A simple and sensitive enzymatic method for cholesterol quantification in macrophages and foam cells

A precise and sensitive method for measuring cellular free and esterified cholesterol is required in order to perform studies of macrophage cholesterol loading, metabolism, storage, and efflux. Until now, the use of an enzymatic cholesterol assay, commonly used for aqueous phase plasma cholesterol assays, has not been optimized for use with solid phase samples such as cells, due to inefficient solubilization of total cholesterol in enzyme compatible solvents. We present an efficient solubilization protocol compatible with an enzymatic cholesterol assay that does not require chemical saponification or chromatographic separation. Another issue with enzyme compatible solvents is the presence of endogenous peroxides that interfere with the enzymatic cholesterol assay. We overcame this obstacle by pretreatment of the reaction solution with the enzyme catalase, which consumed endogenous peroxides resulting in reduced background and increased sensitivity in our method. Finally, we demonstrated that this method for cholesterol quantification in macrophages yields results that are comparable to those measured by stable isotope dilution gas chromatography with mass spectrometry detection. In conclusion, we describe a sensitive, simple, and high-throughput enzymatic method to quantify cholesterol in complex matrices such as cells. A precise and sensitive method for measuring cellular free and esterified cholesterol is required in order to perform studies of macrophage cholesterol loading, metabolism, storage, and efflux. Until now, the use of an enzymatic cholesterol assay, commonly used for aqueous phase plasma cholesterol assays, has not been optimized for use with solid phase samples such as cells, due to inefficient solubilization of total cholesterol in enzyme compatible solvents. We present an efficient solubilization protocol compatible with an enzymatic cholesterol assay that does not require chemical saponification or chromatographic separation. Another issue with enzyme compatible solvents is the presence of endogenous peroxides that interfere with the enzymatic cholesterol assay. We overcame this obstacle by pretreatment of the reaction solution with the enzyme catalase, which consumed endogenous peroxides resulting in reduced background and increased sensitivity in our method. Finally, we demonstrated that this method for cholesterol quantification in macrophages yields results that are comparable to those measured by stable isotope dilution gas chromatography with mass spectrometry detection. In conclusion, we describe a sensitive, simple, and high-throughput enzymatic method to quantify cholesterol in complex matrices such as cells. Cholesterol accumulation in macrophage foam cells is one of the earliest histological features of atherosclerosis in the arterial intima (1Glass C.K. Witztum J.L. Atherosclerosis. The road ahead.Cell. 2001; 104: 503-516Abstract Full Text Full Text PDF PubMed Scopus (2654) Google Scholar). In order to perform studies of macrophage cholesterol loading, metabolism, storage, and efflux, one requires a precise, accurate, and sensitive method for measuring cellular free and esterified cholesterol levels. The most commonly used assays to quantify cholesterol levels can be separated into two groups: 1) analytical methods such as gas-liquid chromatography or liquid chromatography coupled with flame ionization or mass spectrometry detection and quantification (2Klansek J.J. Yancey P. St Clair R.W. Fischer R.T. Johnson W.J. Glick J.M. Cholesterol quantitation by GLC: artifactual formation of short-chain steryl esters.J. Lipid Res. 1995; 36: 2261-2266Abstract Full Text PDF PubMed Google Scholar, 3Cullen P. Fobker M. Tegelkamp K. Meyer K. Kannenberg F. Cignarella A. Benninghoven A. Assmann G. An improved method for quantification of cholesterol and cholesteryl esters in human monocyte-derived macrophages by high performance liquid chromatography with identification of unassigned cholesteryl ester species by means of secondary ion mass spectrometry.J. Lipid Res. 1997; 38: 401-409Abstract Full Text PDF PubMed Google Scholar, 4Paik M.J. Yu J. Hu M.B. Kim S.J. Kim K.R. Ahn Y.H. Choi S. Lee G. Gas chromatographic-mass spectrometric analyses of cholesterol and its precursors in rat plasma as tert-butyldimethylsilyl derivatives.Clin. Chim. Acta. 2008; 396: 62-65Crossref PubMed Scopus (20) Google Scholar); and 2) enzymatic assays, which can be colorimetric (5Gamble W. Vaughan M. Kruth H.S. Avigan J. Procedure for determination of free and total cholesterol in micro- or nanogram amounts suitable for studies with cultured cells.J. Lipid Res. 1978; 19: 1068-1070Abstract Full Text PDF PubMed Google Scholar, 6Heider J.G. Boyett R.L. The picomole determination of free and total cholesterol in cells in culture.J. Lipid Res. 1978; 19: 514-518Abstract Full Text PDF PubMed Google Scholar) or fluorometric (7Amundson D.M. Zhou M. Fluorometric method for the enzymatic determination of cholesterol.J. Biochem. Biophys. Methods. 