418
RECEPTORS, TRANSPORT, AND BINDING PROTEINS
[45]
[45] I n c o r p o r a t i o n of f l - C a r o t e n e into M i x e d Micelles B y L O U I S E M . C A N F I E L D , THOMAS A . F R I T Z ,
and
THOMAS E . T A a A R A
Introduction
Dietary B-carotene is correlated with decreased incidence of a variety of cancers,~ and it may have biological functions independent of its provitamin A activity. 2,3 These discoveries have led to renewed interest in the biological functions of carotene and the need to develop reliable in vitro assay systems. However, owing to its structure (Fig. 1), B-carotene is insoluble in water and has limited solubility in organic solvents. 4 In addition, B-carotene is easily oxidized both by light and by components in biological fluids, for example, metals and active oxygen species) These factors have complicated the study of B-carotene metabolism; in fact, the differential solubility of B-carotene may explain the disparate results obtained by different laboratories. 6 This chapter presents a method for the preparation of mixed micelles containing known quantities of B-carotene. The preparation is stable, reproducible, and simple to prepare. Although these micelles may have general application as a delivery vehicle in a variety of in vitro systems, the procedure was modified from an earlier preparation 7 designed to simulate micelles formed in the lumen of the human small intestine. Thus, the preparation is particularly well suited for the study of intestinal absorption and metabolism of B-carotene. Reagents and Materials Stock Solutions
Bile salts, 0.1 M: A stock solution containing 30 mM sodium glycocholate, 30 mM sodium glycochenodeoxycholate, 15 mM sodium glycodeoxycholate, 10 mM sodium taurocholate, 10 mM sodium taurochenodeoxycholate, and 5 mM sodium taurodeoxyi R. Peto, R. Doll, J. D. Buckley, and M. B. Sporn, Nature (London) 29tl, 201 (1980). 2 T. E. Edes, W. Thornton, and J. Shah, J. Nutr. 119, 796 (1989). 3 A. Bendich, Clin. Nutr. 7, 113 (1988). 4 E. DeRitter and A. E. Purcell, in "Carotenoids as Colorants and Vitamin A Precursors" (J. C. Bauernfeind, ed.), p. 815. Academic Press, New York, 1981. 5 G. W. Burton and K. U. Ingold, Science 224, 569 (1984). 6 D. S. Goodman and J. A. Olson, this series, Vol. 15, p. 462. 7 M. EI-Gorab and B. A. Underwood, Biochem. Biophys. Acta 306, 58 (1973).
METHODS IN ENZYMOLOGY, VOL. 189
Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
[45]
B-CAROTENE MICELLES
419
FIG. 1. Structure of fl-carotene.
cholate is prepared as previously described. 7 The bile salts are mixed at 37° in doubly distilled, deionized water (MilliQ, MiUipore, Bedford, MA) to provide a final bile salt concentration of 0.1 M. All bile salts are certified over 98% pure and are products of Sigma (St. Louis, MO). Stock solutions are stored at - 2 0 ° until use. /3-Carotene: Stock solutions of/3-carotene (Fluka, Buchs, Switzerland), typically 1.5 mM, in HPLC-grade tetrahydrofuran (THF) with 0.25 g/liter butylated hydroxytoluene (BHT) are prepared daily from crystalline /3-carotene and maintained at - 2 0 ° under argon until use. Concentrations and purity are verified spectroscopically. Fatty acids: Fatty acids, certified over 98% pure are products of Sigma. Individual stock solutions, typically 100-150 mM, of triolein, monoolein, oleic acid, and phosphatidylcholine are prepared in HPLC-grade hexane and maintained in darkened glass vials at - 2 0 ° under argon until use. Krebs-Ringer bicarbonate buffer: Krebs-Ringer bicarbonate medium (without calcium chloride and sodium bicarbonate) is purchased from Sigma; 15 mM sodium bicarbonate and 1 mM calcium chloride is added, and the buffer is adjusted to pH 7.4 with NaOH. The final sodium ion concentration in the buffer is 0.14 M. Preparation of Micellar Solutions All manipulations involving carotenoids are performed under subdued light. The use of aluminum foil to cover solutions is strictly avoided as/3carotene is readily photooxidized by reflection of stray light. Appropriate volumes of fatty acid stock solutions in hexane are added to a glass flask to provide final concentrations as follows: triolein (1.13 mM), monoolein (2.50 mM), oleic acid (7.5 mM), and phosphatidylcholine (0.68 mM). Appropriate amounts of/3-carotene from stock solutions are added to the fatty acids to provide concentrations in the final micellar solutions from 20 to 200/zM (Fig. 2). The solvents are evaporated just
420
RECEPTORS, TRANSPORT, AND BINDING PROTEINS
[45]
=,
O
8
/ ,
,
|
,
i
,
,
,
i00 #-CAROTENE
i
,
r
200 1N
M
,
i
,
300 I
~
,
,
i
400
SOLUTION,/JM
FIG. 2. Incorporation of B-carotene into a mixed micellar solution. /3-Carotene was sonicated at 37° for 2 hr in the presence of bile salts and fatty acids as described in the text. The micellar phase was separated by centrifugation at 106,000 g for 18 hr at 4° and analyzed for/3-carotene by the spectrophotometric assay.
