738

METHODS

I

Analysis of Cholesterol and Desmosterol in Cultured Cells Without Organic Solvent Extraction Edward H. Goh*, Debra K. Krauth a n d Scott M. Colles Medical Sciences Program, Indiana University, School of Medicine, Bloomington, Indiana 47405

Cell cultures. (L-M (CCL 1.2}, U-937 (CRL 1593} and PC-12 {CRL 1721} cells were purchased from American Type Culture Collection, Rockville, MD. L-M cells are a line of mouse fibroblasts which synthesize desmosterol instead of cholesterol {9}. L-M cells are cultured in Eagle minimum essential medium (No. M1018} containing Hank's salt mixture, L-glutamine {2 mM}, and non-essential amino acids. The medium is supplemented with sodium bicarbonate (4.2 mM), Bacto-peptone (0.5%}, penicillin I100 mg/L), and streptomycin (100 rag/L). Stock L-M cells are grown in 75 cm 2 flasks maintained in ambient atmosphere at 37 ~C. Cells are dislodged with trypsin (1 mg/mL phosphate~buffered saline {PBS), pH 7.4} when they reach confluency, treated with trypsin inhibitor (1 mg/mL PBS}, and washed with PBS. Aliquots of suspended cells are taken and used either as suspension or to prepare monolayers in a six-well plate. For this purpose, 7.5 • l0 s cells are seeded in each of the wells and allowed to recover for 24 hr. At this time, the monolayers generally reach a 75% confluency containing about 200 ~g of protein and 10 nmoles of desmosterol in each well. The monolayers are washed with PBS before use in Organic solvents are usually used to extract cultured cell experiments. U-937 cells are human monocytes, and they sterols, such as cholesterol and desmosterol, before they are grown as a suspension in RPMI 1640 medium suppleare quantified with microbial cholesterol esterase and oxi- mented with 10% fetal bovine serum and 5% CO2. PC-12 dase. The extraction ensures the accessibility of the cells are rat adrenal pheochromocytoma cells and are sterols to the microbial enzymes and prevents the inter- grown as a suspension in RPMI 1640 supplemented with ference of cellular components in the quantitation of 10% horse serum, 5% fetal bovine serum and 5% CO2. Digestion of cells. Unless otherwise specified, all cells hydrogen peroxide produced by the action of microbial are digested with sodium dodecyl sulfate {SDS) buffer enzymes on the sterols {1-8}. Organic solvent extraction of cultured cells, however, containing 0.1% SDS, lmM {ethylenedinitrilo)tetraacetic also creates difficulties. These include the variable losses (EDTA}, and 0.1M tris{hydroxymethyl}aminomethane of solvent during extraction, the usage and disposal of {Tris}, pH 7.4. SDS digest for cells grown in a monolayer volatile and dangerous chemicals, and the extensive ex- is prepared by adding 1 mL of SDS buffer to the monopenditure of time and effort. For these reasons, a method layer in each of the wells of the six-well plate and the plate to quantify cellular sterols with microbial cholesterol en- incubated at 37~ for 5 rain. The gelatinous mixture in zymes, but without prior extraction with organic solvent, the well is then taken up and discharged through a 19-gauge needle attached to a 1 mL disposable syringe. was investigated. This process is repeated (4-8 times} until a homogenous solution is obtained. SDS digest for suspension cells is MATERIALS AND METHODS prepared by adding concentrated SDS buffer to cell Standards and reagents. Standards and biochemicals were suspension to obtain a 0.1% concentration of SDS. The obtained from Sigma Chemical Co. (St. Louis, MO). Cho- gelatinous mixture is then homogenized as described for lesterol oxidase from Streptornyces species (No. C9406) cells in monolayer. SDS cell digest is stored at 4~ for and cholesterol esterase from Pseudornonas Fluorescens up to 30 days, or longer when frozen. The amounts of (No. C9406) were used. Analytical and high-pressure sterol and protein in the cell digest remain unchanged liquid chromatography (HPLC) grade solvents were ob- when stored under these conditions. Analysis of sterols. Sterols analyzed in this study intained from Fisher (Springfield, N J). Standard solutions of cholesterol, cholesterol oleate, and/3-sitosterol were pr~ clude cholesterol, desmosterol and/3-sitosterol. The latpared in isopropanol and stored in light-proof containers. ter is used as an internal standard for the HPLC analysis of cholesterol and desmosterol. There is no difference in the rate of oxidation of these sterols by the enzymes used *To whom correspondence should be addressed. Abbreviations: EDTA, (ethylenedinitrilo)tetraaceticacid disodium in this study (10}. The oxidized forms of these sterols are salt; HPLC, high-pressureliquidchromatography;PBS, phosphate- identified and quantified by a modified version of a previously described HPLC procedure (10). buffered saline; SDS, sodium dodecylsulfate.