1999; 38: 43-52Crossref PubMed Scopus (216) Google Scholar). However, the chromatographic and mass spectrometry methods require specialized equipment and chemical saponification of cholesterol esters; furthermore, these methods are time consuming and require processing samples one at a time. In contrast, the colorimetric and fluorometric enzymatic methods are simple, sensitive, and relatively fast assays that can process many samples at once. The principal of a sensitive fluorometric assay for the measurement of total cholesterol is shown below, with free cholesterol measured by omitting the first esterase step, and cholesterol mass in cholesterol esters calculated as the difference between the total and free cholesterol contents. Cholesterol esters →Cholesterol Esterase Cholesterol+fatty acids Cholesterol+O2→Cholesterol Oxidase Cholest-4-ene-3-one+H2O2 H2O2+ADHP(non-fluorescent) →HRP resorufin (fluorescent) The enzymatic method is the principal method used for measuring cholesterol levels in plasma, where cholesterol is solubilized in an aqueous solution through its incorporation in lipoproteins. However, in order to accurately measure the cholesterol content of cells or tissue by this method, one must identify a suitable solvent that allows good recovery of free and esterified cholesterol in solution and that is compatible with the enzymes and substrates. Previously, the use of the enzymatic cholesterol assay for cells and tissues has been limited due to the inadequate quantitative recovery of free cholesterol and cholesterol esters in enzyme compatible solvents; in fact, a methods paper has argued that the enzymatic method cannot be used for this purpose due to poor extraction and solubilization, which led to an underestimation of the cholesterol mass compared with an analytical method (8Cullen P. Tegelkamp K. Fobker M. Kannenberg F. Assmann G. Measuring cholesterol in macrophages: comparison of high-performance liquid chromatography and gas-liquid chromatography with enzymatic fluorometry.Anal. Biochem. 1997; 251: 39-44Crossref PubMed Scopus (10) Google Scholar). Here, we have identified a suitable and simple solvent system that yields excellent cholesterol recovery. Although this solvent initially led to high background in the enzymatic cholesterol assay, we discovered that pretreatment of this solvent with the enzyme catalase consumed endogenous peroxides, thus resulting in our ability to measure cellular cholesterol content by the simple fluorometric enzymatic assay. We demonstrate that this method for cholesterol quantification in macrophages yields results that are comparable to those measured by gas chromatography-mass spectrometry (GC-MS). 14CCholesterol, 3Hcholesterol, and 3Hcholesteryl oleate were purchase from Perkin Elmer. Bovine liver catalase, cholesterol oxidase from Streptomyces sp., cholesterol esterase from Pseudomonas sp., horseradish peroxidase (HRP), deuterated cholesterol 2H 2,2,3,4,4,6, and SylonTM HTP (HMDS+TMCS+Pyridine) were purchased from Sigma-Aldrich. 10-acetyl-3,7-dihydroxyphenoxazine (ADHP) was purchased from AnaSpec; ADHP is also commonly known as Amplex® Red (Invitrogen). Black polystyrene 96-well plates were purchased from Whatman. Human LDL (1.019 < d < 1.063 g/ml, adjusted with KBr) was prepared by ultracentrifugation and acetylated as described previously (9Basu S.K. Goldstein J.L. Anderson G.W. Brown M.S. Degradation of cationized low density lipoprotein and regulation of cholesterol metabolism in homozygous familial hypercholesterolemia fibroblasts.Proc. Natl. Acad. Sci. USA. 1976; 73: 3178-3182Crossref PubMed Scopus (823) Google Scholar). Cholesterol and cholesteryl oleate standards were prepared by dissolving 4 mg of desiccated products (Sigma-Aldrich) in 1 ml chloroform. The bicinchoninic acid (BCA) protein assay was performed using the BCA assay kit from Pierce. Acetylated LDL (AcLDL) was incubated for 10 min at 37°C in the presence of 3Hcholesterol. The radiolabeled AcLDL at 100 µg/ml was added for 2 days to RAW264.7 mouse macrophages cultured in 12-well dishes. To determine if solvent extraction of the cellular total cholesterol was quantitative, we compared the recovery of 3H dpm after solvent extraction with the recovery from a whole cell lysate. After two washes with PBS, the cells were extracted by addition of 1 ml hexane:isopropanol (3:2, v:v) directly