until dryness under argon, and a volume of bile salt stock (37 °) to provide a final bile salt concentration of 12 mM is added with shaking to emulsify the fl-carotene. Krebs-Ringer buffer (37°) is added to volume, providing a final sodium concentration of 0.15 M. The resulting suspension is mixed by vigorous shaking until the solution is homogeneous. The preparation is sonicated in glass flasks for 2 hr at 37° in a bath sonicator (Bransonic 220) with occasional shaking. On completion of the sonication procedure, the turbid mixture is transferred to precoated Beckman Ultra-Clear ultracentrifuge tubes 8 and centrifuged at 4 ° for 2-24 hr at 106,000 g using a 50 Ti rotor (Beckman, Palo Alto, CA). Extent of incorporation of fl-carotene did not vary significantly when mixtures were centrifuged for 2, 5, 18, or 24 hr. For convenience, we typically used 2 or 18 hr. Following centrifugation, the upper lipid layer is removed from the clear aqueous solution by aspiration with a Pasteur pipette. Solubilization of/3-carotene in the lower aqueous phase is taken to be evidence of micellar formation. Spectrophotometric
Assay
Concentrations of fl-carotene in micelles are determined by scanning from 250 to 600 nm in a Beckman DU-40 scanning spectrophotometer. The absorption maximum of/3-carotene in the micellar phase is at 460 nm, as opposed to 450-452 nm in organic solutions. 4 B-Carotene concentras L. Holmquist, J. Lipid Res. 23, 1249 (1982).
[45]
B-CAROTENE MICELLES
421
tions are determined at the absorption maximum using an extinction coefficient of 2620 (El%m) 9 after correction for the absorption of a blank micelle solution not containing carotene. To ensure that the absorption being measured in the micellar solutions is not quenched by interaction of lipids in the micelles, and that the extinction coefficient is not significantly altered, B-carotene is extracted from micelles and concentrations verified by HPLC. Chromatographic Assay
Extraction of B-Carotenefrom Micelles. B-Carotene is extracted from micelles with 4 volumes of hexane/ethanol (3 : 1, v/v) containing 0.25 g/ liter BHT and a known concentration of C45 B-carotene as internal standard.~° Samples are vortexed 2 min and phases separated by centrifugation for 10 min at 1000 g. The upper hexane layer is aspirated with a pipette, dried under nitrogen, blanketed with argon, and stored at - 7 0 ° until assayed. The extraction efficiency in a typical procedure in our laboratory was 87.5 - 4.4% (n = 6). Assay of B-Carotene. Samples are resuspended in acetonitrile/THF (85 : 15, v/v) containing 0.25 g/liter BHT, injected onto a Waters (Bedford, MA) HPLC system using a Beckman Ultrasphere ODS 5-/zm C~8 column (4.6 mm x 25 cm), and eluted with acetonitrile/THF (85 : 15, v/v) containing 0.25 g/liter BHT at a flow rate of 2.5 ml/min. 11 Concentrations are determined from a standard curve run daily using C45 B-carotene as the internal standard.
