morphological changes of human erythrocytes induced by cholesterol sulphate

Morphological Changes of Human ErythrocytesInduced by Cholesterol Sulphate

MARIA PRZYBYLSKA,1 MILyOSZ FABER,1 ANDRZEJ ZABOROWSKI,1 JACEK SWIETOSLyAWSKI,2

and MARIA BRYSZEWSKA1

1Institute of Biophysics, University of Lodz, Lodz, Poland, and 2Electron Microscopy Laboratory ofMedical Academy in Lodz, Lodz, Poland

Objectives: Morphological alterations of human erythrocytes in-duced by cholesterol sulphate (5-cholesten-3b-ol sulphate, CS)were studied.Design and Methods: Influence of CS on red blood cell stability (inisotonic conditions) by simultaneous application of flow cytometryand scanning electron microscopy was studied.Results: In isotonic medium CS induces erythrocyte size and shapechanges in dose- and time-dependent manner. Incubation (in vitro)of erythrocytes with CS concentrations from 4 3 1025 mol/dm3 to8 3 1025 mol/dm3 led to a progressive sphero-echinocitic shapetransformation accompanied by a cell size decrease. In contrast tothis, for CS content equal to 1 3 1025 mol/dm3 the maintenance ofthe normal biconcave shape of red blood cells was observed.Conclusions: The results suggest that CS, similarly to numerousevaginating amphiphilic agents, induces a transformation of theerythrocyte normal discoid shape to echinocytic form. This effectmay be caused, at least partly, by an asymmetric expansion of themembrane lipid bilayer due to asymmetric distribution of CS incor-porated into the membrane. The echinocytic shape transformationof erythrocytes indicated that CS intercalates in the outer hemileaf-let of the lipid bilayer leading to membrane externalization.Copyright © 1998 The Canadian Society of Clinical Chemists

KEY WORDS: cholesterol sulphate; erythrocyte; flowcytometry; scanning electron microscope.

Introduction

Sterols are essential lipid constituents of all eukary-otic cells. These compounds not only play a major

role in a variety of biochemical processes associatedwith biological membranes, but also serve as precur-sors for steroid hormones and bile acids.

Literature has indicated that the most importantsterol derivative is cholesterol sulphate (5-cho-lesten-3b-ol sulphate, CS)—a widespread compoundfound in mammalian body fluids and tissues (1–4),especially highly elevated in sperm (5), endome-trium (6,7), and keratinizing tissues such as epider-mis (8,9), nails (10), hair (10,11), and hoof (12).

Most of CS in animal cells resides in the plasmamembrane, where it is believed to be an indispens-able component, necessary to normal cellularfunction (13,14). In erythrocytes, CS participatesin membrane stabilization and protects it againsthypotonic (15) and thermally-induced (15,16) he-molysis. It has also been found to prevent Sendaivirus from fusion to both human erythrocytes andliposomal membranes (17). In the upper layer ofepidermis CS has been thought to play an impor-tant role in determining cohesion, desquamation,and permeability barrier function (18). CS mayalso be involved in the membrane modification ofthe spermatozoa acrosome during maturation pro-cess (5,19,20). It has been considered to be thecrucial participant in the process of the embryoimplantation into endometrium (6,7). It has alsobeen suggested to be involved in the blood co-agulation process by modulation of platelet func-tion (21,22). In adrenal tissue CS serves as aprecursor of 5-3b-hydroxysteroid sulphates (23,24). Besides, CS may regulate lipid metabolicpathways related to growth and differentiation(25). Relatively large amount of CS in bile andfeces suggests that it takes part in cholesterolexcretion (26).

CS has attracted much attention because of sev-eral serious diseases such as recessive X-linkedichthyosis (10,27,28), familial hypercholesterolemia(29), liver cirrhosis (29), homozygous sickle cellanemia (30), diabetes mellitus (31) and palmoplanarkeratoderma (32) where abnormal CS levels havebeen reported.

Although the molecular basis for the essential roleof CS in animal cells has long been the object ofintense interest, its function in the cell membranesis still not well understood. Therefore, the purpose ofthis study was to evaluate the influence of CS on redblood cell stability (in isotonic conditions) by simul-taneous application of flow cytometry and scanningelectron microscopy.

Correspondence: Dr. Maria Przybylska, Department ofBiophysics, University of Lodz, 12/16 Banacha St, 90-237Lodz, Poland. E-mail: [email protected]

Manuscript received April 14, 1997; revised and ac-cepted November 31, 1997.

