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Nutritional regulation of cellular phosphatidylinositol
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similar to the concentrations of myo-inositol found in fetal rat serum during late gestation (Fig. 3). The methods described here for altering intracellular CMP and the availability of myo-inositol have proved useful in the manipulation of the relative rates of synthesis of phosphatidylinositol and phosphatidylglycerol in type 1I pneumonocytes. These methods complement others that have been described (e.g., use of Li ÷) for altering phospb_oinositide metabolism in a variety of cell types.
[21] N u t r i t i o n a l R e g u l a t i o n o f C e l l u l a r P h o s p h a t i d y l i n o s i t o l
By BRUCE J. HOLUB and CLARK M. SKEAFF Introduction A marked surge in interest has developed recently concerning the influence of nutrition on the level and composition of phosphatidylinositol in selected mammalian tissues and cells. This interest has been stimulated by the recognition that, in addition to providing a structural role in mainraining biological membranes and regulating the activity of membranebound enzymes, phosphatidylinositol is a precursor of the intracellular messengers 1,2-diacylglyceroP and inositol trisphosphate 2 (either directly or indirectly via its conversion to phosphatidylinositol 4,5-bisphosphate). The degradation of phosphatidylinositol, which contains predominantly lstearoyl 2-arachidonoyl molecular species in most mammalian cells, 3 also provides for the release of free arachidonic acid for conversion to the eicosanoids. Furthermore, dietary modifications (in myo-inositol levels, type of dietary fat, dietary cholesterol levels, etc.) can significantly influence the amount of phosphatidylinositol as well as its fatty acid and molecular species compositions 3'4 in certain cells; this may be important in the functioning of this phospholipid and its derivatives in both the 1 y. Nishizuka, Trends Biochem. Sci. 8, 13 (1983). 2 M. J. Berridge and R. F. Irvine, Nature (London) 312, 315 (1984). 3 B. J. Holub, Adv. Nutr. Res. 4, 107 (1982). 4 B. J. Holub, in "Inositol and Phosphoinositides: Metabolism and Regulation" (J. E. Bleasdale, J. Eichberg, and G. Hauser, eds.), p. 31. Humana Press, Clifton, New Jersey, 1985.
METHODS IN ENZYMOLOGY, VOL, 141
Copyright © 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.
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resting and stimulated cell. For example, the consumption of fish oils and concentrates containing eicosapentaenoic acid provides for the introduction of novel molecular species (namely, 1-acyl-2-eicosapentaenoylphosphatidylinositol) into circulating human platelets. 4 Nutritional modification of cellular phosphatidylinositol may eventually offer clinical applications in the control of phosphatidylinositol-mediated physiological responses. General Experimental Approaches and Principles Nutritional Considerations
Attention needs to be given to the choice of animal model and diet composition used to study the influence of nutrition on disorders related to phosphatidylinositol metabolism. For example, the female gerbil is highly sensitive toward the development of an intestinal lipodystrophy 5 and the young male rat toward an accumulation of hepatic triacylglycerol when fed inositol-deficient diets in a manner which depends on the type of dietary fat. 3 The use of semipurified diets (inositol deficient and inositol supplemented at fixed concentrations) containing essential nutrients at levels which satisfy the known requirements for the animal employed is recommended. The National Research Council has published the qualitative and quantitative nutrient requirements for laboratory animals 6 including the rat, mouse, gerbil, guinea pig, hamster, vole, and fish. Documents are also available from the National Academy of Sciences which give the specific requirements for other animals. A chow diet is an inappropriate "control diet" since it will likely differ considerably from the experimental diets in the level of almost all nutrients including fat, protein, carbohydrate, vitamins, and minerals in addition to containing endogenous forms of inositol, thereby greatly restricting interpretation of the experimental results. The amount of inositol in the experimental diets should be chemically assessed where possible to confirm the expected values and the suitability of the mixing procedures. For example, significant amounts of endogenous phosphatidylinositol may be present in dietary vegetable oils and other constituents. The routine measurement of food intake, weight gain, and gain:feed ratios of the animals during nutritional studies will help eliminate these three parameters as experimental variables across the dietary groups if they are found not to differ from each other. s D. M. Hegsted, K. C. Hayes, A. Gallagher, and H. Hanford, J. Nutr. 103, 302 (1973). 6 The National Research Council, in "Nutrient Requirements of Laboratory Animals," 3rd ed., No. 10. Natl. Acad. Sci., Washington, D.C., 1978.
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Procedures
Lipid Methodology The extraction of phospholipids from mammalian tissues and cells using chloroform-methanol mixtures as described by Folch et al. 7 and Bligh and Dyer 8 and modifications of these procedures have been described in detail with specific reference to the extraction of phosphatidylinositol.9,~° A number of chromatographic procedures ~°-14 (including column, conventional thin-layer, high-performance thin-layer, and highperformance liquid chromatography) have been described for the separation and isolation of phosphatidylinositol from lipid extracts leading to their quantitation by various approaches (for example, determination of lipid phosphorus, ~5:6 densitometry, ~7 fluorometric analysis, 12 etc.). Onedimensional thin-layer chromatography for the separation of phosphatidylinositol from phosphatidylserine and other phospholipids as outlined js has been frequently claimed to prove difficult. 19,2°One-, 2°,2~ t w o - , 16 and even three-dimensional22 thin-layer chromatographic systems have been reported to facilitate these separations. The direct quantitation of phosphatidylinositol by phosphorus determination in the presence of silica gel following thin-layer chromatography has been documented,~S,16 thereby avoiding elution and subsequent losses of the phospholipid from the gel scrapings. The positional distribution of fatty acyl chains in the 1- and 2-positions of isolated phosphatidylinositol can be determined23 by fatty acid analyses of the reaction products [lyso 7 j. Folch, M. Lees, and G. H. Sloane-Stanley, J. Biol. Chem. 226, 497 (1957). s E. G. Bligh and W. J. Dyer, Can. J. Biochem. Physiol. 37, 911 (1959), 9 F. B. St. C. Palmer, Biochim. Biophys. Acta 231, 134 (1971). l0 W. W. Christie, "Lipid Analysis," 2nd ed. Pergamon, Oxford, 1982. ti j. Stein and G. Stein, "Techniques in Lipid and Membrane Biochemistry," Part I. Elsevier/North-Holland Biomedical Press, Amsterdam, 1982. 12 C. A. Harrington, D. C. Fenimore, and J. Eichberg, Anal. Biochem. 106, 307 (1980). 13A. Karasawa, S. H. Yoshikawa, A. Miyakawa, A. Nishi, and R. Ishitani, J. Chromatogr. 260, 513 (1983). J4 M. Saito, Y. Tanaka, and S. Ando, Anal. Biochem. 132, 376 (1983). is A. F. Rosenthal and S. C. H. Han, J. LipidRes. 10, 243 (1969). 16 G. Rouser, S. Fleischer, and A. Yakamoto, Lipids 5, 494 (1970). ~7M. Goppelt and K. Resch, Anal. Biochem. 140, 152 (1984). is V. P. Skipski, R. F. Peterson, and M. Barclay, Biochem. J. 90, 374 (1964). 19 H. D. Kaulen, Anal. Biochem. 45~ 664 (1972). 20 D. Allan and S. Cockcroft, J. Lipid Res. 23, 1373 (1982). 2~ j. B. Fine and H. Sprecber, J. Lipid Res. 23, 660 (1982). z2 j. K. G. Kramer, R. C. Fouchard, and E. R. Farnworth, Lipids 18, 896 (1983). 23 B. J. Holub and A. Kuksis, Can. J. Biochem. 49, 1347 (1971).