Cultured cell sterols such as cholesterol and desmosterol are usually extracted into organic solvents before they are quantified with cholesterol esterase and oxidase. A method to quantify these cultured cell sterols using cholesterol enzymes without prior organic solvent extraction is described. In this method, a suspension or monolayer of cultured I~M, U-937, or PC-12 cells is digested with 0.1% sodium dodecyl sulfate (SDS), and the digest treated with microbial cholesterol enzymes. The quantity of oxidized sterols produced by the reaction can be measured easily with high-pressure liquid chromatography, when a mixture of sterols is present, or by the production of hydrogen peroxide when only one sterol is present. This method is easier and safer to use than solvent extraction and can greatly expedite the quantitation of cultured cell sterols. Preliminary data show that other lipids such as choline phospholipids, triglycerides, and fatty acids can also be directly quantified in SDS cell digest by using specific enzymes to transform these lipids into hydrogen peroxides. Lipids 25, 738-741 {1990).

LIPIDS,Vol. 25, No. 11(1990)

739

METHODS Typical incubation for HPLC analysis involves the transfer of 50 ~L of isopropanol containing 2 ~g of internal standard, &sitosterol, to a screw-cap tube {1.3 • 100 cm). A 200-~L aliquot of cell digest, digestion buffer, or a mixture of both is added. This is followed by the addition of 50 ~L of an enzyme mixture. For free sterols, the mixture contains 150 mM sodium phosphate, pH 7.0, 30 mM sodium taurocholate, 1.02 mM carbowax 6000, and 0.1 units of cholesterol oxidase. Cholesterol esterase {0.1 units} is added to the enzyme mixture when total sterols are to be measured. The reaction mixture is incubated for 60 rain at 37 ~ and extracted with petroleum hydrocarbon {bp, 35-60~ as described before {10). The organic layer is recovered, solvent is evaporated with nitrogen, and the remaining oxidized sterols are redissolved in acetonitrile for injection into HPLC. The extraction of oxidized sterols from the aqueous mixture is necessary to prevent the aqueous mixture from damaging the column. The extraction process is tedious and an alternative was investigated. It was found, empirically, that a 1:1 iv/v) mixture of 1 M KOH and acet~ nitrile remains separated in two different layers, instead of forming a homogenous solution. This suggests that KOH may be used to cause acetonitrile-containing oxidized sterols to separate from an equal volume mixture of acetonitrile and aqueous reaction mixture containing oxidized sterols. This possibility was tested and the partition in the presence of KOH and acetonitrile was found to be as efficient as with petroleum hydrocarbon in extracting oxidized sterols from the aqueous reaction mixture. Since this approach is less time-consuming and safer, it was adapted for routine use in this study. For this purpose, a 0.2-mL aliquot of KOH {2.5 M) was added to the reaction mixture after incubation at 37~ for 60 min. The mixture was vortexed and 0.5 mL of acetonitrile was added. The resulting biphasic mixture was vortexed vigorously for 30 sec and centrifuged for 1 rain {1,000 X g). An aliquot of the upper layer, acetonitrile, was taken and injected directly into the HPLC. The conditions and the equipment used for HPLC were reported previously {10). Briefly, chromatography is performed at room temperature with an Ultrasphere~XL C18 column {3 ~m, 75 X 4.6 mm) {Beckman Instruments, Fullerton, CA) equipped with a guard cartridge. The column is eluted with a 1:1 {v/v}mixture of methanol and acetonitrile at a flow rate of 1 mL/min. Eluted oxidized sterols are quantitated by ultraviolet absorption at 240 nm. Sterol peak heights are measured and ratios between cholesterol or desmosterol and &sitosterol determined. The ratios obtained are used to calculate the amounts of cholesterol and desmosterol present in the samples. Standard curves were developed for each sterol individually. Hydrogen peroxides produced by cholesterol oxidase are made fluorescent and quantified by fluorometry using previously established procedures {8). For this purpose, a 50-pL aliquot of water containing 0.2 unit of horseradish peroxide and 0.2 mg of p-hydroxyphenylacetic acid is added to the reaction mixture, prepared as described for HPLC analysis. The mixture is incubated as described for HPLC analysis. One mL of 50 mM sodium phosphate, pH 7.4, is added after the incubation. The samples are mixed and fluorescence is read at an excitation wavelength of 325 nm and an emission wavelength of 415 nm