Clinical Biochemistry, Vol. 31, No. 2, 73–79, 1998Copyright © 1998 The Canadian Society of Clinical Chemists

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CLINICAL BIOCHEMISTRY, VOLUME 31, MARCH 1998 73

Materials and methods

Chemicals of analytical grade were purchasedfrom POCH (Gliwice, Poland). Cholesterol sulphate(5-cholesten-3b-ol sulphate) was from Steraloids(Wilton, NH, USA).

BLOOD PREPARATION

Blood samples from adult healthy subjects, ob-tained from the Blood Bank of Lodz, were taken into3% trisodium citrate and centrifuged at 600 3 g for10 minutes. Next red blood cells were washed threetimes with an equal volume of sodium phosphate-buffered saline (PBS: 0.150 mol/dm3 NaCl; 0.0019mol/dm3, NaH2PO4; 0.0081 mol/dm3 Na2HPO4;pH 5 7.4) to remove plasma and white buffy coat.After washing, the packed erythrocytes were resus-pended in medium at 1% hematocrit (1.5 3 108

cells/cm3). To tubes containing 3 cm3 of this cellsuspension, CS in ethanol was added to give finalconcentrations from 0.4 3 1025 mol/dm3 to 8 3 1025

mol/dm3. To the other set of tubes the same volumeof ethanol was added. The final concentration ofethanol, equal in all tubes, was 1%. Additionally,control samples without ethanol were prepared.Next, blood samples were incubated at room temper-ature for 0.5, 1.0, 1.5, or 2.0 hours, respectively.After this time each of these samples was centri-fuged at 600 3 g for 10 minutes and resuspended inthe same medium.

All measurements of this study were performed onfreshly prepared erythrocytes.

FLOW CYTOMETRY

Red blood cells suspensions (0.5 cm3) containingapproximately 106 cells/cm3 were analyzed using aScatron Argus (Norway) arc lamp-based compactflow cytometer, with simultaneous separate detec-tion at low angle (LS1) and right angle (LS2). Thelight scattered near the forward direction (low an-gle) is expected to be proportional to the size (vol-ume) of the particle and is independent of cellrefractive index and shape, whereas scattering atthe right angle depends on cell shape and internalproperties of the scattering particles (33,34). LS1/LS2 is a dual parameter contour plot histogramproportional to the total cell diversity. For each set

of histograms the percentage of altered cells andpeak position (P) was obtained from values collectedby using a standard computer programme for theArgus cytometer.

RED BLOOD CELL SHAPE OBSERVATION

For scanning electron microscope observations,erythrocytes were washed twice with an equal vol-ume of sodium-potassium phosphate buffer (0.164mol/dm3 Na2HPO4, 0.036 mol/dm3 KH2PO4; pH 57.4) and were fixed with 1% glutaraldehyde in thesame buffer to achieve final haematocrit of about50%. Fixed cells were allowed to settle on standardmicroscopic cover glasses. After 1.0 h cover glasseswere washed twice with phosphate buffer. Subse-quently samples were dehydrated with successivewashes in ascending ethanol series (30%, 50%, 70%,85%, 95%; v/v), in 100% acetone and finally driedwith CO2 in the triple-point. Finally, erythrocyteswere coated with gold-palladium and examined in aStereoscan 600 scanning electron microscope (Cam-bridge Instruments, UK).

In each sample 100 erythrocytes were classifiedand mean morphological index (MI) was calculated.Echinocytes were assigned a morphology score of1–5 and discocytes 0, based on Bessi’s nomenclaturefor classification of red blood cells shapes (35).

STATISTICAL ANALYSIS

Data are expressed as means 6 SD. For furtheranalysis of erythrocyte morphology, hierarchicalanalysis of variance was used. Before this proceduredata were transformed as arc sin =xi (LS1, LS2, andLS1/LS2) or log xi (peak position) function (36).

Results

FLOW CYTOMETRY

Series of scans obtained by flow cytometry mea-surements reflect the development of changes inerythrocyte size, shape and membrane surface mor-phology. Typical scattering histograms for controlerythrocytes are presented in Figure 1. The curveplotted on LS1 histogram and on LS2 histogram aresmooth, sharp, and almost symmetric. The surfacecontour plotted on LS1/LS2 histogram is greatlyconcentrated.

Figure 1 — Scanning electron micrograph and scattering histograms of control erythrocytes incubated in PBS.