fl-Carotene in Micellar Solutions As shown in Fig. 2, the maximum concentration of fl-carotene incorporated into micelles using this method is 15/zM, well above physiological concentrations. ~2Thus, this preparation provides a method for delivering fl-carotene within the physiological range for most biological systems. Incorporation offl-carotene into micelles is linear from 20 to 100 /zM added carotene. When fl-carotene is added in concentrations greater than 400 /~M, solutions are turbid and crystals, possibly bile salts, are visible in the solution. Independent measurements of B-carotene extracted from micelles and injected onto the HPLC agree within 12 -+ 5.8% 9 0 . Isler, H. Lindlar, M. Montavon, R. Rugge, and P. Zeller, Heir. Chim. Acta 39, 249 (1956). ~0Gift of Dr. Frederick Khachik, Nutrition Composition Laboratory, U.S. Department of Agriculture, Beltsville, Maryland. n y . M. Peng and J. Beaudry, J. Chromatogr. 273, 410 (1983). n j. Erdman, Clin. Nutr. 7, 101 (1988).
422
RECEPTORS, T R A N S P O R T , A N D B I N D I N G PROTEINS
[45]
r~
O
z
4 0 ]00
200
300
400
/~)~ROTENE IN MICEU.ARSOLUTION,~M
FIG. 3. Percent/3-carotene incorporated into a mixed micellar solution as a function of/3carotene added. Experimental conditions and procedures were as indicated in Fig. 2. (n = 6) o f the spectroscopic measurements. The percent fl-carotene that is incorporated into micelles varies from about 4 to 13% of the initial concentration, and the percent incorporation decreases with increasing concentrations (Fig. 3). When stored at 4 °, the micelles are stable for 24 hr as determined by spectrophotometric assay ( < 5 % decrease in absorption). By 48 hr, absorption at 460 nm decreases by about 10%, apparently indicating degradation of fl-carotene and/or micelles. Acknowledgment The work was supported in part by Contract N01 HD2992, grant #CA-27502, and grant #CA-23074 from the NIH, Bethesda, MD.
RECEPTORS, TRANSPORT, AND BINDING PROTEINS
[45]
[45] I n c o r p o r a t i o n of f l - C a r o t e n e into M i x e d Micelles B y L O U I S E M . C A N F I E L D , THOMAS A . F R I T Z ,
and
THOMAS E . T A a A R A
Introduction
Dietary B-carotene is correlated with decreased incidence of a variety of cancers,~ and it may have biological functions independent of its provitamin A activity. 2,3 These discoveries have led to renewed interest in the biological functions of carotene and the need to develop reliable in vitro assay systems. However, owing to its structure (Fig. 1), B-carotene is insoluble in water and has limited solubility in organic solvents. 4 In addition, B-carotene is easily oxidized both by light and by components in biological fluids, for example, metals and active oxygen species) These factors have complicated the study of B-carotene metabolism; in fact, the differential solubility of B-carotene may explain the disparate results obtained by different laboratories. 6 This chapter presents a method for the preparation of mixed micelles containing known quantities of B-carotene. The preparation is stable, reproducible, and simple to prepare. Although these micelles may have general application as a delivery vehicle in a variety of in vitro systems, the procedure was modified from an earlier preparation 7 designed to simulate micelles formed in the lumen of the human small intestine. Thus, the preparation is particularly well suited for the study of intestinal absorption and metabolism of B-carotene. Reagents and Materials Stock Solutions
Bile salts, 0.1 M: A stock solution containing 30 mM sodium glycocholate, 30 mM sodium glycochenodeoxycholate, 15 mM sodium glycodeoxycholate, 10 mM sodium taurocholate, 10 mM sodium taurochenodeoxycholate, and 5 mM sodium taurodeoxyi R. Peto, R. Doll, J. D. Buckley, and M. B. Sporn, Nature (London) 29tl, 201 (1980). 2 T. E. Edes, W. Thornton, and J. Shah, J. Nutr. 119, 796 (1989). 3 A. Bendich, Clin. Nutr. 7, 113 (1988). 4 E. DeRitter and A. E. Purcell, in "Carotenoids as Colorants and Vitamin A Precursors" (J. C. Bauernfeind, ed.), p. 815. Academic Press, New York, 1981. 5 G. W. Burton and K. U. Ingold, Science 224, 569 (1984). 6 D. S. Goodman and J. A. Olson, this series, Vol. 15, p. 462. 7 M. EI-Gorab and B. A. Underwood, Biochem. Biophys. Acta 306, 58 (1973).
