Physical studies of myelin I. Thermal analysis

BIOCHIMICA

ET BIOPHYSICA

101

ACTA

nn.4 55472

PHYSICAL

STUDIES

I. THERMAL

ANALYSIS

B. D. LADBROOKE, Unilever Research (Received

OF MYELIN

T. J. JENKINSON,

Laboratory,

April Ioth,

The Frythe,

1’. B. KAMAT Welwyn,

Hert.

AND

(Great

D. CHAPMAN Britain)

1968)

SUMMARY

I. Myelin isolated from ox brain white matter and the lipids extracted from this material have been examined by thermal analysis techniques including differential thermal analysis and differential scanning calorimetry. The results provide information about the organisation of lipids and water in the myelin structure. 2. On drying myelin a crystallisation and precipitation of the lipid and the cholesterol takes place. Endothermic transitions associated with the lipid and cholesterol can then be observed. 3. The total lipid extract in water does not show a detectable endothermic transition but the cholesterol-free lipid does. In the absence of cholesterol, part of the myelin lipid is crystalline at body temperature. 4. With wet myelin no thermal transitions are detectable. In this case the lipids and cholesterol appear to be organised into a single phase. The presence and organisation of the cholesterol in the membrane appear to prevent the lipids from crystallising. 5. To maintain the organisation of the lipid in the myelin there appears to be a critical amount of water required. This water is unfreezable at o0 and may correspond to “bound” water.

INTRODUCTION

A number of physical and chemical studies have been carried out with the nerve myelin sheath and purified myelin fractions. These studies have been reviewed by several authors rp2.They provide information on many gross structural parameters of the myelin. Recent studies in this laboratory have shown that differential thermal analysis and differential scanning calorimetry are useful for examining the thermotropic and lyotropic transitions which occur with phospholipid+-6 on transforming from the crystalline or gel phase to the liquid crystalline phase, and also the interactions between phospholipids, water and cholesterol 6. Here we apply these techniques to a natural membrane, i.e. the myelin membrane, asking the questions: Biochim.

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B. D. LADBROOKE et al.

(a) Do thermal transitions similar to those which we observe with the phospholipids also occur with natural membranes? (b) If they do occur, what is the relationship between these transition temperatures and the membrane environmental temperatures ? (c) If they do not occur, is this because of interactions between lipid and cholesterol or lipid and protein? A subsequent communication will report nuclear magnetic resonance and infrared spectroscopic studies on this material. ZvIATERIALS AND METHODS

ofmyelin The method of EICHBERG,WHITTAKERAND DAWSON~was modified as follows to yield a single myelin fraction consisting of light and heavy fragments. Ox brains collected immediately after slaughter and stored frozen at -20 “C were allowed to thaw; the white matter was carefully dissected free of the grey matter and rinsed with ice-cold 0.32 M sucrose. A 10% homogenate of the white matter in 0.32 M sucrose was made with a motor-driven Potter-Elvehjem homogeniser fitted with a teflon pestle and surrounded by crushed ice. The homogenate was centrifuged at 17000 xg for 45 min in rotor SW 25-2 in the Spinco preparative ultracentrifuge model L250. The pellet consisting of nuclei, mitochondria, synaptosomes, and large and small myelin fragments was washed once with 0.32 M sucrose and resuspended in the same medium. This suspension was layered over z vol. 0.88 M sucrose in lusteroid tubes which fit in Rotor SW.25-2, and spun at 6ooooxg for I h. The material at the barrier between 0.88 and 0.32 M sucrose was carefully collected, brought to the density of 0.32 M sucrose, and recycled through the 0.88 M barrier, as before, at 60000 xg for 2 h. The barrier material was collected and washed free of sucrose by ultracentrifugation with ice-cold distilled and deionised water. All centrifugation procedures were carried out between o and 5 “C. The final washed material, unless stated otherwise, was freeze dried and stored at -20 “C in a desiccator over silica gel. The freeze dried material was gg + 1% soluble in chloroform-methanol (2 : I, v/v)~. PreParation