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NUTRITIONAL REGULATION OF CELLULAR PI
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(1-acyl) phosphatidylinositol and free fatty acids] following digestion with phospholipase A2. The chemical classes of cellular phosphatidylinositol can be fractionated according to unsaturation by argentation thin-layer chromatography of the intact phospholipid 24 or its chemical derivatives (e.g., 1,2-diacylglycerol acetates following phospholipase C digestion and acetylation of 1,2-diacylglycerols,Z5 dimethylphosphatidic acidsZ6). The individual molecular species can be determined following quantitation of the various classes and their characterization by gas-liquid chromatography and further analyses of the positional distributions of the constituent fatty acids as required. The specific series of procedures as outlined in detail below have been developed and used in our laboratory to provide a simultaneous determination of the mass of cellular phosphatidylinositol and its fatty acid composition. This approach employs the combined use of one-dimensional thin-layer chromatography and gas-liquid chromatography of the fatty acid methyl esters (including methyl pentadecanoate as an internal standard) formed from the separated phosphatidylinositol.
Thin-Layer Chromatographic Separation of Phospholipids Separation of the individual phospholipids in the lipid extract is achieved using a simple and rapid one-dimensional thin-layer chromatography system. The thin-layer chromatography plates used are Merck silica gel 60 glass precoated 20 × 20 cm plates (without fluorescein indicator) with 0.25-ram layer thickness (Art 5721-7 British Drug House, Toronto, Ontario, Canada). Plates are kept at room temperature in the manufacturer's storage box and are removed Gust) prior to each use. All solvents used throughout the described procedures were purchased from Fisher Scientific (Toronto, Ontario, Canada) and were of ACS certified grade. Using a 25-pA Hamilton syringe, the lipid extract, dissolved in 25-50/xl of chloroform-methanol, 2 : 1 (v/v), is spotted in a straight band 3 cm long approximately 1.5-2 cm above the bottom of the plate. During the sample application, a gentle stream of nitrogen is blown across the origin in order to facilitate the spotting of a tight lipid band. Normally, 4-5 lanes can be spotted per plate. The best chromatographic separations are produced when 150-350/zg of lipid/cm or less is applied to the thin-layer chromatography plate. Spotting lipid samples in excess of 650/xg/cm frequently results in the overlapping of the sphingomyelin and phosphatidylcholine
24 B. J. Holub and A. Kuksis, J. Lipid Res. 12, 510 (1971). 25 V. G. Mahadevappa and B. J. Holub, Biochim. Biophys. Acta 713, 73 (1982). 2~ M. G. Luthra and A. Sheltawy, Biochem. J. 126, 1231 (1972).
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bands but does not compromise the separation of the other phospholipids. We have found that possible streaking of the phospholipids during development is eliminated if the nitrogen drying is continued for 3-5 min after spotting has been completed. The solvent system, consisting of chloroform-methanol-acetic acidwater, 50:37.5:3.5:2 (v/v/v/v), is mixed in a stock bottle and added to the development chamber [length, 22 cm; height, 22 cm; width, 10.5 cm (Desaga, Heidelberg, West Germany)]. The chamber is then lined completely on three sides with Whatman chromatography paper (0.18 mm thickness) and allowed to equilibrate until the solvent has run the entire height of the chromatography paper (usually 45-60 min). For optimal phospholipid separation, the plate should be placed in the development chamber shortly after equilibration is completed. For chromatographic separation, the plate is then placed as vertically as possible in the development chamber and allowed to develop until the solvent has run to within 0.5 cm of the top (an etched horizontal line can be drawn if desired) of the plate (usually 1.5-2 hr). When development is complete, the plate is removed and air dried at room temperature for 5-10 min. Using an atomizer with nitrogen gas flow, the gel surface is sprayed with a saturated solution of 2,7-dichlorofluorescein in methanol-water, 1 : 1 (v/v), until a slight yellow color is visible. After exposing the plate to ammonia vapor by placing it in a raised bottom developing chamber containing 50-100 ml of 8 N ammonia, the phospholipid bands are visualized under ultraviolet light at a wavelength of 366 nm. The color of the bands can be intensified, if necessary, by successive exposure to acetic acid and ammonia vapor. Figure 1 illustrates the typical chromatographic separation pattern obtained from a tissue/cellular lipid extract. In this case, rat liver extract was used for separation. The five major phospholipids, sphingomyelin, phosphatidylcholine, phosphatidylserine, phosphatidylinositol, and phosphatidylethanolamine, are completely separated. Lysophosphatidylcholine (Rf 0.03), although not visible in Fig. I, runs above the origin and below sphingomyelin. Cardiolipin runs above phosphatidylethanolamine while the neutral lipid fraction runs with the solvent front. The lower portion of the neutral lipid band contains mainly cholesterol and free fatty acids whereas the upper portion is composed mainly of triacylglycerols and cholesterol esters with some monoacylglycerols and diacylglycerols. We have routinely used this solvent system for the separation of the individual phospholipids from blood platelets of various species, including human. Phospholipids in lipid extracts from goat and rat lung lamellar bodies as well as from fetal calf serum and human skin fibroplasts have also been
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NUTRITIONAL REGULATION OF CELLULAR PI
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~NL
CL
PE
PI
PS PC SPH 0 FIG. I. Thin-layer chromatogram demonstrating the phospholipid separations of a rat liver extract as described in the text. The photograph was taken under ultraviolet light at 366 nm using a camera equipped with a 55-mm lens and #237 CID filter (Lee Filters, Ltd., Andover, Hants, UK). The following bands have been identified: sphingomyelin (SPH), R~. 0.06; phosphatidylcholine (PC), Rf 0.10; phosphatidylserine (PS), Rf 0.27; phosphatidylinositol (PI), Rf 0.38; phosphatidylethanolamine (PE), Rr 0.57; cardiolipin (CL), Rf 0.73; neutral lipid (NL). O, Origin.