to the wells. The extracts were transferred to scintillation vials and dried overnight. Cells in other wells were dissolved in 1 ml of 0.2N sodium hydroxide, incubated at 37°C for 3 h, and 200 µL was transferred to a vial and the pH neutralized by the addition of acetic acid. Four milliliters of scintillation fluid were added to the dried cholesterol extract or cell lysate and radioactivity assessed using a LS6500 scintillation counter (Beckman). Following the extraction of cellular total cholesterol in hexane:isopropanol, the total cholesterol must be resolubilized in a solvent system that is compatible with the assay enzymes and substrates. After testing several solvent systems, we selected isopropanol:Nonidet P-40 (NP40; 9:1, v:v). In order to assess the recovery of total cholesterol in this solvent, known amounts of 14Ccholesterol and 3Hcholesteryl oleate were added to the hexane:isopropanol extract of cholesterol loaded but unlabeled RAW264.7 cells and these were dried down together in microfuge tubes. One milliliter of isopropanol:NP40 was added to the microfuge tubes and vortexed. The free cholesterol and cholesteryl oleate radioactivity recovered in an aliquot of this solvent was determined by scintillation counting and compared with the added radioactivity. RAW 264.7 mouse macrophages were plated in 6-well dishes unless otherwise specified and cholesterol loaded by incubation with the specified concentration of AcLDL for 24 h. Unloaded cells were used as a control. Total cell cholesterol was extracted from the cells using 1 ml hexane:isopropanol (3:2, v:v). The extracts were transferred to microfuge tubes and dried. After solvent extraction, any residual solvent remaining on the cells was evaporated at room temperature. Then the protein from the same wells was dissolved by addition of 1.4 ml of 0.2N NaOH. The plate was incubated at 37°C for 3 h then rocked for 5 min at room temperature. The protein lysates were transferred to microfuge tubes and protein concentration was determined using the BCA assay. Cholesterol standards, samples, and blanks were dissolved in isopropanol:NP40 (9:1, v:v) and treated similarly, using 1 ml to redissolve each well of cells from a 6-well dish. In a black 96-well plate, 10 µL of a 100 U/ml catalase solution was distributed in each well and 40 µL of each sample was mixed followed by 15 min incubation at 37°C in order to eliminate any peroxides present in reagents or samples. Then 150 µL of reagent A (0.1 M potassium phosphate buffer, pH 7.4, 0.25 M NaCl, 5 mM cholic acid, 0.1% Triton X-100, 0.3 U/ml cholesterol oxidase, 1.3 U/ml HRP, and 0.4 mM ADHP) was added and mixed in each well. The plate was incubated at 37°C for an additional 15 min and fluorescence was read at an excitation wavelength of 530 nm and an emission wavelength of 580 nm. The cholesterol mass of the 40 μl aliquot of the unknown samples was determined by linear regression using the fluorescence emission of the blanks and the 40 μl cholesterol standards (20 to 800 ng range). The final cholesterol mass in the 1 ml samples was calculated by multiplying by 25. The same procedure was followed for total cholesterol except that reagent A was supplemented with 0.67 U/ml cholesterol esterase, yielding a final concentration of 0.5 U/ml. The cholesterol mass in cholesterol esters was determined by subtracting the free cholesterol values from the total cholesterol values. Macrophages were scraped from each well of a 6-well plate and were resuspended in 400 µL water and 100 µL of 1 µg/ml deuterated internal cholesterol standard (2H 2,2,3,4,4cholesterol) in isopropanol. Two milliliters of hexane:isopropanol (3:2) and 20 μl of acetic acid were then added to the cell suspension. After vortexing and centrifugation, cholesterol and its derivatives present in the organic phase were collected. The aqueous phase was reextracted by the addition of 1ml hexane followed by vortexing and centrifugation. The hexane layer was collected and combined with the previous organic phase. The remaining aqueous phase containing the extracted cells was used to determine protein concentration by the BCA assay. The combined organic extract was divided into two parts. One part was dried under nitrogen for free cholesterol quantification. The other part was dried under nitrogen and cholesterol esters were saponified in 100 µL 0.5 M potassium hydroxide in methanol for 1 h at 37°C followed by adding 100 µL of 1 M hydrochloric acid, 300 µL water, and 1 ml isopropanol:hexane:acetic acid (40:10:1, v:v:v). The total cholesterol was extracted by two successive additions of 1 ml hexane and dried under nitrogen. 