METHODS IN ENZYMOLOGY, VOL. 189
Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
[45]
B-CAROTENE MICELLES
419
FIG. 1. Structure of fl-carotene.
cholate is prepared as previously described. 7 The bile salts are mixed at 37° in doubly distilled, deionized water (MilliQ, MiUipore, Bedford, MA) to provide a final bile salt concentration of 0.1 M. All bile salts are certified over 98% pure and are products of Sigma (St. Louis, MO). Stock solutions are stored at - 2 0 ° until use. /3-Carotene: Stock solutions of/3-carotene (Fluka, Buchs, Switzerland), typically 1.5 mM, in HPLC-grade tetrahydrofuran (THF) with 0.25 g/liter butylated hydroxytoluene (BHT) are prepared daily from crystalline /3-carotene and maintained at - 2 0 ° under argon until use. Concentrations and purity are verified spectroscopically. Fatty acids: Fatty acids, certified over 98% pure are products of Sigma. Individual stock solutions, typically 100-150 mM, of triolein, monoolein, oleic acid, and phosphatidylcholine are prepared in HPLC-grade hexane and maintained in darkened glass vials at - 2 0 ° under argon until use. Krebs-Ringer bicarbonate buffer: Krebs-Ringer bicarbonate medium (without calcium chloride and sodium bicarbonate) is purchased from Sigma; 15 mM sodium bicarbonate and 1 mM calcium chloride is added, and the buffer is adjusted to pH 7.4 with NaOH. The final sodium ion concentration in the buffer is 0.14 M. Preparation of Micellar Solutions All manipulations involving carotenoids are performed under subdued light. The use of aluminum foil to cover solutions is strictly avoided as/3carotene is readily photooxidized by reflection of stray light. Appropriate volumes of fatty acid stock solutions in hexane are added to a glass flask to provide final concentrations as follows: triolein (1.13 mM), monoolein (2.50 mM), oleic acid (7.5 mM), and phosphatidylcholine (0.68 mM). Appropriate amounts of/3-carotene from stock solutions are added to the fatty acids to provide concentrations in the final micellar solutions from 20 to 200/zM (Fig. 2). The solvents are evaporated just
420
RECEPTORS, TRANSPORT, AND BINDING PROTEINS
[45]
=,
O
8
/ ,
,
|
,
i
,
,
,
i00 #-CAROTENE
i
,
r
200 1N
M
,
i
,
300 I
~
,
,
i
400
SOLUTION,/JM
FIG. 2. Incorporation of B-carotene into a mixed micellar solution. /3-Carotene was sonicated at 37° for 2 hr in the presence of bile salts and fatty acids as described in the text. The micellar phase was separated by centrifugation at 106,000 g for 18 hr at 4° and analyzed for/3-carotene by the spectrophotometric assay.
until dryness under argon, and a volume of bile salt stock (37 °) to provide a final bile salt concentration of 12 mM is added with shaking to emulsify the fl-carotene. Krebs-Ringer buffer (37°) is added to volume, providing a final sodium concentration of 0.15 M. The resulting suspension is mixed by vigorous shaking until the solution is homogeneous. The preparation is sonicated in glass flasks for 2 hr at 37° in a bath sonicator (Bransonic 220) with occasional shaking. On completion of the sonication procedure, the turbid mixture is transferred to precoated Beckman Ultra-Clear ultracentrifuge tubes 8 and centrifuged at 4 ° for 2-24 hr at 106,000 g using a 50 Ti rotor (Beckman, Palo Alto, CA). Extent of incorporation of fl-carotene did not vary significantly when mixtures were centrifuged for 2, 5, 18, or 24 hr. For convenience, we typically used 2 or 18 hr. Following centrifugation, the upper lipid layer is removed from the clear aqueous solution by aspiration with a Pasteur pipette. Solubilization of/3-carotene in the lower aqueous phase is taken to be evidence of micellar formation. Spectrophotometric
Assay
Concentrations of fl-carotene in micelles are determined by scanning from 250 to 600 nm in a Beckman DU-40 scanning spectrophotometer. The absorption maximum of/3-carotene in the micellar phase is at 460 nm, as opposed to 450-452 nm in organic solutions. 4 B-Carotene concentras L. Holmquist, J. Lipid Res. 23, 1249 (1982).
