. 1,2) Cholesterol also exisits in a buried form in phospholipid bilayer in the biomembrane.1,2) It is suggested that the interactions of cholesterol with phospholipids are based on the hydrophobic interaction. Therefore, it is presumed that cyclodextrins (CDs), cyclic oligosaccharides that have a hydrophobic cavity, interact with cholesterol to form inclusion complexes in aqueous solution. Several studies on the interactions of CDs with cholesterol have been reported. For example, intravenous administration of 2-hydroxypropyl-b-CD (HP-b-CD) in rats leads to a trasient decrease in plasma cholesterol levels.3) This phenomenon is explained as follows: in blood, high cholesterol concentration is available due to the complex formation with HP-b-CD, and this complex can be transported rapidly from the intravascular to the extravascular compartment. In other studies, it has been reported that cholesterol can be extracted from both cell and monolayer membranes by CDs.2,4,5) However, all studies concerning these interactions are qualitative and, to the best of our knowledge, no studies on the thermodynamic parameters of interaction of cholesterol with CDs in aqueous solution have been reported.We therefore investigated the interactions of cholesterol with CDs in aqueous solution quantitatively using the solubility measurement now common in pharmaceutical field, measurement of proton nuclear magnetic resonance ( 1 H-NMR), and Corey-Pauling-Koltun (CPK) atomic models. As CDs, a-CD, b-CD, HP-b-CD, g-CD, and heptakis (2,6-di-Omethyl)-b-CD (DOM-b-CD) were used. a-CD, b-CD, and g-CD contain six, seven, and eight glucopyranose units, respectively. The structure of the CDs is that of a truncated cone with average cavity diameters of 0.57, 0.78 and 0.95 nm for a-CD, b-CD, and g-CD, respectively. These three CDs are less hydrophobic, and HP-b-CD with the same cavity diameter as b-CD is even less hydrophobic. On the other hand, DOM-b-CD has a deeper cavity and is more hydrophobic than b-CD. ExperimentalMaterials a-CD, b-CD, g-CD, and DOM-b-CD, purchased from Nacalai Tesque Co. (Kyoto, Japan) were used after recrystallization from water. HP-b-CD was purchased from Aldrich Chemical Company, Inc. The average degree of the 2-hydroxylpropyl group per b-CD was 5.6. The CDs were dried for 12 h at 110°C in a vacuum before use. Cholesterol purchased from Sigma (St. Louis, MO, U.S.A.) was used without further purification. The water purified with Milli-Q Labo (Ͼ18 MW · cm) was used throughout experiment.Solubility Method (1): Solubility of Cholesterol First, the solubility of cholesterol in water was determined at 10, 25, 37, and 45Ϯ0.05°C. Excess cholesterol was added in L-type test tubes containing water and the test tubes were stoppered. The solutions containing excess cholesterol were shaken 100 times/min for 8 d to achieve the solubilitiy equilibrium. The solution was then filtered through a membrane filter (0.22 mm, PVDF Membrane, IWAKI) (Tokyo, Japan) and the filtrate (100.0 ml) was concentrated completely in rotary evaporater at 40°...

Heptakis (2,3,6-tri-O-methyl)-b-cyclodextrin (TOM-b-CyD) has recently been used to alter the membrane cholesterol content. For example, using TOM-b-CyD, cholesterol of the myometrial plasma was selectively depleted from the myometrial plasma membrane.1) TOM-b-CyD has also been used to clarify whether membrane proteins exist at an association with specialized microdomains called lipid rafts by depleting cholesterol contained in them.2) It is clear that cholesterol forms a soluble complex with TOM-b-CyD in aqueous solution. Although the interactions of cholesterol with TOM-b-CyD are particularly important, to the best of our knowledge, no studies on the thermodynamic parameters of the interaction in aqueous solution have been reported.We therefore investigated the interactions of cholesterol with TOM-b-CyD in aqueous solution quantitatively using the solubility measurement method now common in the pharmaceutical field, measurement of proton nuclear magnetic resonance ( 1 H-NMR), and Corey-Pauling-Koltum (CPK) atomic models. As cholesterol exists in a buried form in the phospholipid bilayer in biomembranes 3,4) the interactions of cholesterol with phospholipids are suggested to be based on the hydrophobic interaction. On the other hand, TOM-b-CyD has a deeper and more hydrophobic cavity than the parent cyclodextrin.5) Therefore, it is suggested that the interaction of cholesterol with TOM-b-CyD is based on the hydrophobic interaction in aqueous solution. In a previous paper, 6) we reported that heptakis (2,6-di-O-methyl)-b-cyclodextrin (DOM-b-CyD), which like TOM-b-CyD has a deeper and more hydrophobic cavity than the parent cyclodextrin, forms soluble complexes with cholesterol in aqueous solution.However, the inclusion behaviors of these hydrophobic cyclodextrins differ in general. For example, DOM-b-CyD penetrates the matrix of liposomes and extracts phospholipid from liposomes to form a soluble complex, whereas only a small amount of TOM-b-CyD penetrates the matrix of liposomes to remain there and therefore, TOM-b-CyD has very week ability to form a soluble complex with phospholipids. 7,8) For these reasons, TOM-b-CyD is used to alter membrane cholesterol content as mentioned above, although DOM-b-CyD is not used for this purpose. As another example, the formation constant of the complex between TOMb-CyD 9) and 8-anilino-1-naphthalenesulfonate (ANS) is smaller than that between DOM-b-CyD 10) and ANS. Furthermore, each incluson mode is different in the TOM-b-CyD-ANS complex and DOM-b-CyD-ANS complex. Therefore the comparison of the interaction between TOM-b-CyD and cholesterol with that between DOM-b-CyD and cholesterol in present paper would be informative and pertinent. ExperimentalMaterials TOM-b-CyD purchased from Nacalai Tesque Co. (Kyoto, Japan) was used after recrystallization from water and dried for 12 h at 110°C in a vacuum before use. Cholesterol purchased from Sigma (St. Louis, MO, U.S.A.) was used without further purification. Water purified with Milli-Q Labo (Ͼ18 MW · cm) was used throughout the ex...