Fluidity Parameters of Lipid Regions Determined by Fluorescence Polarization

Membrane lipid composition and cellular function.

Alpha-, beta- and gamma-cyclodextrins are cyclic hexamers, heptamers, and octamers of glucose, respectively, and thus are hydrophilic; nevertheless, they have the ability to solubilize lipids through the formation of molecular inclusion complexes. The volume of lipophilic space involved in the solubilization process increases with the number of glucose units in the cyclodextrin molecule and, consequently, cyclodextrins were found to have different effects on human erythrocytes: (a) in the induction of shape change from discocyte to spherocyte the potency was observed to be alpha greater than gamma, but with beta-cyclodextrin hemolysis occurred before the change was complete; (b) in the increase of fluorescence intensity of 1-anilinonaphthalene-8-sulfonate in cyclodextrin-pretreated membranes, the observed potency was beta much greater than gamma greater than alpha; (c) in the release of potassium and hemoglobin, the potency was beta greater than alpha greater than gamma. The potencies of cyclodextrin for solubilizing various components of erythrocytes were alpha greater than beta much greater than gamma for phospholipids, beta much greater than gamma greater than alpha for cholesterol and beta much greater than gamma greater than alpha for proteins. The solubilization potencies were derived from concentration/final-effect curves. The above processes occurred without entry of solubilizer into the membrane, since (a) beta-[14C]cyclodextrin did not bind to erythrocytes and (b) cyclodextrins did not enter the cholesterol monolayer. A study of the [3H]cholesterol in erythrocytes indicated that beta-cyclodextrin extracted this lipid from membrane into a new compartment located in the aqueous phase which could equilibrate rapidly with additional erythrocytes. Therefore, the effects of cyclodextrins differ from those of detergents which first incorporate themselves into membranes then extract membrane components into supramolecular micelles.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1989.tb15171.x

Differential effects of α‐, β‐ and γ‐cyclodextrins on human erythrocytes

Mechanisms and consequences of cellular cholesterol exchange and transfer

Cholesterol transport between cells and high-density lipoproteins.

Factors influencing the lipid composition and fluidity of red cell membranes in vitro: production of red cells possessing more than two cholesterols per phospholipid

Acanthocytic red cells in patients with abetalipoproteinemia are morphologically similar to the red cells in spur cell anemia. Fluidity of membrane lipids is decreased in spur cells due to their excess cholesterol content. Acanthocyte membranes have an increased content of sphingomyelin and a decreased content of lecithin. To assess the effect of this abnormality of acanthocyte membrane lipid composition on membrane fluidity, we studied red cells from five patients with abetalipoproteinemia and four obligate heterozygote family members.

Membrane fluidity was measured in terms of microviscosity (¯η) at 37°C, assessed by means of the fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene. It was increased from 3.2±0.1 poise in normals to 4.01-4.14 poise in acanthocytes. This was associated with an increase in the sphingomyelin/lecithin ratio from 0.84±0.08 in normals in 1.45-1.61 in acanthocytes. The ¯η of acanthocyte membranes was not influenced by the degree of vitamin E deficiency. Similar changes in ¯η were observed in liposomes prepared from red cell lipids. Heterozygotes had normal sphingomyelin/lecithin ratios and normal values for ¯η. The flow activation energy for viscosity, a measure of the degree of order in the hydrophobic portion of the membrane, was decreased from 8.3 kcal/mole in normal red cells to 7.2 kcal/mole in acanthocytes, indicating that acanthocyte membrane lipids are more ordered. Variations in the sphingomyelin/lecithin mole ratio of liposomes prepared from brain sphingomyelin and egg lecithin with equimolar cholesterol caused similar changes in both ¯η and activation energy. The deformability of acanthocytes, assessed by means of filtration through 3-μm filters, was decreased.

These studies indicate that the increased sphingomyelin/lecithin ratio of acanthocytes is responsible for their decreased membrane fluidity. As in spur cells and in red cells enriched with cholesterol in vitro, this decrease in membrane fluidity occurs coincidentally with an abnormality in cell contour and an impairment in cell deformability.