using an Aminco SPF-500 {American Instrument Co., Silver Spring, MD). Other analyses. Aliquots of SDS cell digest were taken and protein was measured with bicinchoninic acid reagent marketed by Pierce {Rockford, IL}. Statistical methods. The results are presented as means + SEM. The significance of difference between group means is evaluated by Student's t-test. RESULTS AND DISCUSSION

Cultured cell sterols, such as cholesterol and desmosterol, are usually extracted into organic solvents before they are quantified with cholesterol esterase and oxidase (1-8). Organic solvent extraction of cultured cells is laborious, dangerous, and impractical when a large number of samples is involved. The extraction, however, is needed in order to allow cholesterol enzymes to gain complete access to the cellular sterols and to remove nonlipid cellular components which may interfere with the subsequent quantitation of enzymatic products, oxidized sterols and hydrogen peroxide. Instead of extraction with organic solvents, the accessibility of cultured cell cholesterol can be ensured by digesting the cells with a detergent which is compatible with the microbial enzymes. The search for this detergent was initiated by testing the ability of several common detergents at commonly used concentrations to dissolve cultured L-M cells. Individual solutions of Triton X-100 {1%}, sodium tanrocholate {1%}, Kyro EOB {0.5%}, Carbowax 6000 {10 mM), and SDS {0.1%} were prepared in Tris buffer {0.1 M, pH 7.4) with 1 mM EDTA. One-mL aliquots of each detergent were added to a confluent monolayer of L-M cells in each well of a six-well plate. The plates were incubated for 5 min at 37~ and examined under a light microscope. Recognizable cell remnants were observed in all wells except the one treated with SDS. The ability of 0.1% SDS to digest cells was clearly superior and hence was further tested. The optimal concentration of SDS for the digestion of cells was tested by the accessibility of cell sterol to the action of microbial enzymes. For this purpose, cholesterol oxidase and esterase were added to a suspension of L-M cells together with various concentrations of SDS. The suspensions were incubated under standard conditions and the amounts of L-M cell desmosterol measured by HPLC. The results are shown in Figure 1. Maximal amounts of desmosterol were detected with SDS at concentrations equal to or greater than 0.1%. Because of this, the concentration of SDS for cell digestion was kept at 0.1%. The compatibility of 0.1% SDS with microbial cholesterol oxidase and esterase was tested by assaying various amounts of free cholesterol or cholesteryl oleate standards in the absence and presence of 0.1% SDS. The amounts of oxidized cholesterol recovered in incubations with and without SDS under standard incubation conditions are identical for free cholesterol and cholesteryl oleate. The rates of oxidation for each type of cholesterol are linear up to 25 nmoles in the presence of 0.1% SDS and 50 nmoles in the absence of SDS {data not shown}. The difference is probably caused by the reduction in the activity of microbial enzymes by SDS. Since HPLC can detect as little as 10 pmoles of oxidized sterols {10), LIPIDS,Vol. 25, No. 11 (1990)

740

METHODS TABLE 2 Recovery of Cholesterol Standards from S D S Digest of L-M Cells a

80._=

Sterols measured

~

2.5 nmoles free cholesterol added Free cholesterol Free desmosterol

N.D. 1.61 - 0.03

2.47 _ 0.04 1.63 + 0.02

NE

3.0 nmoles cholesterololeate added Esterified cholesterol Esterified desmosterol

N.D. 0.i0 + 0.12

3.15 _ 0.02 0.06 _+ 0.15

--0

E 60e..

