Study on erythrocyte membrane fluidity by laser Raman spectroscopy

Cell Biology

International

Reports,

STIJDY ON ERYTHRCCYTE CHENG Beijing, CHEN

MEMBRANE

Shi . Department China Yul ing .

Vol. 12, No. 3, March

of

Institute

FLUIDITY

Biophysics,

of

BY LASER Beijing

Biophysics,

205

1988

RAMAN SPECTRCSCCP‘; Medical

Academic

Llniversi

Sinica,

ty,

Beijing,

&ina ABSTRACT Differences between patients with cirrhosis of liver, nephrotlc and normal subjects in laser Raman syndrome, coronary heart disease In regions of spectra of erythrocyte membranes have been found. 1000-1140 cm and 2840-3000 c.m , the ratios 1113ti /IIo8o and I289o ‘I2850

in

patient

membranes

are

higher

than

those

in

normal

ones

respectively. These results mean that erythrocyte membrane fluidity of the se patients is reduced. This reduction may be attributed to the possibility that erythrocyte membrane of patients get more from their plasma and resulted in the modification cholesterol of the ratio of cholesterol/phospholipids in the membranes. II’!!RODUZTIC@J erythrocyte membrane has been attractive As a ncmbrane model, for investigation. A special property of this membrane is an exchange of lipids existing between membrane and their surrounding medium (d’hcllander and Chevallier, 1972; Gottlief, 1980; Schick Hence constituents and properties of erythroc.yte and Schick, 1985). membrane lipids would be influenced and changed as the 1 ipid metabolism in living organism is in disorder. Three kinds of diseases disease and coronary heart these patients are complicated

cirrhosis of liver, have been chosen, with abnormal lipid

nephrotic because metabolism.

syndrome mst of

Being highly sensitive and quickly reactive, laser Rarn;tn spectroscopy is a good means to invest igate membrane without any disturbing of the original structure by probing. Using Paman spectroscopy, we have studied and compared erythrocyte membrane fluidity of these patients. PAT1 ENTS , INSTRLMEIVI’S

AND MWllCDS

Diagnosis of four patients teaching hospital of by 3rd. cholesterol contents in their 375mg/dl and 97mg/dl respectively. 22Omg/dl). Diagnosis of four 0309-I

651/881030205-7/$03.00/O

with Beijing plasma

cirrhosis of liver was made The Medical University. were 155mg/dl, 164rig/dl, (norma 1 standard being IZOpatients with nephrot ic syndrome 0 1988 Academic

Press

Ltd.

206

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Vol. 12, No. 3, March

1988

was made by 1st. teaching hospital of Beijing hJedica1 University. The cholesterol contents were 326mg/dl, 217mg/dl, 324mgJdl and 264mg/dl respectively. (normal standard 75-165mg/dl). V.vo patients with coronary heart disease was made by An-Zhen hospital, and cholesterol were 233mg/dl and 363mg/dl respectively. (normal standard 120-200mg/dl). All of these latter six patients were complicated with hypercholesterolemia.

was was

Laser Raman at 514.5mr-1, Zcm/sec.

Spectrometer from argon

SPEX laser.

1403 U.S.A. Excitation wavelenth Laser power was 8OOmv and scan

Preparation of erythrocyte membrane (ghost ); Erythrocytes were seperated from the whole blood, rinsed with saline and put them into hypotonic (5n-M) phosphate buffer (pH7.9) to form membrane. The membranes were then rinsed with the buffer twice and double distilled water twice followed by centrifugalization (100,OOOg). A white pellet (compressed membranes) was obtained and ready for use. RESULTS Raman spectra of human erythrocyte membrane in 5 norma 1 subjects, 4 patients with cirrhosis of liver, 4 patients with nephrotic syndrome and 2 patients with coronary heart disease were obtained. We have found that the structurally sensitive bands have significantly changed in regions of 1000-1140 cm and 28483100 cm for 10 patients with the three kinds of diseases (figure l-8). Spectra in the region of 1000-1140 cm represent conformation and their changes in fatty acid hydrocarbon. 1130 cm intensity indicates the quantity of all-trans in backbone and 1087 cm” of gauche conformation. 1982; Lippert and Peticolas, 1971). (Tu, From these figures it is clear that there are more of the alltrans and less of the gauche conformation in the c-c backbone of In all these their erythrocyte membranes than normal subjects. patients, I l130/Ilo87:~~l, while Il130/Ilo87-~~l for the normal subjects (figure l-4). Spectra in the region of 2800-3100 cm’ are Caber and also associated with membrane fluidity ( Tu, 1982; Peticolas, 1977). The 2890 cm’ Raman band is from asynmetrical is increased CH stretching vibration of methylene (-CH-) and I289o The 2850 cm band when lateral packing between chains is tight. which is from synmetrical CH stretching vibration of methylene Spectra (figures 5-8) show that ( -CH- ) is relatively constant. the ratios in 10 patients is markedly higher than those Iz 890 “2850 in norma 1 subjects, also indicating that erythrocyte membranes of 10 patients are less fluid than that of normal control, DISCUSSION

Raman

Both Bulkin (1972) spectra of human

and Lippert erythrocyte

(1975) independently membranes. This

obtained result was

Ceil Biology

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Reports,

Vol. 12, No. 3, March

1988

207

fig l-4 wavenumber fig 1. subjects’ I

1088

11134

9.60B

03 L_____r_L_I--------.-

=0.2:

(cm-‘! .,

r--T\--.-

I

spectrum

R emen --

-

-

sh

r

r

=4.4:1

1 1140.00

1076.cHJ

1012.00

r

i/ LL--

I

03 j.-

of

of wi t h liver

1083=537 =2378.8 1135

11135’110s3

04

1

1140.00

1070.00

s,.21781 s+: it

I

,.40E

i

=233.6

fig 2. patients cirrhosis

9.20B

norm spectrwx

I! ‘I

1000.00 03

1ow

=1028

11134’Ilo*8

7.0qiY

in

i 3

-

[cm-‘1

--

--

.