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Decreased Fluidity of Red Cell Membrane Lipids in Abetalipoproteinemia

Progressive hemolytic anemia occurred in a 4 1/2-year-old girl with familial intrahepatic cholestasis; a peripheral smear contained bizarre spiculated "spur" red cells. Analysis of this patient's fresh red cells revealed a 59% increase in cholesterol content with a normal phospholipid content and therefore an increase in the cholesterol/phospholipid molar ratio to 1.35 (normal = 0.92). A similar abnormality of lipid composition was present in serum lipoproteins. The lipid abnormality in red cell membrane was associated with a decrease in membrane fluidity, as assessed by the fluorescence polarization of the hydrophobic probe 1,6-diphenyl-1,3,5-hexatriene. Following incubation with patient's plasma, normal cells acquired a spur-shaped morphology with an associated decrease in osmotic fragility and a 25% increase in cholesterol content. The patient's cells, during incubation with normal plasma, acquired morphologic features of spiculated spherocytes with an increase in osmotic fragility and a 21% decrease in cholesterol content. Chenodeoxycholate and lithocholate were present in markedly elevated concentrations in serum. These studies show that a process identical to spur cell anemia in alcoholic cirrhosis may accompany severe liver disease in children with intrahepatic cholestasis.

Increased Cholesterol and Decreased Fluidity of Red Cell Membranes (Spur Cell Anemia) in Progressive Intrahepatic Cholestasis

The molecular defect responsible for the red cell abnormality in hereditary spherocytosis (HS) remains elusive. A number of workers have qucstiond whether the defect in HS results from an abnormality of either lipid composition or lipid organization in the membrane. However, direct measurements of the cholesterol/phospholipid mole ratio of HS membranes have been normal (Cooper 8( Jandl, 1969a). Although early measurements suggested that phospholipid composition was abnormal a number of laboratories have established that phospholipid composition is normal in HS (e.g. Reed & Swisher, 1966). Similarly, a suggestion that fatty acid composition is abnormal (Kuiper 8( Livne, 1972) has not been confirmed (Zail & Pickering, 1979). Three findings which appear to be established arc that HS red cells have less lipid per cell than normal both before and after splenectomy (Reed & Swisher, 1966; Cooper & Jandl, I%%), that they have less surface area per unit lipid (Cooper & Jandl, 1969a), and that lipid is lost from HS cell membranes a t a rate more rapid than normal under conditions of metabolic deprivation (Reed & Swisher, 1966; Cooper & Jandl, 1969b). Fluidity is a characteristic Lvhich reflects the organization of lipid in cell membranes (Cooper, 1977). It is influenced by factors such as the length and saturation of membrane fatty acids, the composition of phospholipids, the presence of lysophosphatides, and the relative amounts of cholesterol and phospholipid. Membrane proteins may also influence the fluidity of lipids in membranes. Aloni et a1 (1975) have reported that membrane fluidity is decreased in HS. Because studies in a number of laboratories have established that HS membrane lipid composition is normal, we undertook to reinvestigate the question of membrane fluidity in HS. This report represents a pooling of data obtained in two separate laboratories on two continents utilizing red cells from 13 patients with HS. It includes both children and adults, and it includes patients with and without spleens. Ghost membranes were prepared from freshly obtained red cells by the method of Ilodge et a1 (1963). The fluorescent probe, 1 ,&diphenyl1,3,5-hexatriene (IDPH) (Aldridge Chemical Company, Milwaukee, WI), was used to label ghosts. DPH was kept as a stock solution in tetrahydrofuran at a concentration of 2 x lop3 M. Immediately prior to use, it was diluted 132000 in 0.155 M NaCl with vigorous mixing. One volume of dilute DPH dispersion was added to one volume of red cell ghosts suspended in 0.155 M NaCl at a ghost concentration of 1 x 10X/ml and the mixture was incubated a t 37°C for 30 min. Measurements of fluorescence polarization in Philadelphia were performed at 37°C with an Elscint MV-1 microviscosimeter (Elscint Corporation, Hackensack, NJ), as described previously (Cooper et a l , 1978). Measurements of fluorescence polarization in Melbourne were performed at 25°C with a Hitachi-Perkin-Elmer MPM spectrofluorometer equipped with a thermostated cell block and polarization accessory (Thulborn &

Mary H. Leslie

Papers

Factors influencing the lipid composition and fluidity of red cell membranes in vitro: production of red cells possessing more than two cholesterols per phospholipid

Decreased Fluidity of Red Cell Membrane Lipids in Abetalipoproteinemia

Increased Cholesterol and Decreased Fluidity of Red Cell Membranes (Spur Cell Anemia) in Progressive Intrahepatic Cholestasis

Normal fluidity of red cell membranes in hereditary spherocytosis