0.0

aValues for esterified sterols are obtained by subtracting the amount of free sterols from the amount of total sterols found in the samples. Amounts are given in nmoles/assay (n >/5); N.D., not detected.

I

I

0.1 0.2 gm/100 ml CONCENTRATION OF SDS

FIG. 1. The effect of S D S concentrations on the amounts of desmosterol detected in cultured L-M cell suspension (n >/5L

TABLE 1 Sterol Content of Cell Digests Without and with Organic Solvent Extraction a

L-M U-937 PC-1 2

With standard

LU----- 7 0 -

crQ.

Cell lines

Without standard

Without extraction

With extraction

FS

TS

FS

TS

65.43 +0.29 60.73 __-0.55

65.17 -+0.41 61.69 -+0.42

65.50 -+0.21 60.72 _+1.00

65.53 --+0.33 62.55 __-0.58

38.66 9 +0.13

39.30 -+0.18

38.53 -+0.27

38.86 +0.05

aIn nmoles/mg cell protein (n >t 4); FS, free sterol; TS, total sterol.

further a t t e m p t s to extend the linearity of the a s s a y are unnecessary. The differences in the a m o u n t s of sterols between SDS cell digests and organic solvent e x t r a c t s of cell digests were i n v e s t i g a t e d n e x t in three different cell lines. For this study, cell digests were either incubated directly with microbial enzymes or e x t r a c t e d with chloroform and methanol as described b y A m e s (11). Total lipids in the cell digest were recovered, dissolved in isopropanol, recons t i t u t e d with 0.1% SDS and microbial enzymes, and inc u b a t e d as usual. The results are given in Table 1. There is no difference between the a m o u n t s of free and t o t a l sterols m e a s u r e d directly in cell digests and indirectly in the e x t r a c t s from the digests. This is a further indication t h a t microbial enz y m e s are able to access all sterols in the SDS digest. In addition, the lack of difference in the amount of free sterol between cell digest and the e x t r a c t also suggests t h a t endogenous cellular cholesterol esterifying and hydrolyzing enzymes present in SDS cell digests are inactive during LIPIDS,Vol. 25, No. 11 (1990)

the 60 min incubation of SDS digest with microbial enz y m e s at 37~ To confirm this, 50 pL of isopropanol containing/Jsitosterol and either free cholesterol or cholesterol oleate s t a n d a r d s were added and incubated with L-M cell S D S digest for 3 hr at 37~ before the usual incubation with the microbial enzymes. The results are shown in Table 2. The recovery of free cholesterol and cholesteryl oleate was quantitative after 3 hr of incubation with SDS cell digest. Thus, endogenous cellular enzymes m e d i a t i n g the synthesis and hydrolysis of steryl esters are inactive in cell digest during incubation with microbial enzymes. This is necessary in order to calculate the a m o u n t of esterified sterols present in cell digest which is obtained b y subt r a c t i n g the a m o u n t of free sterols f r o m the a m o u n t of t o t a l sterols found in the digest. The inactivity of endogenous cellular cholesterol enzymes suggests t h a t endogenous cellular enzymes capable of altering the amount of hydrogen peroxide produced b y exogenous microbial cholesterol enzymes m a y also be inactive in SDS cell digest. Their inactivity would allow hydrogen peroxide produced b y microbial cholesterol enzymes to be measured directly and equated to the amount of sterols present in the cell digest. Quantifying hydrogen peroxide in SDS cell digest with f l u o r o m e t r y is less laborious than quantifying oxidized sterols b y HPLC. The former m a y be advantageous when only one t y p e of sterol is present in cultured cells. The possible use of hydrogen peroxides to quantify cellular sterols in SDS cell digest is examined b y comparing the quantities of sterols as reported b y fluorometry and b y H P L C . The comparison is m a d e with the slopes of their regression line. Perfect agreement is represented b y a slope of 1. The results are shown in Table 3. The

TABLE 3 Comparison Between HPLC (Y) and Fluorescence (X) Analyses of Cultured Cell Total Sterols in Cell Digest a Cell lines

L-M PC-12 U-937 a(n >/8).