50 µL Sylon™ HTP was added to the dried cholesterol preparations and trimethylsilyl (TMS) derivatives were formed in a 1 h incubation at 90°C. Calibration curves were prepared after TMS derivatization of varying cholesterol amounts plus 100 ng of stable deuterium-labeled cholesterol internal standard. One microliter of the TMS-derivatized sample or calibrator was injected onto 6890/5973 GC-MS equipped with an automatic liquid sampler (Agilent Technologies) using the positive ion chemical ionization mode with methane as the reagent gas. The source temperature was set at 230°C. The electron energy was 240 eV and the emission current was 300 µA. The cholesterol TMS ethers were separated on a J and W Scientific (Folsom, CA) DB-1 column (20 m, 0.18 mm inner diameter, 0.18 µm film thickness). The injector and the transfer line temperatures were maintained at 250°C. The initial GC oven temperature was set at 230°C and increased 20°C/min to 270°C followed by increases of 4°C/min to 300°C. The total ion mass spectra of TMS derivatives were recorded in the mass range m/z 200 to 500. The GC chromatograms were extracted at m/z = 329 and 335 for cholesterol and 2H 2,2,3,4,4cholesterol, respectively, and the peak areas were integrated. Then cholesterol content in each sample was calculated by stable isotope dilution analysis. In order to further validate the enzymatic assay, we performed both analyses on the same cell extracts using two internal standards. RAW 264.7 cells were untreated or cholesterol loaded by 24 h incubation with varying amounts of AcLDL as described above. Cells were washed in DMEM twice. One milliliter of hexane:isopropanol (3:2, v:v), 2 nCi of 3Hcholesterol, and 100 ng of deuterated internal cholesterol standard (2H 2,2,3,4,4cholesterol) were added to each well. After mixing for 5 min, half of the extract was transferred to a microfuge tube for processing using the enzymatic assay as described above. The remaining half of the extract was transferred to a glass tube for processing using the GC-MS assay as described above. The 3Hcholesterol standard was used to calculate the fraction of the total cellular extract that was input into the enzymatic assay. The deuterated cholesterol standard was used for the GC-MS cholesterol calculations as described above. We first validated that hexane:isopropanol (3:2) yielded quantitative recovery of total 3Hcholesterol from cholesterol loaded RAW264.7 cells. The extracted dpm represented 98.6 ± 2% (N = 4 ± SD) of the total cells lysate control. For the enzymatic cholesterol assay, the dried hexane:isopropanol extract must then be redissolved in a solvent compatible with the enzymes and substrates. Cullen et al. (8Cullen P. Tegelkamp K. Fobker M. Kannenberg F. Assmann G. Measuring cholesterol in macrophages: comparison of high-performance liquid chromatography and gas-liquid chromatography with enzymatic fluorometry.Anal. Biochem. 1997; 251: 39-44Crossref PubMed Scopus (10) Google Scholar) reported that isopropanol, ethanol, or 10% triton X-100, all suitable for enzyme activity, allowed at best 50% free cholesterol recovery and 20% for cholesterol esters. We tested the suitability of isopropanol:NP40 (9:1) for sterol extraction by adding it to a dried cell hexane:isopropanol extract containing known amounts of 14Ccholesterol and 3Hcholesteryl oleate. We observed that the isopropanol:NP40 mixture yielded 102 ± 3% and 96 ± 7% recovery of free cholesterol and cholesterol esters, respectively (N = 4, ± One of the for the use of our enzymatic method on the of the isopropanol:NP40 mixture with endogenous peroxides, a with resulting in high fluorescence background of sample In order to this we the standards, and samples with a final concentration of 20 U/ml of catalase for 15 min at 37°C cholesterol quantification. reduced the background fluorescence by at curves were prepared in presence or of catalase and the results are shown in the of catalase to 40 U/ml not the results not 20 U/ml was as the standard The that catalase not reduced background but also improved the sensitivity of the assay as shown by the with this We to determine the of cells that our was sensitive to We measured total cholesterol in of cells recovered from a to and we both protein ± and total cholesterol ± 10 in cells, yielding ± total cell To determine the of this assay, one and one AcLDL loaded cell extract were to total and free cholesterol assays using The of for the of these assays from to thus excellent In this the and AcLDL loaded cells yielded and total cell In order to quantify total cholesterol esters to be into free cholesterol by cholesterol we to that the concentration of cholesterol esterase as well as the incubation time were to the of all cholesterol esters into free the cholesteryl oleate we 400 ng cholesterol to ng cholesteryl in the