[45]
B-CAROTENE MICELLES
421
tions are determined at the absorption maximum using an extinction coefficient of 2620 (El%m) 9 after correction for the absorption of a blank micelle solution not containing carotene. To ensure that the absorption being measured in the micellar solutions is not quenched by interaction of lipids in the micelles, and that the extinction coefficient is not significantly altered, B-carotene is extracted from micelles and concentrations verified by HPLC. Chromatographic Assay
Extraction of B-Carotenefrom Micelles. B-Carotene is extracted from micelles with 4 volumes of hexane/ethanol (3 : 1, v/v) containing 0.25 g/ liter BHT and a known concentration of C45 B-carotene as internal standard.~° Samples are vortexed 2 min and phases separated by centrifugation for 10 min at 1000 g. The upper hexane layer is aspirated with a pipette, dried under nitrogen, blanketed with argon, and stored at - 7 0 ° until assayed. The extraction efficiency in a typical procedure in our laboratory was 87.5 - 4.4% (n = 6). Assay of B-Carotene. Samples are resuspended in acetonitrile/THF (85 : 15, v/v) containing 0.25 g/liter BHT, injected onto a Waters (Bedford, MA) HPLC system using a Beckman Ultrasphere ODS 5-/zm C~8 column (4.6 mm x 25 cm), and eluted with acetonitrile/THF (85 : 15, v/v) containing 0.25 g/liter BHT at a flow rate of 2.5 ml/min. 11 Concentrations are determined from a standard curve run daily using C45 B-carotene as the internal standard.
fl-Carotene in Micellar Solutions As shown in Fig. 2, the maximum concentration of fl-carotene incorporated into micelles using this method is 15/zM, well above physiological concentrations. ~2Thus, this preparation provides a method for delivering fl-carotene within the physiological range for most biological systems. Incorporation offl-carotene into micelles is linear from 20 to 100 /zM added carotene. When fl-carotene is added in concentrations greater than 400 /~M, solutions are turbid and crystals, possibly bile salts, are visible in the solution. Independent measurements of B-carotene extracted from micelles and injected onto the HPLC agree within 12 -+ 5.8% 9 0 . Isler, H. Lindlar, M. Montavon, R. Rugge, and P. Zeller, Heir. Chim. Acta 39, 249 (1956). ~0Gift of Dr. Frederick Khachik, Nutrition Composition Laboratory, U.S. Department of Agriculture, Beltsville, Maryland. n y . M. Peng and J. Beaudry, J. Chromatogr. 273, 410 (1983). n j. Erdman, Clin. Nutr. 7, 101 (1988).
422
RECEPTORS, T R A N S P O R T , A N D B I N D I N G PROTEINS
[45]
r~
O
z
4 0 ]00
200
300
400
/~)~ROTENE IN MICEU.ARSOLUTION,~M
FIG. 3. Percent/3-carotene incorporated into a mixed micellar solution as a function of/3carotene added. Experimental conditions and procedures were as indicated in Fig. 2. (n = 6) o f the spectroscopic measurements. The percent fl-carotene that is incorporated into micelles varies from about 4 to 13% of the initial concentration, and the percent incorporation decreases with increasing concentrations (Fig. 3). When stored at 4 °, the micelles are stable for 24 hr as determined by spectrophotometric assay ( < 5 % decrease in absorption). By 48 hr, absorption at 460 nm decreases by about 10%, apparently indicating degradation of fl-carotene and/or micelles. Acknowledgment The work was supported in part by Contract N01 HD2992, grant #CA-27502, and grant #CA-23074 from the NIH, Bethesda, MD.