Total lipid

extract

A solution of freeze-dried myelin in chloroform-methanol (I : I, v/v) was passed through sintered glass (maximum pore diameter, 5-15 p) and the filtrate evaporated to dryness on a rotary evaporator at 40 “C. The residue was extracted with boiling chloroform until there was no further loss in weight. The combined extracts were passed through sintered glass and dried to constant weight. Total lipids accounted for 77 f 2% (by wt.) of the freeze-dried material. In some instances total lipid extract was prepared according to the method of AUTILIO,NORTONANDTERRY~.Thin-layer chromatography of the lipid extracts prepared by these two methods was virtually identical and was in agreement with the reported thin-layer chromatographic data of THOMSONAND KIES~.

of lipid co&ituents The total lipid extract was fractionated by silicic acid chromatography according to the elution scheme of NORTONAND AUTILIO10.Fractions corresponding to (i) Separation

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164 (1968)

Ior-

THERMAL

ANALYSIS

103

OFMYELIN

cholesterol, (ii) cholesterol-free lipids, (iii) total galactolipids, and (iv) total phospholipids were finally obtained from chloroform-methanol (z:I, v/v) solution and thoroughly dried in vacua. Preparation

of dehydrated nzyelin samples Myelin was prepared as above, but, prior to freeze-drying, the aqueous suspension was packed into a tight pellet by centrifugation for ro6oooxg for 16 h. This pellet contained 70-80% water. Homogeneous samples of lower water content were prepared by equilibration of portions of the pellet over a series of saturated salt solutions which gave atmospheres of known relative humidityI’. The time required for equilibration was long (3-4 days at 25 “C) owing to the tendency for a dry surface layer to be formed which impeded loss of water from the centre of the specimen. The water content of each sample was determined by a dry-weight determination after evacuation under vacuum (IO-~ torr) at go “C for 4 h. All weighings were carried out using a Cahn electrobalance model G. Thermal

analysis (a) Diferential thermal analysis. Samples (S-IO mg) in 4-mm diameter glass tubes were examined using a du Pont 900 differential thermal analyser. A scan speed of IO “C/min was used with a AT sensitivity of 0.1 “C/in. (b) Diferential scanning calorimetry. Samples (10-12 mg) were encapsulated in aluminium volatile sample holders and examined in a Perkin-Elmer differential scanning calorimeter DSC-I. Sensitivity was increased by using an improved sample holder in conjunction with a 5-mV chart recorder giving an effective sensitivity of 0.2 mcal/sec per inch. A scan speed of 16 “C/min was used. Peak areas were measured with a planimeter. RESULTS

Apart from a large endothermic peak near o “C due to the melting of ice, the wet myelin pellet containing 70--80~/~ water shows no thermal transitions between -40 “C and IOO “C. On removal of some water an endothermic transition is initially observed at 35 “C. As more water is removed this peak moves to higher temperatures until it reaches a limit at 55 “C. An additional peak also occurs at 36 “C. In Fig. I typical differential thermal analysis curves are shown. The ice peak disappears in the composition range 30-15O/~water and this is also the point at which the endothermic peak first appears. Using the differential scanning calorimeter, the variation in size of the ice peak and the endothermic peak were followed quantitatively. The values obtained are plotted in Fig. 2 and show that the disappearance of the ice peak coincides with the appearance of the transition peak at a water content of roughly 20%. As the water content is further decreased the size of the transition peak steadily increases. When the water content is reduced below 5% there is a large increase in the heat absorbed in the transition. This corresponds to the occurrence of two thermal transitions observed with the differential thermal analysis equipment (Fig. re). It is not possible to resolve these separate transitions with the differential scanning calorimetric inBiochim.

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B. D. LADBROOKE et ai.