240
INOSITOL PHOSPHOLIPIDS AND METABOL1TES
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separated with success using this thin-layer chromatography system (J. Kirkland and T. Bray, S. Karmiol and W. Bettger, respectively, Department of Nutritional Sciences, University of Guelph, Ontario, Canada). Phospholipid separation of the lamellar body lipid extract has demonstrated that phosphatidylglycerol migrates between phosphatidylethanolamine and cardiolipin. In the agonist-stimulated cell, where phosphatidic acid may accumulate to significant levels, possible overlap of this phospholipid with phosphatidylethanolamine may slightly compromise the phosphatidylethanolamine determinations. We have found this one-dimensional thin-layer chromatography method for phospholipid separation to be highly reproducible. Furthermore, we have not experienced difficulty using this system with varying external humidity and temperature, factors which frequently render consistent phospholipid separations difficult. However, we have not tested these separations under extreme environmental conditions.
Preparation of Fatty Acid Methyl Esters The individual phospholipid bands are scraped into separate 16 x 100 mm screw-top glass test tubes containing 3 ml of 6% concentrated H2SO4 in methanol by volume and 5-10/.~g of monopentadecanoate (containing 15:0) as an internal standard. Other methylation procedures are available 1°,~1although control experiments need to be conducted when applied in the presence of gel scrapings. The future commercial availability of dipentadecanoylphosphatidylinositol will allow its addition as an internal standard during the lipid extraction phase. This use of phosphatidylinositol as an internal standard will eliminate the need to correct for any minor lipid losses incurred throughout the isolation procedure of phosphatidylinositol samples, whereas the use of the monopentadecanoate internal standard only accounts for lipid losses subsequent to the methylation phase. The amount of 15 : 0 internal standard used depends on the quantity of phospholipid being analyzed and should ideally represent approximately 20% of the total fatty acids present in the phospholipid. The tubes are tightly sealed with Teflon-lined caps, in order to minimize the evaporation of methylating reagent, vortexed for 60 sec, and then placed in an oven at 80 ° for 6-14 hr. When analyzing sphingomyelin, the longer methylation times of 12-16 hr are required while shorter times of 3-6 hr are generally sufficient for methylating the fatty acids in phosphatidylserine, phosphatidylcholine, phosphatidylinositol, and phosphatidylethanolamine. Upon completion, the tubes are removed from the oven and allowed to cool to room temperature. Following the addition of 2 ml of petroleum ether, each tube is vortexed for 60 sec. One milliliter of water is
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added and the tubes are vortexed again for 30 sec. The upper petroleum ether phase, containing the eluted fatty acid methyl esters, is removed and transferred to a 2-ml minivial. The vial is placed in a sand bath at 40° while the petroleum ether is evaporated to dryness under a gentle stream of nitrogen gas. The fatty acid methyl esters are then immediately dissolved in 25 ~1 of gas-liquid chromatography grade hexane.
Analysis of Fatty Acid Methyl Esters Using Gas-Liquid Chromatography The fatty acid methyl esters are analyzed using a 30 m x 0.53 mm i.d. DB-225 (25% cyanopropylphenyl) J&W Scientific megabore column (Chromatographic Specialities, Inc., Brockville, Ontario, Canada) installed on a Hewlett Packard 5890 gas liquid chromatograph with flame ionization detector. The column head pressure is set at 65 kPa and the column, split vent, and purge vent flow rates are adjusted to 6.8, 20, and 6.0 cc of helium per minute, respectively. The injection port, equipped with a glass split insert, and the detector are maintained at 250 °. The medical air, hydrogen, and nitrogen flow rates through the flame ionization detector are set at 360, 38, and 36 cc per minute, respectively. Normal injection volume of the fatty acid methyl esters dissolved in hexane is 1-2 /xl. When detecting trace amounts of fatty acid methyl esters in the sample, it may be essential to inject large quantities of sample onto the gas-liquid chromatograph. This can be achieved by evaporating, prior to injection, some of the 25 ~1 of hexane in which the fatty acid methyl esters are dissolved. The megabore column provides the superior resolving power of the capillary column while allowing for the quantification of trace amounts of fatty acid methyl esters that may otherwise be undetected by the capillary column due to its limited sample capacity. With instrument range 4 and attentuation 0 the chromatograph is run at an oven temperature of 210° producing complete fatty acid methyl ester separation within 25 min. The peak areas are measured using a Spectral Physics (SP4100) integrator and identified by comparing the retention times with known standards. Using the peak area and the known weight of the monopentadecanoate internal standard, the weight and moles of the individual fatty acids can be calculated. These results can be used to determine the moles of phospholipid. It is important that blank regions of the thin-layer plate corresponding to the phospholipid bands be scraped, methylated in the presence of an internal standard, and analyzed by gasliquid chromatography so that the sample peak areas can be corrected for any possible gel or solvent background contamination. Accuracy of the detector response is regularly verified by injection and quantification of a
242
INOSITOL PHOSPHOLIPIDS AND METABOLITES
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25 minutes Z
i TM
FIG. 2. The chromatogram represents the fatty acid composition of human platelet phosphatidylinositol taken from a subject consuming a dietary fish oil supplement containing eicosapentaenoic acid (20 : 5n-3) for 22 days. The 16 : 0, 18 : 0, 18 : 1, 18 : 2n-6, 20 : 4n-6, and 20:5n-3 represent 1.4, 42.9, 4.3, 0.6, 45.4, and 0.8 tool%, respectively, of the total fatty acids identified. Based on corresponding analysis of blank gel regions of the thin-layer chromatography plate, most of the unidentified peaks are due to background contamination.
