxidase in the absence of substrate. Catalase provides protection during the cholesterol oxidation. Microemulsions are very good media in which to perform enzyme catalyzed reactions with substrates of low water solubility. Their use for the reproducible determination of cholesterol should be examined.     

Application of an insolubilized hepatic microsomal drug oxidase to the continous production of N-oxides

The optimum operating temperature and pH have been determined for a catalyst prepared by the insolubilization of a hepatic microsomal mixed-function oxidase on glass beads. Temperature is a more significant variable than pH in determining potential catalyst yield (total product/unit catalyst). An operating range of about 25–28°C is optimal with respect to catalyst life at pH 7.6. Like the purified soluble oxidase, the glass bead-mounted enzyme catalyzes the NADPH- and oxygen-dependent oxidation of a variety of amines, hydrazines, and thioureylenes. The catalyst has been used in a simple slurry reactor to prepare mg quantities of N-oxide metabolites of prochlorperazine, guanethidine, and ethylmorphine.     

Continuous steroid biotransformations in microchannel reactors

The use of microchannel reactor based technologies within the scope of bioprocesses as process intensification and production platforms is gaining momentum. Such trend can be ascribed a particular set of characteristics of microchannel reactors, namely the enhanced mass and heat transfer, combined with easier handling and smaller volumes required, as compared to traditional reactors. In the present work, a continuous production process of 4-cholesten-3-one by the enzymatic oxidation of cholesterol without the formation of any by-product was assessed. The production was carried out within Y-shaped microchannel reactors in an aqueous-organic two-phase system. Substrate was delivered from the organic phase to aqueous phase containing cholesterol oxidase and the product formed partitions back to the organic phase. The aqueous phase was then forced through a plug-flow reactor, containing immobilized catalase. This step aimed at the reduction of hydrogen peroxide formed as a by-product during cholesterol oxidation, to avoid cholesterol oxidase deactivation due to said by-product. This setup was compared with traditional reactors and modes of operation. The results showed that microchannel reactor geometry outperformed traditional stirred tank and plug-flow reactors reaching similar conversion yields at reduced residence time. Coupling the plug-flow reactor containing catalase enabled aqueous phase reuse with maintenance of 30% catalytic activity of cholesterol oxidase while eliminating hydrogen peroxide. A final production of 36 m of cholestenone was reached after 300 hours of operation.     

Turnover Rate of Brain Acetylcholine Using HPLC Separation of the Transmitter

A simple, reliable method was developed for measuring brain acetylcholine (ACh) turnover using HPLC methodology. Mice were injected intravenously with [3H]choline ([3H]Ch), and the turnover rate of ACh was calculated from the formation of [3H]ACh. Ch and ACh were separated from phosphorylcholine and from other radioactive compounds using tetraphenylboron extraction and counterion/reverse-phase chromatography. Endogenous Ch and ACh were quantified electrochemically through hydrogen peroxide production in a postcolumn reactor containing covalently bonded ACh esterase and Ch oxidase. Labeled Ch and ACh were quantified in the same sample by collecting the chromatographic fractions for radioactive content determinations. The method is rapid, well adapted to large series, and highly reproducible, with recoveries of 72.1% for Ch and 79.3% for ACh. The turnover value in mouse cerebral hemispheres was 16.02 nmol g-1 min-1 and decreased to 9.94 nmol g-1 min-1 in mice treated with oxotremorine.     

Diurnal variations of rat liver enzymes catalyzing cholesterol ester hydrolysis

Cholesterol ester hydrolase activity was determined at 3 h time intervals over 24 h in lysosomes, cytosol and microsomes from ad libitum-fed and 24 h food-deprived female rat liver. Diurnal rhythms were identified for the acid and neutral esterases, which were strikingly changed by fasting. In fed animals, lysosomal esterase specific activity exhibited a peak at noon and a sustained medium rate at early darkness, whereas total esterase was maximal at midnight. The circadian patterns of the cytosolic and the microsomal esterases paralleled each other, though the amplitude of rhythms differed, showing higher activities around midnight. After fasting, cholesterol esterase activity from all cell fractions reached a maximum near dark onset. These results are the first to indicate that cholesteryl ester hydrolysis may play a role in generating the diurnal rhythm of hepatic cholesterol.     

Production of recombinant cholesterol oxidase containing covalently bound FAD in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Background  

Cholesterol oxidase is an alcohol dehydrogenase/oxidase flavoprotein that catalyzes the dehydrogenation of C(3)-OH of cholesterol. It has two major biotechnological applications, i.e. in the determination of serum (and food) cholesterol levels and as biocatalyst providing valuable intermediates for industrial steroid drug production. Cholesterol oxidases of type I are those containing the FAD cofactor tightly but not covalently bound to the protein moiety, whereas type II members contain covalently bound FAD. This is the first report on the over-expression in Escherichia coli of type II cholesterol oxidase from Brevibacterium sterolicum (BCO).     

High-sensitive chemiluminescent assay for cholesterol

Chemiluminescent measurement of cholesterol can be performed in various biological tissues and fluids. The method described in this study has a sensitivity of 54 pmol. The tissue samples used for the determination of cholesterol can be reduced to as little as 1 mg and assay can be performed on diluted biological fluids, allowing sampling of plasma or serum as little as 5 μl. Cholesterol is solubilized in sodium cholate and aliquots are added to a reaction mixture containing cholesterol oxidase, luminol and peroxidase. Cholesterol oxidase, in the presence of cholesterol yields H₂O² which produces light in presence of luminol and peroxidase. Emitted light is quantified at a wavelength of 420 nm by means of a photomultiplier. Optimal conditions of the assay were determined and examples of cholesterol determinations, in blood plasma and nervous tissues, are presented.     

Microbial cholesterol oxidases: bioconversion enzymes or signal proteins?

Cholesterol oxidases (3beta-hydroxysterol oxidases; EC 1.1.3.6), serve as catalysts for the initial step in the degradation of cholesterol, and probably other natural sterols, that are used as carbon sources for growth of different bacteria. Because of their suitability for attacking cholesterol they have been widely used for the quantification of cholesterol in clinical and food specimens. Cholesterol oxidase has also found application as a probe for membrane structure, as an insecticide, and has been implicated in bacterial pathogenesis. Recently, we have found that a Streptomyces cholesterol oxidase is required for the biosynthesis of the antifungal polyene pimaricin, apparently acting as an antifungal sensor. Here we describe our current understanding of these fascinating enzymes.