104

a

1,’

0

10 Myelln

20 30 composition

40 i%

50 60 70 by wt. of water

80

, 90

:I0 100

1

Fig. I. Dehydration of myelin. Differential thermal analysis heating curves for samples of myelin previously equilibrated at different relative humidities. i\pproximate final water contents (wt.% water) are: (a) 30%~ (b) 15%~ (c) IO%, (d) 5%, (e) 3%. Fig. 2. Dependence of heats of transition on the water content of myelin. Values obtained by integration of the peak areas on the differential scanning calorimetric recordings. c?, heat absorbed in the ice-melting transition at 0” expressed as Cal/g total material (right-hand ordinate scale) ; ----, the variation expected if no water is bound to the myelin. The point at which the experimental curve extrapolates to zero gives the composition of hydrated myelin containing its full complement of unfreezable (bound) water. Inset: x , heat

strument

“C. This may represent tightly bound water, probably associated with protein, which is not readily removed, or may be due to some decomposition at 90 “C. It was 90

not possible to obtain a value for the heat change which unequivocally corresponded to 100% myelin. The influence of cholesterol on the lipid transitions is reflected by the behaviour of the lipid extracts in water. The differential scanning calorimetric heating curve for a 70% dispersion of the total lipid extract in water (Fig. 3a) shows no transition apart from the ice-melting peak. The cholesterol-free lipid, however, (Figs. 3b and 3c) does show an endothermic transition. This transition clearly occurs over an extended temperature range which is perhaps best defined by the onset temperature on heating as the lower limit (25 “C) and the onset temperature on cooling as the upper limit (50 “C). It is, however,.clear.that in the absence of cholesterol, part of the myelin lipid in water is crystalline at body temperature. The heat absorbed in the transition is 2.8-3.0

Cal/g lipid. Differential thermal

Biochim. Biophys.

analysis

Acta, 164 (1968) ror-rag

heating

curves

for myelin

specimens

dried in

THERMAL ANALYSIS

220

240

260

OF MYELIN

280 300 Temperature

320

-20 0 20 40 60 80 100120 Sample temperature cc1

360

340

l”K1

140

Fig. 3. Differential scanning calorimetric curves of lipid extracts of myelin in excess water. This figure is drawn to a different convention from that used in Figs. I, 4 and 3 in order to emphasise the difference between differential scanning calorimetric and differential thermal analysis data (see ref. 5, p. 447). (a) Total lipid extract @us 30% water, heating curve; (b) cholesterol-free lipid extract plus 30% water, heating curve; (c) as (b), cooling curve. (N.B. on the cooling curve the exothermic peak due to the freezing of water does not occur until 258 “K (- 15 “C) owing to supercooling. Fig. 4. Differential thermal analysrs heating curves for dried membrane and dried lipid fractions from beef-brain myelin. (a) Myelin dried in O~CUOover P,O,; (b) freeze-dried myelin pattern A; (c) freeze-dried myelin pattern B; (d) total lipid extract; (e) cholesterol fraction; (f) cholesterolfree lipid fraction; (g) galactolipid fraction; (h) phospholipid fraction. The broken tie-lines indicate transition attributable to crystalline cholesterol (36 “C) and the phospholipid chain-melting transition (49 “C).

various

ways are shown in Figs. 4a-4c.

peaks in the temperature

range 36-60

and there are also differences

between

With

freeze-dried

myelin,

the sequence

of

“C differs from that of myelin dried over P,O, various

freeze dried specimens.

Broadly,

two

kinds of behaviour were encountered. These are typified in Figs. 4b and 4c. The reason for the difference appears to be associated with variability in the freeze drying process.

Both patterns

are observed

with different

preparations

from the same mem-

brane material. The large endothermic peak at about IZO “C which is obtained with freeze-dried myelin is attributed to loss of water vapour since it is not observed if the material is thoroughly dried under high vacuum. If the dried sample is then exposed to the atmosphere for 2 h, the peak is again obtained. The sequence of peaks between 35 and 60 “C for both A and B types of freeze-dried myelin does not change after further drying in vacua. Dried myelin shows further transitions above 180 “C associated with extensive chemical degradation, but this region has not been studied in detail. Differential thermal analysis heating curves for the lipid fractions of myelin are shown in Figs. 4d-4h. Complementary microscopic observations show that the phospholipid fraction (Fig. 4h) transforms from an anisotropic crystalline solid to an Biochim.

Biophys.

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164 (1968)

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B. D. LADBROOKE

106 anisotropic

liquid-crystalline

The galactolipid 110-115

fraction

phase of the “neat”

(Fig. 4g) is an anisotropic

“C and transforms

to an anisotropic,

type at 48-50 crystalline

“C (m.p.,

et al.