PRZYBYLSKA ET AL.

74 CLINICAL BIOCHEMISTRY, VOLUME 31, MARCH 1998

For CS content equal to 1 3 1025 mol/dm3, curvesplotted on both scattering histograms, the dualparameter contour plot and peak position (P) incomparison with control ones remain almost un-changed even for the longest time (2h) of incubation(Figures 1 and 2).

For lower (0.4 3 1025 mol/dm3) and higher (4–8 31025 mol/dm3) (Figure 2) CS concentrations thecurves become more flat, jagged, asymmetric, andeventually double-peaked, and the peak position (P)is significantly changed. The extension of changes is

especially significant for the highest (8 3 1025

mol/dm3) CS concentration used (Figure 2).The diversity in the scattering erythrocyte prop-

erties, expressed as a percentage of altered cells andpeak position (P) induced by CS as a function of timeand CS concentration was summarized on Figure 3.Hierarchical analysis of variance (with time of incu-bation as a nested factor within cholesterol sulphateconcentration) showed that both factor have signif-icant effects on the morphology of human erythro-cytes (Table 1).

Figure 2 — Scattering histograms of erythrocytes incubated in PBS containing cholesterol sulphate, (A) 0.4 3 1025

mol/dm3, (B) 1 3 1025 mol/dm3, (C) 4 3 1025 mol/dm3, (D) 8 3 1025 mol/dm3.

CHOLESTEROL SULPHATE-INDUCED CHANGES IN HUMAN RED BLOOD CELLS


RED BLOOD CELL SHAPE OBSERVATION

As it is shown in Figure 4, erythrocytes sub-jected to CS concentration of 0.4 3 1025 mol/dm3

for 0.5 to 2.0 h underwent only few morphologicalchanges, in comparison with control cells (Figure1). The increasing CS content transforms redblood cells from discocytes into sphero-echinocitic

forms with intercellular bridges indicating asticky nature of the cell surfaces and leads pro-gressively to a decrease of a cell size (Figure 4).The greatest degree of erythrocyte shape and sizemodification has occurred at CS concentration of8 3 1025 mol/dm3, especially for longer incubationtime (2 h). Only for CS concentration equal to 1 31025 mol/dm3 the maintenance of the normal

Figure 3 — Flow cytometric analysis of cholesterol sulphate effect on erythrocytes morphological parameters (A) 0.4 31025 mol/dm3, (B) 1 3 1025 mol/dm3, (C) 4 3 1025 mol/dm3, (D) 8 3 1025 mol/dm3 of cholesterol sulphate). Data areexpressed as mean 6 SD (n 5 4).

TABLE 1Hierarchical Analysis of Variance of Erythrocyte Morphological Features

Source of Variation

LS1 LS2 LS1/LS2 Peak Position

MS df F MS df F MS df F MS df F

CS concentration 6068.8 3 424.0a 5758.0 3 290.5a 5059.0 3 310.8a 0.445 3 405.3a

Time of incubation 250.0 12 17.5a 231.9 12 11.7a 204.3 12 12.6a 0.028 12 25.5a

Residual 14.3 48 19.8 48 16.3 48 0.001 48

Data were transformed as arcsin =xi (LS1, LS2, and LS1/LS2) or log xi (peak position (P).a p , 0.001.

PRZYBYLSKA ET AL.


smooth biconcave shape of red blood cells wasobserved (Figure 4).

The degree of erythrocyte echinocytic transforma-tions, expressed as morphological indexes (MI), forincreasing incubation times and CS concentrationsare summarised in Figure 5.

Discussion

The normal human erythrocyte is a flexible bicon-cave disk. Numerous studies have shown that eryth-rocytes respond to various treatments by alteringtheir morphological features (37–40).

The comparison of series of scattering histogramsobtained by flow cytometry (Figures 1 and 2) andcorresponding SEM images (Figures 1–4) indicatesthat in isotonic solution cholesterol sulphate, simi-larly to a variety of anionic amphiphilic agents(37–40), causes erythrocyte size and shape changes,in dose- and time-dependent manner (Figure 3).

We found that the effect of this compound onerythrocyte stability is distinctly biphasic. Only in anarrow range of CS content (1 3 1025 mol/dm3),equal to its physiological levels (14), erythrocytesmaintained their native, smooth, biconcave shape,even for longer incubation time (2 h). Contrary tothis, the incubation of red blood cells with lower(0.4 3 1025 mol/dm3) and higher (4–8 3 1025

mol/dm3) CS concentrations, promotes substantialmorphological alterations.