standard solution containing five known fatty acid methyl esters (of varying chain length and unsaturation) in equal quantities. Results indicate an accuracy to within 0.2-0.3% of actual values. Figure 2 illustrates a typical chromatogram obtained by injecting, un-
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der the conditions previously described, the fatty acid methyl esters derived from 12 nmol of human platelet phosphatidylinositol. A total of 20 fatty acids were identified, representing 98% of the total fatty acids detected. Overall quantitative recovery of a known phosphatidylinositol standard following thin-layer chromatography, methylation, and gas-liquid chromatography was 95%.
Elution of Phosphatidylinositol from Silica Gel The elution of both radioactive and nonradioactive phosphatidylinositol from thin-layer chromatograms is often necessary when subsequent work requires a highly purified compound. Such applications may include the subsequent use of phosphatidylinositol for the determination of the positional distribution of the constituent fatty acids and characterizing the molecular species compositions, the direct use of phosphatidylinositol as a substrate for membrane/cytosolic enzyme assays in vitro (phospholipase C, phospholipase A2 and A~, phosphatidylinositol kinase, etc.), the formation of chemical derivatives (lysophosphatidylinositols, 1,2-diacylglycerols), and chemical analyses or physicochemical investigations. Although different elution procedures can be employed, our laboratory has applied the Arvidson procedure, 27 as originally outlined for phosphatidylcholine and phosphatidylethanolamine, to the elution of phosphatidylinositoF 8 from silica gel H thin-layer chromatograms sprayed with dichlorofluorescein for detection purposes. This method also allows for the removal of dichlorofluorescein. A brief description of a specific procedure which has been used in our laboratory for the elution of phosphatidylinositol from a 4 x 2.5 cm band of gel scrapings from a precoated and developed plate containing silica gel H (0.25 mm thick) is given herein. Following the scraping of the silica gel into a test tube with the aid of a razor blade, 3 ml of chloroform-methanol-acetic acid-water, 50 : 39 : 1 : 10 (v/v/v/v) is added followed by vortex mixing for 1 rain. After sedimentation by low-speed centrifugation, the solvent is removed via a Pasteur pipet and transferred to a second test tube. An additional 3-ml aliquot of the elution mixture is added to the residual gel scrapings, mixed, and subsequently pooled with the original extract after centrifugation to allow gel sedimentation. To the combined eluent, 2 ml of 4 N ammonium hydroxide is added and, after vortex mixing for 15 sec, the upper phase is removed by Pasteur pipet, discarded, and replaced by 2 ml of methanol-water, 1 : 1 (v/v). After mixing, 27 G. A. E. A r v i d s o n , Eur. J. Biochem. 4, 478 (1968). 28 B. J. Holub, Biochim. Biophys. Acta 369, 111 (1974).
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INOSITOL PHOSPHOLIPIDS AND METABOLITES
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the lower chloroform phase containing phosphatidylinositol is removed and the remaining upper phase is extracted again with 3 ml of chloroform to remove an additional amount of the phospholipid. Using this procedure, the combined chloroform portions (a total of approximately 6 ml) were found to contain an average of 87% of the original phosphatidylinositol applied to the origin of the thin-layer plates just prior to development. Larger solvent volumes than those described above relative to gel areas and multiple extractions can be used to provide recoveries >87% if desired. Acknowledgments We wish to thank Mr. D. G. Hamilton for his photographic expertise as well as Mrs. L. Thomas and Mrs. F. Graziotto for assistance in the manuscript preparation. The research was supported by the Medical Research Council of Canada and the Heart and Stroke
Foundation of Ontario.
[22] M o d u l a t i o n o f P h o s p h a t i d y l i n o s i t o l T u r n o v e r b y Liposomes Containing Phosphatidylinositol
By NABILA M. WASSEF and CARL R. ALVING The discovery of increased phosphatidylinositol (PI) turnover associated with receptor-agonist interaction was first made in 1953~ and since then numerous other instances of enhanced PI metabolism have been describedf1-8 In recent years the PI metabolic cycle has been recognized as part of a system that generates "messenger" molecules that influence a variety of cellular activities. 9,1° Although dozens of stimuli have been I M. R. Hokin and L. E. Hokin, J. Biol. Chem. 203, 967 (1953). 2 L. E. Hokin, Int. Rev. Cytol. 23, 187 (1968). 3 R. H. Michell, Biochim. Biophys. Acta 415, 81 (1975). 4 R. H. Michell, Trends Biochem. Sci. 4, 128 (1979). 5 R. H. Michell and C. J. Kirk, Trends Pharmacol. Sci. 2, 86 (1981). R. F. Irvine, R. M. C. Dawson, and N. Freinkel, in "Contemporary Metabolism" (N. Freinkel, ed.), Vol. 2, p. 301. Plenum, New York. 7 R. V. Farese, Metab., Clin. Exp. 32, 628 (1983). 8 j. Bleasdale, J. Eichberg, and G. Hauser, eds., "Inositol and Phosphoinositides: Metabolism and Regulation." Humana Press, Clifton, New Jersey, 1985. 9 M. J.Berridge and R. F. Irvine, Nature (London) 312, 315 (1984). 10 y . Nishizuka, Science 225, 1365 (1984).