191 “C).

solid which softens at phase at 140’

fluid, liquid-crystalline

(m.p., 195 “C). The X-ray diffraction pattern of this material at temperatures below IIS “C shows only integral orders of a principal long spacing (55.5 A) in the low angle region, and a sharp Bragg line at 4.4 A in the high angle region. Above long spacing decreases

140 “C the

to 39.4 A_ and in the high angle region a diffuse line at 4.5 :i

is obtained. In the intermediate temperature range (115-140 “C) both patterns appear to be present. There was no evidence of any change in the X-ray diffraction pattern corresponding curve.

to the transition

One explanation

heterogeneously, crystalline

the separate

condition

a resemblance

at 75 “C seen on the differential

of these

components

at different

between

this

changes

is that

in the mixture

temperatures.

curve

the galactolipid

and that

thermal

analytic

fraction

behaves

transforming

Alternatively,

to a liquid

we note that there is

for a saturated

phosphatidylethanol-

amine4.

Fig. 5. Differential thermal analysis heating curves for myelin preparations of human origin (dried in V~CUDover P&I,). (a) Human femoral nerve (peripheral nervous system) ; (b) human posterior columns of spinal cord (central nervous system).

Finally,

in Fig.

5 we show differential

thermal

human myelin, dried over P,O, from the peripheral

analysis

heating

curves

for

(Fig. 5a) and the central (Fig. 5b)

nervous system. DISCUSSION Whilst thermal transitions are not observed with wet, fresh myelin, they are observed with freeze-dried material and on the removal from the wet myelin of increasing amounts of water (see Fig. I).Since there is a close resemblance between the differential thermal analysis curves of the dried myelins (Figs. 4a-4c) and the total lipid extract (Fig. 4d), we associate the thermal transitions in the temperature range 35-60”

with transitions of the lipid components. Thermal transitions have been observed previously with anhydrous phospholipids by CHAPMAN AND COLLINS and CHAPMAN, BYRNE AND SHIPLEY~ and with cholesterol by SPIER AND VAN SENDEN 12. With the phospholipids the transition temperature of the lipid is dependent upon the nature of the polar head group and on

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ANALYSIS

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the length and degree of unsaturation of the hydrocarbon chains and on the presence or absence of water. The transition has been associated with a phase change from crystalline to a liquid-crystalline form in which the hydrocarbon chains have considerable mobility. With pure cholesterol an endothermic transition was observed at 40 “C and X-ray studies showed that this is caused by a lattice transformation of the crystalline solid. The cholesterol fraction from myelin (Fig. 4e) shows a transition at 35 “C. This is somewhat lower than the transition temperature observed with pure cholesterol, but this may be clue to the presence in this fraction of some cholesterol esters13. The total lipid extract (Fig. 4cl) and the rigorously dried myelin (Fig. 4a) both show a thermal transition at this temperature. As the presence of this peak is indicative of crystalline cholesterol, it can be seen that one consequence of drying the membrane is the precipitation of some free cholesterol. Consistent with this conclusion is the work of BEAR, PALMER AND SCHMITT~~and ELKES AND FINEAN~~*~~ who reported that they sometimes observed a reflection at 34.5 A in the low angle X-ray diffraction patterns of dried nerves which they associated with the separation of a “cholesterol phase”. The thermal transition at 49 “C observed with the total lipid extract can be compared with a similar transition with the phospholipid extract (Fig. 4h). This transition can be attributed to a crystalline liquid-crystalline transition involving melting of the hydrocarbon chains of the phospholipids. This transition is very broad, as might be expected from a heterogeneous mixture; the temperature at which the peak minimum occurs is probably best regarded as the temperature above which all the chains are fluid. The galactolipid fraction, however, shows several transitions and the material is not completely liquid crystalline until 140 “C. It seems most likely that the separate transitions are of different components in the mixture. With the cholesterol-free lipid extract (i.e. phospholipids plus galactolipids, Fig. 4f) it appears that galactolipid transitions occur at lower temperatures while the phospholipid transition is again obtained at 49 “C. With the total lipid extract no separate transitions attributable to galactolipids are observed. Possibly the transition temperatures are lowered still further in the presence of cholesterol and are obscured by the dominant phospholipid transition. A detailed comparison of the behaviour of the total lipid extract with myelin dried in various ways (Figs. 4a-4c) is complicated by three factors (i) the presence of trace amounts of residual water, (ii) the effect of lipid-protein interactions, and (iii) the mixed lipid phases formed on drying the membrane will not necessarily be the same as those obtained by crystallisation of the lipid extract from organic solvents. The freeze-dried preparations evidently contain water giving rise to the large endothermic peak at IZO “C. However, vacuum drying of these preparations, so that the peak at 120 “C is removed, does not result in any shift of the lipid transitions. This water is evidently not associated with the lipid; it may represent water bound to the protein and the reason why it occurs with the freeze-dried myelin but not with that dried over P,O, is probably due to the larger surface area of the freeze-dried material. A similar effect has been reported for dry fibrous proteins21+ and polypeptides23. ‘4 comparison between the differential thermal analysis curves of the dried myelin from beef brain (Fig. 4a) with those of the myelin specimens of human origin Biochim.