Flow cytometrical scans reflect these changesshowing significant differences in erythrocyte size(volume), shape and surface structure upon the

Figure 4 — Scanning electron micrographs of erythrocytes incubated in PBS containing cholesterol sulphate, (A) 0.4 31025 mol/dm3, (B) 1 3 1025 mol/dm3, (C) 4 3 1025 mol/dm3, (D) 8 3 1025 mol/dm3.

Figure 5 — The degree of erythrocyte echinocytic trans-formations, expressed as the morphological indexes (MI),obtained with increasing CS concentration. Data are ex-pressed as mean 6 SD.



influence of CS. Jagged, asymmetric and double-peaked scattering curves suggesting the existence oftwo subpopulations of cells (Figure 2) are well con-firmed by the results obtained by SEM showing theformation of spicules, vesicles and numerous inter-cellular links (Figure 4).

It is noteworthy that these morphological changestake place at CS concentration above physiologicallevel, but in the range found in patients sufferingfrom certain diseases such as recessive X-linkedichthyosis, where CS concentration in plasma variesfrom 5.8 3 1025 to 8.6 3 1025 mol/dm3 (41).

Morphological alterations induced by CS can beexplained, in terms of Sheetz and Singer (42) bilayercouple hypothesis. According to it, shape alterationsarise from a differential expansion of amphipaticmolecules into two monolayers of cell membranes.Echinocytogenic amphiphiles such as CS, arethought to be preferentially intercalated in the outerhemileaflet of the lipid bilayer and, at sublyticconcentrations, cause red blood cells transformationfrom discoid to echinocytic forms (42).

However, for low concentrations (0.4 –1 3 1025

mol/dm3) this sterol does not involve substantialperturbation of the plasma membrane, which maybe, at least partly, explained by the ability of thisamphipathic molecule to increase membrane sur-face pressure and hydration (due to the presenceof ionised hydroxyl group in physiological condi-tions) (43).

Our results are in agreement with earlier studieson the interaction of amphiphiles with the erythro-cyte membrane, which have suggested that theerythrocyte membrane can incorporate definiteamount of foreign amphiphiles without losing itsintegrity and barrier properties (39).

The interesting point of our observations is thatthe comparison of the degree of echinocytic transfor-mation, expressed in terms of the morphologicalindex (Figure 5) with data which present the per-centage of altered cells and peak position (P) posi-tions (Figure 3) obtained by cytometric measure-ments, leads to the conclusion that both methodsprovide convergent information. It is worth empha-sizing that the cytometric method seems to be farbetter than the others. It almost completely allowsto avoid drastic, time consuming sample prepara-tion, which may alter red blood cells morphologicalfeatures and provides significantly better data re-currence (compare standard deviation values onFigures 3 and 5). In conclusion, it may be consideredas a useful method for carrying out measurements ofvarious agents effect on cell morphology and mem-brane properties.

In this context, it is important to note that indi-cating a biphasic behavior cholesterol sulphate mayhave particular relevance to many phenomena incell biology that involve shape changes, includingcell locomotion, cell-cell and cell-virus fusion, secre-tion, phagocytosis and the others. Due to its uniqueproperties CS may be considered to be a potentmodulator of liposome stability essential for drug

dosage control and release properties in liposome-based therapeutic agent (44). Further study of mo-lecular mechanisms involved in a protective abilityof CS against cell membrane destabilization mayhelp develop a new target for antiviral therapy (17).

Acknowledgement

The authors wishes to thank Dr. Miroslyaw Przybylskifor his advice and help in statistical analysis of data.

References

1. Moser HW, Moser AB, Orr J. Preliminary observa-tions on the occurrence of cholesterol sulfate in man.Biochim Biophys Acta 1966; 116: 146–55.

2. Bleau G, Chapdelaine A, Roberts KD. The assay ofcholesterol sulfate in biological material by enzymaticradioisotopic displacement. Can J Biochem 1972; 50:277–86.

3. Iwamori M, Moser HW, Kishimoto Y. Cholesterolsulfate in rat tissues. Tissue distribution, develop-mental change and brain subcellular localization. Bio-chim Biophys Acta 1976; 441: 268–79.

4. Veares MP, Evershed RP, Prescott MC, Goad LJ.Quantitative determination of cholesterol sulphate inplasma by stable isotope dilution fast atom bombard-ment mass spectrometry. Biomed Environ Mass Spec-trom 1990; 19: 583–8.