METHODS 1N ENZYMOLOGY.VOL. 141
Copyright© 1987by AcademicPress. Inc. All rightsof reproductionin any formreserved.
INOSITOL
PHOSPHOLIPIDS
AND METABOLITES
[21]
similar to the concentrations of myo-inositol found in fetal rat serum during late gestation (Fig. 3). The methods described here for altering intracellular CMP and the availability of myo-inositol have proved useful in the manipulation of the relative rates of synthesis of phosphatidylinositol and phosphatidylglycerol in type 1I pneumonocytes. These methods complement others that have been described (e.g., use of Li ÷) for altering phospb_oinositide metabolism in a variety of cell types.
[21] N u t r i t i o n a l R e g u l a t i o n o f C e l l u l a r P h o s p h a t i d y l i n o s i t o l
By BRUCE J. HOLUB and CLARK M. SKEAFF Introduction A marked surge in interest has developed recently concerning the influence of nutrition on the level and composition of phosphatidylinositol in selected mammalian tissues and cells. This interest has been stimulated by the recognition that, in addition to providing a structural role in mainraining biological membranes and regulating the activity of membranebound enzymes, phosphatidylinositol is a precursor of the intracellular messengers 1,2-diacylglyceroP and inositol trisphosphate 2 (either directly or indirectly via its conversion to phosphatidylinositol 4,5-bisphosphate). The degradation of phosphatidylinositol, which contains predominantly lstearoyl 2-arachidonoyl molecular species in most mammalian cells, 3 also provides for the release of free arachidonic acid for conversion to the eicosanoids. Furthermore, dietary modifications (in myo-inositol levels, type of dietary fat, dietary cholesterol levels, etc.) can significantly influence the amount of phosphatidylinositol as well as its fatty acid and molecular species compositions 3'4 in certain cells; this may be important in the functioning of this phospholipid and its derivatives in both the 1 y. Nishizuka, Trends Biochem. Sci. 8, 13 (1983). 2 M. J. Berridge and R. F. Irvine, Nature (London) 312, 315 (1984). 3 B. J. Holub, Adv. Nutr. Res. 4, 107 (1982). 4 B. J. Holub, in "Inositol and Phosphoinositides: Metabolism and Regulation" (J. E. Bleasdale, J. Eichberg, and G. Hauser, eds.), p. 31. Humana Press, Clifton, New Jersey, 1985.
METHODS IN ENZYMOLOGY, VOL, 141
Copyright © 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.
[21]
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resting and stimulated cell. For example, the consumption of fish oils and concentrates containing eicosapentaenoic acid provides for the introduction of novel molecular species (namely, 1-acyl-2-eicosapentaenoylphosphatidylinositol) into circulating human platelets. 4 Nutritional modification of cellular phosphatidylinositol may eventually offer clinical applications in the control of phosphatidylinositol-mediated physiological responses. General Experimental Approaches and Principles Nutritional Considerations
Attention needs to be given to the choice of animal model and diet composition used to study the influence of nutrition on disorders related to phosphatidylinositol metabolism. For example, the female gerbil is highly sensitive toward the development of an intestinal lipodystrophy 5 and the young male rat toward an accumulation of hepatic triacylglycerol when fed inositol-deficient diets in a manner which depends on the type of dietary fat. 3 The use of semipurified diets (inositol deficient and inositol supplemented at fixed concentrations) containing essential nutrients at levels which satisfy the known requirements for the animal employed is recommended. The National Research Council has published the qualitative and quantitative nutrient requirements for laboratory animals 6 including the rat, mouse, gerbil, guinea pig, hamster, vole, and fish. Documents are also available from the National Academy of Sciences which give the specific requirements for other animals. A chow diet is an inappropriate "control diet" since it will likely differ considerably from the experimental diets in the level of almost all nutrients including fat, protein, carbohydrate, vitamins, and minerals in addition to containing endogenous forms of inositol, thereby greatly restricting interpretation of the experimental results. The amount of inositol in the experimental diets should be chemically assessed where possible to confirm the expected values and the suitability of the mixing procedures. For example, significant amounts of endogenous phosphatidylinositol may be present in dietary vegetable oils and other constituents. The routine measurement of food intake, weight gain, and gain:feed ratios of the animals during nutritional studies will help eliminate these three parameters as experimental variables across the dietary groups if they are found not to differ from each other. s D. M. Hegsted, K. C. Hayes, A. Gallagher, and H. Hanford, J. Nutr. 103, 302 (1973). 6 The National Research Council, in "Nutrient Requirements of Laboratory Animals," 3rd ed., No. 10. Natl. Acad. Sci., Washington, D.C., 1978.