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H. D. LADBROOKE

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(Figs. 5a and 5b) shows a very close resemblance between the materials from the central nervous system, but that from the peripheral nervous system is quite different. This is consistent with the finding that there is a greater difference in the lipid composition of myelin from the peripheral nervous system and myelin from the central nervous system of a given species than between myelin from the central nervous system of different species24925. With the hydrated myelin the differential scanning calorimetric results show that the first 20% of water is unfreezable. This corresponds to “bound” water similar to that previously observed with phospholipid-water systemsb. No lipid transitions are observed with myelin containing 20% or more of water. The effect is analogous to the behaviour of phosphatidylcholine-cholesterol mixtures in watere. Addition of cholesterol to dipalmitoyllecithin in water causes the chain-melting transition temperature and also the heat absorbed in the transition to decrease. When an equimolar ratio of lecithin to cholesterol is reached the transition is no longer observable. This ratio corresponds to the maximum amount of cholesterol which can be incorporated into the lecithin lamellae. Complementary X-ray diffraction6 and nuclear magnetic resonance studiesI have been interpreted to show that cholesterol controls the fluidity of the hydrocarbon chains by restricting the motion of the fluid chains and by preventing the chains from crystallising. In the absence of cholesterol, the myelin lipids, even in the presence of water, can crystallise at body temperature (see Fig. 3). In view of this close correspondence between the behaviour of the lipids in myelin and the model system, it would seem that transitions due to the lipid are genuinely absent in hydrated myelin, and that instrument sensitivity is not a limiting factor. However, we cannot be equally certain that thermal effects due to protein denaturation are really absent. Several studies have been carried out with protein so1utions26~27 using differential thermal analysis and denaturation endotherms are commonly observed in the temperature range 30-80 “C. With myelin, since only 20% due to denaturation would be of the dry weight is proteinlO, any heat absorption very small. These results are consistent with the X-ray diffraction studies of intact18 and isolatedIg myelin from the central nervous system which showed that drying of myelin results in the crystallisation of the lipid components to give a multiphase system. The amount of “bound” water (20%) as determined in our study is rather less than the estimate for the essential water in myelin (30-40~/~) obtained from the X-ray studies. In this connection it is interesting that with myelin samples containing 20-30~/~ water, an ice peak is obtained but other transitions below o “C are also observed. Similar behaviour occurs in simple lipid-water systems containing a small amount of free water. This is attributed to the formation of cubic and vitreous forms of ice as has been observed in gelatin gels 20, This effect may be due to the small amount of free water distributed in the form of a thin film or microscopic channels throughout the sample which affect the crystalline phase of the ice. ACKNOWLEDGEMENTS

We wish to thank Dr. R. M. WILLIAMS Dr. S. STRICH of the Institute of Psychiatry, samples Biochin.

of human Biophys,

myelin.

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164 (1968)

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for X-ray Maudsley

studies Hospital,

on the lipids and London

for

the

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ANALYSIS

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Biophys. Acta, 164 (1968) nor-log