5. Langlais J, Zollinger M, Plante L, Chapdelaine A,Bleau G, Roberts KD. Localization of cholesteryl sul-fate in human spermatozoa support of a hypothesis forthe mechanism of capacitation. Proc Natl Acad SciUSA 1981; 78: 7266–70.

6. Momoeda M, Taketani Y, Mizuno M, Iwamori M,Nagai Y. Characteristic expression of cholesterol sul-phate in rabbit endometrium during the implantationperiod. Biochem Biophys Res Commun 1991; 178:145–50.

7. Nicollier M, Beck L, Mahfoudi A, Coosemans V,Adessi GL. Effect of progesterone on hydrophobiccell-associated proteoglycans bound to cholesterol sul-fate in glandular epithelial cells of guinea-pig endo-metrium. Biochim Biophys Acta 1994; 13: 125–31.

8. Serizawa S, Osawa K, Togashi K, et al. Relationshipbetween cholesterol sulfate and intercellular cohesionof the stratum corneum: Demonstration using a push-pull meter and an improved high-performance tin-layer chromatographic separation system of all majorstratum corneum lipids. J Invest Dermatol 1992; 99:232–6.

9. Wertz PW. Epidermal lipids. Semin Dermatol 1992;11: 106–13.

10. Serizawa S, Nagai T, Ito M, Sato Y. Cholesterolsulphate levels in the hair and nails of patients withrecessive X-linked ichthosis. Clin Exp Dermatol 1990;15: 13–15.

11. Brosche T, Gollwitzer J, Platt D. Cholesterol andcholesterol sulphate concentration in the cell mem-brane complex of human scalp hair—a biomarker ofaging? Arch Gerontol Geriatr Suppl 1994; 4: 19–30.

12. Wertz PW, Downing DT. Cholesterol sulfate: the ma-jor polar lipid of horse hoof. J Lipid Res 1984; 25:1320–3.

13. Bleau G, Lalumierre G, Chapdelaine A, Roberts KD.Red cell surface structure. Stabilization by cholesterol

PRZYBYLSKA ET AL.


sulfate as evidenced by scanning electron microscopy.Biochim Biophys Acta 1975; 375: 220–3.

14. Bleau G, Bodley FH, Longprre J, Chapdelaine A,Roberts KD. Cholesterol sulfate. I. Occurrence andpossible biological function as an amphipathic lipid inthe membrane of the human erythrocyte. BiochimBiophys Acta 1974; 352: 1–9.

15. Bryszewska M, Koter M, Epand RM. Effect of choles-terol sulphate on haemolysis of human erythrocytes.Med Sci Res 1991; 19: 791–2.

16. Przybylska M, Bryszewska M, Epand RM. Effect ofcholesterol sulphate on the thermosensitivity andfluidity of human erythrocytes. Biomed Lett 1994; 49:113–17.

17. Cheetham JJ, Epand RM, Andrews M, Flanagan TD.Cholesterol sulfate inhibits the fusion of Sendai virusto biological and model membranes. J Biol Chem1990; 265: 12404–9.

18. Williams ML, Rutherford SL, Feingold KR. Effects ofcholesterol sulfate on lipid metabolism in culturedhuman keratinocytes and fibroblasts. J Lipid Res1987; 28: 955–67.

19. Cheetham JJ, Chen RJB, Epand RM. Interaction ofcalcium and cholesterol sulphate induces membranedestabilization and fusion: implications for the acrosomereaction. Biochim Biophys Acta 1990; 1024: 367–72.

20. Davis BK, Byrne R, Bedigian K. Studies on the mecha-nism of capacitation. Albumin-mediated changes inplasma membrane lipids during in vitro incubation ofrat sperm cells. Proc Natl Acad Sci USA 1980; 77:1546–1550.

21. Blache D, Becchi M, Davignon J. Occurrence andbiological effects of cholesterol sulfate on blood-plate-lets. Biochim Biophys Acta 1995; 1259: 291–6.

22. Blache D. Enhanced arachidonic acid and calciummetabolism in cholesteryl sulfate-enrched rat plate-lets. J Lipid Med Cell Signal 1996; 13: 127–38.

23. Roberts KD, Bandi L, Calvin HI, Drucker WD, Lieber-man S. Evidence that steroid sulfates serve as biosyn-thetic intermediates. IV. Conversion of cholesterolsulfate in vivo to urinary C19 and C21 steroid sulfates.Biochem 1964; 15: 1983–1988.