236 Experimental
INOSITOL PHOSPHOLIPIDS AND METABOLITES
[21]
Procedures
Lipid Methodology The extraction of phospholipids from mammalian tissues and cells using chloroform-methanol mixtures as described by Folch et al. 7 and Bligh and Dyer 8 and modifications of these procedures have been described in detail with specific reference to the extraction of phosphatidylinositol.9,~° A number of chromatographic procedures ~°-14 (including column, conventional thin-layer, high-performance thin-layer, and highperformance liquid chromatography) have been described for the separation and isolation of phosphatidylinositol from lipid extracts leading to their quantitation by various approaches (for example, determination of lipid phosphorus, ~5:6 densitometry, ~7 fluorometric analysis, 12 etc.). Onedimensional thin-layer chromatography for the separation of phosphatidylinositol from phosphatidylserine and other phospholipids as outlined js has been frequently claimed to prove difficult. 19,2°One-, 2°,2~ t w o - , 16 and even three-dimensional22 thin-layer chromatographic systems have been reported to facilitate these separations. The direct quantitation of phosphatidylinositol by phosphorus determination in the presence of silica gel following thin-layer chromatography has been documented,~S,16 thereby avoiding elution and subsequent losses of the phospholipid from the gel scrapings. The positional distribution of fatty acyl chains in the 1- and 2-positions of isolated phosphatidylinositol can be determined23 by fatty acid analyses of the reaction products [lyso 7 j. Folch, M. Lees, and G. H. Sloane-Stanley, J. Biol. Chem. 226, 497 (1957). s E. G. Bligh and W. J. Dyer, Can. J. Biochem. Physiol. 37, 911 (1959), 9 F. B. St. C. Palmer, Biochim. Biophys. Acta 231, 134 (1971). l0 W. W. Christie, "Lipid Analysis," 2nd ed. Pergamon, Oxford, 1982. ti j. Stein and G. Stein, "Techniques in Lipid and Membrane Biochemistry," Part I. Elsevier/North-Holland Biomedical Press, Amsterdam, 1982. 12 C. A. Harrington, D. C. Fenimore, and J. Eichberg, Anal. Biochem. 106, 307 (1980). 13A. Karasawa, S. H. Yoshikawa, A. Miyakawa, A. Nishi, and R. Ishitani, J. Chromatogr. 260, 513 (1983). J4 M. Saito, Y. Tanaka, and S. Ando, Anal. Biochem. 132, 376 (1983). is A. F. Rosenthal and S. C. H. Han, J. LipidRes. 10, 243 (1969). 16 G. Rouser, S. Fleischer, and A. Yakamoto, Lipids 5, 494 (1970). ~7M. Goppelt and K. Resch, Anal. Biochem. 140, 152 (1984). is V. P. Skipski, R. F. Peterson, and M. Barclay, Biochem. J. 90, 374 (1964). 19 H. D. Kaulen, Anal. Biochem. 45~ 664 (1972). 20 D. Allan and S. Cockcroft, J. Lipid Res. 23, 1373 (1982). 2~ j. B. Fine and H. Sprecber, J. Lipid Res. 23, 660 (1982). z2 j. K. G. Kramer, R. C. Fouchard, and E. R. Farnworth, Lipids 18, 896 (1983). 23 B. J. Holub and A. Kuksis, Can. J. Biochem. 49, 1347 (1971).
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(1-acyl) phosphatidylinositol and free fatty acids] following digestion with phospholipase A2. The chemical classes of cellular phosphatidylinositol can be fractionated according to unsaturation by argentation thin-layer chromatography of the intact phospholipid 24 or its chemical derivatives (e.g., 1,2-diacylglycerol acetates following phospholipase C digestion and acetylation of 1,2-diacylglycerols,Z5 dimethylphosphatidic acidsZ6). The individual molecular species can be determined following quantitation of the various classes and their characterization by gas-liquid chromatography and further analyses of the positional distributions of the constituent fatty acids as required. The specific series of procedures as outlined in detail below have been developed and used in our laboratory to provide a simultaneous determination of the mass of cellular phosphatidylinositol and its fatty acid composition. This approach employs the combined use of one-dimensional thin-layer chromatography and gas-liquid chromatography of the fatty acid methyl esters (including methyl pentadecanoate as an internal standard) formed from the separated phosphatidylinositol.
Thin-Layer Chromatographic Separation of Phospholipids Separation of the individual phospholipids in the lipid extract is achieved using a simple and rapid one-dimensional thin-layer chromatography system. The thin-layer chromatography plates used are Merck silica gel 60 glass precoated 20 × 20 cm plates (without fluorescein indicator) with 0.25-ram layer thickness (Art 5721-7 British Drug House, Toronto, Ontario, Canada). Plates are kept at room temperature in the manufacturer's storage box and are removed Gust) prior to each use. All solvents used throughout the described procedures were purchased from Fisher Scientific (Toronto, Ontario, Canada) and were of ACS certified grade. Using a 25-pA Hamilton syringe, the lipid extract, dissolved in 25-50/xl of chloroform-methanol, 2 : 1 (v/v), is spotted in a straight band 3 cm long approximately 1.5-2 cm above the bottom of the plate. During the sample application, a gentle stream of nitrogen is blown across the origin in order to facilitate the spotting of a tight lipid band. Normally, 4-5 lanes can be spotted per plate. The best chromatographic separations are produced when 150-350/zg of lipid/cm or less is applied to the thin-layer chromatography plate. Spotting lipid samples in excess of 650/xg/cm frequently results in the overlapping of the sphingomyelin and phosphatidylcholine
24 B. J. Holub and A. Kuksis, J. Lipid Res. 12, 510 (1971). 25 V. G. Mahadevappa and B. J. Holub, Biochim. Biophys. Acta 713, 73 (1982). 2~ M. G. Luthra and A. Sheltawy, Biochem. J. 126, 1231 (1972).
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bands but does not compromise the separation of the other phospholipids. We have found that possible streaking of the phospholipids during development is eliminated if the nitrogen drying is continued for 3-5 min after spotting has been completed. The solvent system, consisting of chloroform-methanol-acetic acidwater, 50:37.5:3.5:2 (v/v/v/v), is mixed in a stock bottle and added to the development chamber [length, 22 cm; height, 22 cm; width, 10.5 cm (Desaga, Heidelberg, West Germany)]. The chamber is then lined completely on three sides with Whatman chromatography paper (0.18 mm thickness) and allowed to equilibrate until the solvent has run the entire height of the chromatography paper (usually 45-60 min). For optimal phospholipid separation, the plate should be placed in the development chamber shortly after equilibration is completed. For chromatographic separation, the plate is then placed as vertically as possible in the development chamber and allowed to develop until the solvent has run to within 0.5 cm of the top (an etched horizontal line can be drawn if desired) of the plate (usually 1.5-2 hr). When development is complete, the plate is removed and air dried at room temperature for 5-10 min. Using an atomizer with nitrogen gas flow, the gel surface is sprayed with a saturated solution of 2,7-dichlorofluorescein in methanol-water, 1 : 1 (v/v), until a slight yellow color is visible. After exposing the plate to ammonia vapor by placing it in a raised bottom developing chamber containing 50-100 ml of 8 N ammonia, the phospholipid bands are visualized under ultraviolet light at a wavelength of 366 nm. The color of the bands can be intensified, if necessary, by successive exposure to acetic acid and ammonia vapor. Figure 1 illustrates the typical chromatographic separation pattern obtained from a tissue/cellular lipid extract. In this case, rat liver extract was used for separation. The five major phospholipids, sphingomyelin, phosphatidylcholine, phosphatidylserine, phosphatidylinositol, and phosphatidylethanolamine, are completely separated. Lysophosphatidylcholine (Rf 0.03), although not visible in Fig. I, runs above the origin and below sphingomyelin. Cardiolipin runs above phosphatidylethanolamine while the neutral lipid fraction runs with the solvent front. The lower portion of the neutral lipid band contains mainly cholesterol and free fatty acids whereas the upper portion is composed mainly of triacylglycerols and cholesterol esters with some monoacylglycerols and diacylglycerols. We have routinely used this solvent system for the separation of the individual phospholipids from blood platelets of various species, including human. Phospholipids in lipid extracts from goat and rat lung lamellar bodies as well as from fetal calf serum and human skin fibroplasts have also been
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~NL
CL
PE
PI
PS PC SPH 0 FIG. I. Thin-layer chromatogram demonstrating the phospholipid separations of a rat liver extract as described in the text. The photograph was taken under ultraviolet light at 366 nm using a camera equipped with a 55-mm lens and #237 CID filter (Lee Filters, Ltd., Andover, Hants, UK). The following bands have been identified: sphingomyelin (SPH), R~. 0.06; phosphatidylcholine (PC), Rf 0.10; phosphatidylserine (PS), Rf 0.27; phosphatidylinositol (PI), Rf 0.38; phosphatidylethanolamine (PE), Rr 0.57; cardiolipin (CL), Rf 0.73; neutral lipid (NL). O, Origin.