Equations for linear regression

r

Y = 0.36 + 0.96X Y = 0.38 + 0.14X Y = 0.01 + 0.87X

0.999 0.972 0.993

741

METHODS

amounts reported by fluorescence are equal to or less than the amounts reported b y HPLC, depending on the cell types. Since the amounts reported b y fluorescence strongly correlate with the amounts reported b y HPLC, lesser amounts reported b y fluorescence can be corrected with the regression equations between the two methods. No correction is necessary for direct fluorescence m e a s u r e ment of digests obtained from L-M cells. H y d r o g e n peroxide is the end-product for enzymatic analysis of several lipid classes. The feasibility of quantifying hydrogen peroxide directly in SDS cell digest suggests t h a t other cellular lipids m a y also be quantified directly without prior organic solvent extraction. In preliminary expe "nments, enzymes used to analyze choline phospholipids {12}, triglycerides {13}, and free f a t t y acids {14} are found to be compatible with SDS. Direct analysis of these lipids in SDS cell digest is currently under investigation. The results clearly show t h a t cultured cell sterols can be quantified directly with microbial enzymes after the digestion of cultured cells with 0.1% SDS. This method can be used in place of organic solvent extraction and thereby eliminate a significant source of error in the quantitation of cultured cell cholesterol. This, in turn, will facilitate studies on the metabolism and t r a n s p o r t of lipids in cultured cells. ACKNOWLEDGMENT Financial support for this project is provided, in part, by the American Heart Association, Indiana Affiliate, Inc.

REFERENCES

1. Lange, Y., Culter, H.B., and Steck, T.L. {1980}J. BioL Chem. 255, 9331-9337. 2. Lange, Y., and Ramos, B.V. {1983} J. Biol. Chem. 258, 15130-15134. 3. Robertson, D.L., and Poznansky, M.J. {1985}Biochem. J. 232, 553-557. 4. Glick, J.M., Adelman, S.J., and Rothblat, G.H. {1987} Atherosclerosis 64, 223-230. 5. Slotte, J.P., Lundberg, B., and Bjorkerud, D.S. {1984}Biochim. Biophys. Acta 79~ 423-428. 6. Fielding, P.E., Fielding, C.J., and Havel, R.J. {1983}J. Cli~ Invest. 71, 449-460. 7. Orekhov, A.N., Tertov, V.V., Porkrovsky, N., Adamova, I.Y., Martsenyuk, O.N., Lyakishev, A.A., and Smirnov, V.N. {1988} Circulation Res. 64, 421-429. 8. Heider, J.G., and Boyett, R.L. {1978}J. LipidRes. 19, 514-518. 9. Rothblat, G.H., Bums, C.H., Conner, R.L., and Landry, J.R. {1970} Science 169, 880-882. 10. Goh, E.H., Colles, S.M., and Otte, K.D. {1989} Lipids 24, 652-655. 11. Ames, G.F. {1968}J. BacterioL 95, 833-843. 12. Takayama, M., Itoh, S., Nagaski, T., and Tanimizu, I. {1977} Clin. Chim~ Acta 79, 93-98. 13. Mandy, A.J., Cabeza, C., and Hsia, S.L. {1986}Anal. Biochem. 156, 386-389. 14. Matsubara, C., Nishikawa, Y., Yosida, Y., and Takamura, K. {1983}Anal. Biochem. 130, 128-133. [Received February 12, 1990; Revision accepted August 27, 1990]

LIPIDS, Vol. 25, No. 11 (1990)