24. Roberts KD, Bandi L, Calvin HI, Drucker WD, Lieber-man S. Evidence that steroid sulfate is a precursor ofsteroid hormones. J Am Chem Soc 1964; 86: 958–9.

25. Woscholski R, Kodaki T, Palmer RH, Waterfield MD,Parker PJ. Modulation of the substrate specificity ofthe mammalian phosphatidiloinositol 3-kinase bycholesterol sulfate and sulfatide. Biochemistry 1995;36: 11489–93.

26. Chen L, Imperato TJ, Bolt RJ. Enzymatic sulfation ofbile salt by sulfotransferase from rat kidney. BiochimBiophys Acta 1978; 522: 443–51.

27. Muskiet FAJ, Jansen G, Wolthers BG, Marinkovic-Ilsen A, Van Voorst Vader P. Gas-chromatographicdetermination of cholesterol sulfate in plasma anderythrocytes, for the diagnosis of recessive X-linkedichthyosis. Clin Chem 1983; 29: 1404–7.

28. Shwayder T, Ott F. All about ichthyosis. Ped ClinNorth Am 1991; 38: 835–57.

29. Tamasawa N, Tamasawa A, Takebe K. Higher levelsof plasma cholesterol sulphate in patients with livercirrhosis and hipercholesterolemia. Lipids 1993; 28:833–6.

30. Muskiet FD, Muskiet FAJ. Lipids, fatty acids andtrace elements in plasma and erythrocytes of pediatricpatients with homozygous sickle cell disease. ClinChim Acta 1984; 142: 1–10.

31. Przybylska M, Bryszewska M, Nowicka U, SzoslandK, Kedziora J, Epand RM. Estimation of cholesterolsulphate in blood plasma and in erythrocytes mem-branes from individuals with Down’s syndrome ordiabetes mellitus type I. Clin Biochem 1995; 28(6):593–7.

32. Barlag KE, Goerz G, Ruzicka T, Schurer NY. Palmo-planar keratoderma with an unusual composition ofstratum-corneum and serum sterol derivatives—anew entity. Br J Dermatol 1995; 133: 639–43.

33. Salzman GC, Singham SB, Johnston RG, Bohren CF.Light scattering and cytometry. In: Flow Cytometryand Sorting. (Eds. Melamed MR, Mullaney PF, Men-delsohn MR), pp. 81–107. New York: Wiley-Liss, Inc.1990.

34. Visser JWM, van den Engh GJ, van Bekkum DW.Light scattering properties of murine hemopoeticcells. Blood Cells 1980; 6: 391–407.

35. Bessis M. Red cell shapes: an illustrated classificationand its rationale. In: Red Cell Shape. (Eds. Bessis M,Weed RI, Le Blond PF), pp. 1–23. New York: Springer-Verlag, 1973.

36. Zar JH. Biostatistical analysis. London: Prentice-Hall, 1984.

37. Seeman P, Kwant WO, Sauks T, Argent W. Mem-brane expansion of intact erythrocytes by anesthetics.Biochim Biophys Acta 1969; 183: 490–8.

38. Seeman P. The membrane actions of anesthetics andtranquilizers. Pharmacol Rev 1972; 24: 583–633.

39. Hagerstrand H, Isoma B. Vesiculation induced byamphiphiles in erythrocytes. Biochim Biophys Acta1989; 982: 179–86.

40. Reinhart WH, Rohner F. Effect of amiodarone onerythrocyte shape and membrane properties. Clin Sci1990; 79: 387–91.

41. Bergner EA, Shapiro LJ. Increased cholesterol sulfatein plasma and red blood cell membranes of steroidsulfatase deficient patients. J Clin Endocrinol Metab1981; 53: 221–3.

42. Sheetz MP, Singer SJ. Biological membranes as bi-layer couples. A molecular mechanism of drug-eryth-rocyte interactions. Proc Natl Acad Sci USA 1974; 71:4457–61.

43. Faure C, Tranchant JF, Dufourc EJ. Comparativeeffects of cholesterol and cholesterol sulfate on hydra-tion and ordering of dimyristoylphosphatidylo-cholinemembranes. Biophys J 1996; 70: 1380–90.

44. Viani P, Cervato G, Gatti P, Cestaro B. Plasmadependent pH sensitivity of liposomes containing sul-fatide. Biochim Biophys Acta 1993; 8: 73–80.



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