presence of 0.5 U/ml We ± ng to ± of the the esterase used to ng of cholesteryl oleate into free cholesterol in our assay. To the and of the enzymatic assay with the GC-MS assay, we performed with cells in which or wells were for cholesterol by each method. The total cholesterol levels were ± and ± cell protein for the enzymatic and GC-MS assays, respectively not In these cells, the of the cholesterol was as free cholesterol with values in both assays Although the cholesterol esters levels were low in these cells, were in the GC-MS this difference was not In order to the enzymatic and GC-MS assays a range of cholesterol loading, we incubated RAW 264.7 cells with or µg/ml AcLDL in two internal standards for as described in the each well was for total and free cholesterol by both the enzymatic and GC-MS AcLDL led to increases in cellular total cholesterol that are due to increases in cholesterol esters In this the GC-MS assay yielded values the enzymatic assay, with total cholesterol levels observed in the cells loaded with µg/ml However, in a the two methods using cells loaded with 100 µg/ml we observed the with total cholesterol values by the enzymatic assay ± total cell compared with the GC-MS method ± 5 total cell In the cells treated with varying of we the values of and esterified cholesterol for each well determined by the enzymatic GC-MS methods The total cholesterol levels were < with a of ± The in this assay was due to the which was of a The range of free cholesterol levels not as but were well between the two methods = = ± = The cholesterol esters levels were between these two methods < = ± = of cholesterol values by the enzymatic and GC-MS The are from the wells of cells that a range of cholesterol levels as described in the of Total cholesterol levels = ± = cholesterol levels = ± = Cholesterol esters levels = ± = In this we describe a sensitive and method to quantify total and free cholesterol content in cultured cells, which does not require chemical saponification or specialized equipment other a fluorescence plate We demonstrated efficient extraction of cellular and good recovery by in an isopropanol:NP40 solution that is compatible with the enzymatic assay. We also that pretreatment of the samples and standards with catalase to endogenous peroxides in the isopropanol:NP40 solution was in the background and We determined that was to the catalase the enzymatic cholesterol which is A also used catalase measurement of cholesterol esters by an enzymatic assay, and these also not to the catalase A method of measurement for the enzymatic determination of cholesteryl esters.J. Lipid Res. Full Text Full Text PDF PubMed Scopus Google Scholar). The that catalase does not to be is most due to at low at 37°C and of catalase in and tissues of and PubMed Scopus Google Scholar, M. of liver catalase to for of the enzyme and PubMed Scopus Google and the of catalase for J. Yu with on a film and PubMed Scopus Google Scholar, P. P. and of G. Scholar). To the of the enzymatic method, we compared it with stable isotope dilution the method for free cholesterol and cholesterol ester quantification. the the results of the high-throughput enzymatic assay were with the GC-MS assay. However, in assays we one method yielding cholesterol levels the other method. In with cells, the two methods on yielded comparable results that the not one assay the In two with cholesterol loaded cells, one yielded levels for the enzymatic assay, the other yielded levels for the GC-MS assay. In the where varying amounts of AcLDL were used to a range of cholesterol loading, our that the two methods were in and esterified cholesterol However, in this the GC-MS method yielded values the enzymatic method, which was in the values the was both methods amounts of total and free We that the difference be due to a by use of cholesterol standards, using and of calculations used to determine the cholesterol For the enzymatic assay a cholesterol standard in the linear range to calculate the cholesterol mass in unknown samples. However, the GC-MS results are calculated using an internal deuterated cholesterol standard in each sample that is present at of the sample cholesterol a in the of the internal standard peak to a in the calculated cholesterol In conclusion, we described a sensitive, simple, and method to quantify cholesterol in cultured method allowed the detection of cholesterol in cells to cholesterol with the use of 96-well this assay can process a of samples which further be through assay assay be to all of cells and tissues after determination that the isopropanol:NP40 mixture all cholesterol in the solvent acetylated low density lipoprotein 10-acetyl-3,7-dihydroxyphenoxazine bicinchoninic acid P-40