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separated with success using this thin-layer chromatography system (J. Kirkland and T. Bray, S. Karmiol and W. Bettger, respectively, Department of Nutritional Sciences, University of Guelph, Ontario, Canada). Phospholipid separation of the lamellar body lipid extract has demonstrated that phosphatidylglycerol migrates between phosphatidylethanolamine and cardiolipin. In the agonist-stimulated cell, where phosphatidic acid may accumulate to significant levels, possible overlap of this phospholipid with phosphatidylethanolamine may slightly compromise the phosphatidylethanolamine determinations. We have found this one-dimensional thin-layer chromatography method for phospholipid separation to be highly reproducible. Furthermore, we have not experienced difficulty using this system with varying external humidity and temperature, factors which frequently render consistent phospholipid separations difficult. However, we have not tested these separations under extreme environmental conditions.
Preparation of Fatty Acid Methyl Esters The individual phospholipid bands are scraped into separate 16 x 100 mm screw-top glass test tubes containing 3 ml of 6% concentrated H2SO4 in methanol by volume and 5-10/.~g of monopentadecanoate (containing 15:0) as an internal standard. Other methylation procedures are available 1°,~1although control experiments need to be conducted when applied in the presence of gel scrapings. The future commercial availability of dipentadecanoylphosphatidylinositol will allow its addition as an internal standard during the lipid extraction phase. This use of phosphatidylinositol as an internal standard will eliminate the need to correct for any minor lipid losses incurred throughout the isolation procedure of phosphatidylinositol samples, whereas the use of the monopentadecanoate internal standard only accounts for lipid losses subsequent to the methylation phase. The amount of 15 : 0 internal standard used depends on the quantity of phospholipid being analyzed and should ideally represent approximately 20% of the total fatty acids present in the phospholipid. The tubes are tightly sealed with Teflon-lined caps, in order to minimize the evaporation of methylating reagent, vortexed for 60 sec, and then placed in an oven at 80 ° for 6-14 hr. When analyzing sphingomyelin, the longer methylation times of 12-16 hr are required while shorter times of 3-6 hr are generally sufficient for methylating the fatty acids in phosphatidylserine, phosphatidylcholine, phosphatidylinositol, and phosphatidylethanolamine. Upon completion, the tubes are removed from the oven and allowed to cool to room temperature. Following the addition of 2 ml of petroleum ether, each tube is vortexed for 60 sec. One milliliter of water is
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added and the tubes are vortexed again for 30 sec. The upper petroleum ether phase, containing the eluted fatty acid methyl esters, is removed and transferred to a 2-ml minivial. The vial is placed in a sand bath at 40° while the petroleum ether is evaporated to dryness under a gentle stream of nitrogen gas. The fatty acid methyl esters are then immediately dissolved in 25 ~1 of gas-liquid chromatography grade hexane.
Analysis of Fatty Acid Methyl Esters Using Gas-Liquid Chromatography The fatty acid methyl esters are analyzed using a 30 m x 0.53 mm i.d. DB-225 (25% cyanopropylphenyl) J&W Scientific megabore column (Chromatographic Specialities, Inc., Brockville, Ontario, Canada) installed on a Hewlett Packard 5890 gas liquid chromatograph with flame ionization detector. The column head pressure is set at 65 kPa and the column, split vent, and purge vent flow rates are adjusted to 6.8, 20, and 6.0 cc of helium per minute, respectively. The injection port, equipped with a glass split insert, and the detector are maintained at 250 °. The medical air, hydrogen, and nitrogen flow rates through the flame ionization detector are set at 360, 38, and 36 cc per minute, respectively. Normal injection volume of the fatty acid methyl esters dissolved in hexane is 1-2 /xl. When detecting trace amounts of fatty acid methyl esters in the sample, it may be essential to inject large quantities of sample onto the gas-liquid chromatograph. This can be achieved by evaporating, prior to injection, some of the 25 ~1 of hexane in which the fatty acid methyl esters are dissolved. The megabore column provides the superior resolving power of the capillary column while allowing for the quantification of trace amounts of fatty acid methyl esters that may otherwise be undetected by the capillary column due to its limited sample capacity. With instrument range 4 and attentuation 0 the chromatograph is run at an oven temperature of 210° producing complete fatty acid methyl ester separation within 25 min. The peak areas are measured using a Spectral Physics (SP4100) integrator and identified by comparing the retention times with known standards. Using the peak area and the known weight of the monopentadecanoate internal standard, the weight and moles of the individual fatty acids can be calculated. These results can be used to determine the moles of phospholipid. It is important that blank regions of the thin-layer plate corresponding to the phospholipid bands be scraped, methylated in the presence of an internal standard, and analyzed by gasliquid chromatography so that the sample peak areas can be corrected for any possible gel or solvent background contamination. Accuracy of the detector response is regularly verified by injection and quantification of a
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25 minutes Z
i TM
FIG. 2. The chromatogram represents the fatty acid composition of human platelet phosphatidylinositol taken from a subject consuming a dietary fish oil supplement containing eicosapentaenoic acid (20 : 5n-3) for 22 days. The 16 : 0, 18 : 0, 18 : 1, 18 : 2n-6, 20 : 4n-6, and 20:5n-3 represent 1.4, 42.9, 4.3, 0.6, 45.4, and 0.8 tool%, respectively, of the total fatty acids identified. Based on corresponding analysis of blank gel regions of the thin-layer chromatography plate, most of the unidentified peaks are due to background contamination.