”

fig 3. spectrum of patients with nephro t i c syndrome I 1082=1617 I

i126

=1976

208

Cell Biology International

Reports, Vol. 12, No. 3, March 7988

fig 4. spectrum of patients with coronary heart disease I

1084

I

1128

=3658 =3979 =l.l:l

Ill28’IlO84

3.0-m

04

5-8 in wavenumber fig

fig 5. subjects’

high

norm 1 spectrum

I 2852 =1710 I 2890 = 935 =0.6:1

‘2890”2852

29920.00

Reman

shift

3000.00

(cm-‘)

fig 6. patients cirrhosis I I

2850

spectrum of

=1201

=1398 2882 I 2882/12850=l.2:l

of with liver

Cell Biology

International

Reports,

Vol. 12, No. 3, March

‘i

i

1988

fig 7. patients nephrot

‘2888 ,

_~_

I.

2800.00

.A.

_____.

.-.

2900.00

Raman

--

spectrum ic

of wi ih syndrom

I 2856 =1210 i

I

209

=4320

‘2888’I2856

=3.6:1

..3ooo.00

(cm-‘1

sSift

-.-I-

f?sn;c?

later the wa s in and has

j000.00

2900.00

2800.00

sh

/ ft

Icm-‘1

supported by Milanovich et al (1976). In our Raman spectra of normal human erythrocyte membranes almost the same as that they observed. According Paman bands during phase transition of lipids (Tu, Peticolas, 1977; Lippert and Peticolas, 1971) the been deduced that these patients’ membrane fluidity

experiments, we obtained to changes 1982; Gaber conclusion is reduced.

Erythrocytes are unable to synthesize cholesterol and lack receptor for cholesterol-carrying lipoproteins. Actual ly, 1 ipid composition of erythrocyte membrane depends on plasma lipids and thus they are deficient in a peculiar adaptabi 1 i ty to control membrane fluidity. Hypercholesterolemia is a complication of some patients with nephrotic syndrome and coronary heart disease and cholesterol contents in their plasma are remarkably higher than nor-ma 1 subjects. Cirrhosis of liver is complicated by 1 ipid metabolic disorders. Although cholesterol contents are not high

Cell Biology

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Reports,

Vol. 12, No. 3, March

1988

in some patients, the ratios of cholesterol/phospholipids are higher Therefore erythrocyte than those of normal ones (Cooper, 1975). membranes of 10 patients got mOre cholesterol from their plasma lipids and the ratios of lipid compositions were modified. Membrane fluidity is significantly influenced by composition of membrane lipids. It is we1 1 known that cholesterol increases phospholipids fluidity in gel phase and decrease it in 1 iquid(Chapman and Benga, 1984; Yeagle, 1985). If crystalline phase. cholesterol acyl chains movement erythrocyte membranes get m3re Reduc t ion of fluidity reduced. will be restricted and membrane The membrane fluidity is due to deceleration of lipid movement. abnormalities of molecular behaviour may probably render the whole erythrocytes decrease in its deformability, and thus it is difficult for them to pass through small vesseles, on which we are still studying.

PEFERENCES Bulkin,

B. J. (1972) Raman spectroscopic study of human erythrocyte membranes. Biochemica et Biophysics Acta, 274,649-651. Chapman, D and Benga, G. ( 1984 ) Biomembrane fluidity-Studies of model and natural biomembranes. In: Biological Nimbranes. D. Chapman ed. pp. 19-24. Academic Press, London, New York. :Cooper, R.A. (1975) tidification of cell membrane structure by cholesterol-rich lipid dispersions, a model for the primary spur cell defect. Journal of Clinical Investigation, 55,115126. Caber, B.P. and Pet icolas, On the quantitative of W.L. (1977) biomembrane structure by Raman spectroscopy. Biochemica et Biophysics Acta, 465,260-274. Cottlief, M.H. (1980) Rates of cholesterol exchange between human erythrocytes and plasma 1 ipoproteins. Biochemica et Biophysics Acta, 600.530-541. d’llol lander, F. and Chevallier, F. (1972) Movement of Cholesterol in vitro in rat blood and quantitation of the exchange of free cholesterol between plasma and erythrocytes. Journa 1 of Lipid Research, 13,733-744. Lippert, J.L. and Peticolas, W.L. (1971) Laser Raman investigation of the effect of cholesterol on conformational changes in dipalnitoyl lecithin multilayers. Proceedings of the National Academy of Sciences USA, 68,1572-1576. Lippert, J.L., Gerczyca, L.E., and h&aiklejehn, G. (1975) A laser Raman spectroscopic investigation of phosphol ipid and protein configurations in hemoglobin-free erythrocytes. Biochemica et Biophysics Acta, 382.51-57. Tu, A.T. (1982) Lipids and biological membranes. In: Raman SPeCtrOSCOpy in Biology (principle and Applications). A.T.T~ ed. Ch. 7, pp.187-233. New York, Chichster, Brishare, Toronto, Singapor? ~

Cell Biology

Yeagle, et

Received:

International

Reports,

Vol. 12, No. 3, March

P.L. (1985) Cholesterol Biophysics Acta, 822,267-287.

12.10.87

and

Accepted:

ccl 1

1988

membrane.

12.1.88

211

Biochemica