standard solution containing five known fatty acid methyl esters (of varying chain length and unsaturation) in equal quantities. Results indicate an accuracy to within 0.2-0.3% of actual values. Figure 2 illustrates a typical chromatogram obtained by injecting, un-
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der the conditions previously described, the fatty acid methyl esters derived from 12 nmol of human platelet phosphatidylinositol. A total of 20 fatty acids were identified, representing 98% of the total fatty acids detected. Overall quantitative recovery of a known phosphatidylinositol standard following thin-layer chromatography, methylation, and gas-liquid chromatography was 95%.
Elution of Phosphatidylinositol from Silica Gel The elution of both radioactive and nonradioactive phosphatidylinositol from thin-layer chromatograms is often necessary when subsequent work requires a highly purified compound. Such applications may include the subsequent use of phosphatidylinositol for the determination of the positional distribution of the constituent fatty acids and characterizing the molecular species compositions, the direct use of phosphatidylinositol as a substrate for membrane/cytosolic enzyme assays in vitro (phospholipase C, phospholipase A2 and A~, phosphatidylinositol kinase, etc.), the formation of chemical derivatives (lysophosphatidylinositols, 1,2-diacylglycerols), and chemical analyses or physicochemical investigations. Although different elution procedures can be employed, our laboratory has applied the Arvidson procedure, 27 as originally outlined for phosphatidylcholine and phosphatidylethanolamine, to the elution of phosphatidylinositoF 8 from silica gel H thin-layer chromatograms sprayed with dichlorofluorescein for detection purposes. This method also allows for the removal of dichlorofluorescein. A brief description of a specific procedure which has been used in our laboratory for the elution of phosphatidylinositol from a 4 x 2.5 cm band of gel scrapings from a precoated and developed plate containing silica gel H (0.25 mm thick) is given herein. Following the scraping of the silica gel into a test tube with the aid of a razor blade, 3 ml of chloroform-methanol-acetic acid-water, 50 : 39 : 1 : 10 (v/v/v/v) is added followed by vortex mixing for 1 rain. After sedimentation by low-speed centrifugation, the solvent is removed via a Pasteur pipet and transferred to a second test tube. An additional 3-ml aliquot of the elution mixture is added to the residual gel scrapings, mixed, and subsequently pooled with the original extract after centrifugation to allow gel sedimentation. To the combined eluent, 2 ml of 4 N ammonium hydroxide is added and, after vortex mixing for 15 sec, the upper phase is removed by Pasteur pipet, discarded, and replaced by 2 ml of methanol-water, 1 : 1 (v/v). After mixing, 27 G. A. E. A r v i d s o n , Eur. J. Biochem. 4, 478 (1968). 28 B. J. Holub, Biochim. Biophys. Acta 369, 111 (1974).
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the lower chloroform phase containing phosphatidylinositol is removed and the remaining upper phase is extracted again with 3 ml of chloroform to remove an additional amount of the phospholipid. Using this procedure, the combined chloroform portions (a total of approximately 6 ml) were found to contain an average of 87% of the original phosphatidylinositol applied to the origin of the thin-layer plates just prior to development. Larger solvent volumes than those described above relative to gel areas and multiple extractions can be used to provide recoveries >87% if desired. Acknowledgments We wish to thank Mr. D. G. Hamilton for his photographic expertise as well as Mrs. L. Thomas and Mrs. F. Graziotto for assistance in the manuscript preparation. The research was supported by the Medical Research Council of Canada and the Heart and Stroke
Foundation of Ontario.
[22] M o d u l a t i o n o f P h o s p h a t i d y l i n o s i t o l T u r n o v e r b y Liposomes Containing Phosphatidylinositol
By NABILA M. WASSEF and CARL R. ALVING The discovery of increased phosphatidylinositol (PI) turnover associated with receptor-agonist interaction was first made in 1953~ and since then numerous other instances of enhanced PI metabolism have been describedf1-8 In recent years the PI metabolic cycle has been recognized as part of a system that generates "messenger" molecules that influence a variety of cellular activities. 9,1° Although dozens of stimuli have been I M. R. Hokin and L. E. Hokin, J. Biol. Chem. 203, 967 (1953). 2 L. E. Hokin, Int. Rev. Cytol. 23, 187 (1968). 3 R. H. Michell, Biochim. Biophys. Acta 415, 81 (1975). 4 R. H. Michell, Trends Biochem. Sci. 4, 128 (1979). 5 R. H. Michell and C. J. Kirk, Trends Pharmacol. Sci. 2, 86 (1981). R. F. Irvine, R. M. C. Dawson, and N. Freinkel, in "Contemporary Metabolism" (N. Freinkel, ed.), Vol. 2, p. 301. Plenum, New York. 7 R. V. Farese, Metab., Clin. Exp. 32, 628 (1983). 8 j. Bleasdale, J. Eichberg, and G. Hauser, eds., "Inositol and Phosphoinositides: Metabolism and Regulation." Humana Press, Clifton, New Jersey, 1985. 9 M. J.Berridge and R. F. Irvine, Nature (London) 312, 315 (1984). 10 y . Nishizuka, Science 225, 1365 (1984).
METHODS 1N ENZYMOLOGY.VOL. 141
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