Nov 28, 1983 - the lipid reflects a qualitative difference in the proteins. Multiple sclerosis (MS) is a demyelinating disease affecting primarily the white matter of ...
Proc. Natl. Acad. Sci. USA Vol. 81, pp. 1871-1874, March 1984
Neurobiology
Alteration of lipid-phase behavior in multiple sclerosis myelin revealed by wide-angle x-ray diffraction L. S. CHIA*, J. E. THOMPSON+, AND M. A. MOSCARELLO* *Department of Biochemistry, The Hospital for Sick Children, Toronto, Ontario, Canada M5G Waterloo, Ontario, Canada N2L 3G1
1X8; and tDepartment of Biology, University of Waterloo,
Communicated by J. Tuzo Wilson, November 28, 1983
was separated from the grey matter. Myelin was prepared as described in an earlier report (10). X-Ray Diffraction of Myelin. Myelin samples for x-ray diffraction were prepared as in ref. 10. Wide-angle x-ray diffraction patterns were recorded at various temperatures by using CuKa radiation from a point-focused x-ray tube (type PW 2103/01) on a diffraction camera (Philips type 1030) under conditions in which the samples retained 75% moisture with respect to final dry weight. The lipid-phase transition temperature, defined as the highest temperature at which gel-phase lipid can be detected, was determined to within 1PC. Densitometer tracings of the diffraction patterns were made with a Clifford model 345 densitometer. Lipid Extraction. Total lipids were extracted from the isolated myelin according to a modified procedure of Nichols (11), and the sample was washed by the method of Papahadjopoulos and co-workers (12). A volume of lipid extract containing at least 2 gmol of phospholipid phosphorous was evaporated to dryness under N2, dissolved in 2 ml of chloroform/methanol, 2:1 (vol/vol), and divided into two equal proportions, one portion for total fatty acid analysis and the other for free fatty acid analysis. X-Ray Diffraction of Liposomes. Liposomes for x-ray diffraction were prepared from the total lipid extract of myelin. Ten milligrams of lipid was evaporated to dryness in a conical reaction vial under N2. Residual solvent was removed in a vacuum desiccator and the lipid residue was weighed. An equivalent weight of 40 mM Tris acetate buffer (pH 7.5) was added, and the sample was equilibrated at room temperature. The resulting liposomes were transferred to a sample holder and sealed with polyethylene windows, and x-ray diffraction was carried out as above. Total Fatty Acid Analysis. The sample was evaporated to dryness under N2, dissolved in 1 ml of 14% BF3 in methanol (J. T. Baker), sealed under N2, and heated at 90'C for 90 min. The methyl esters were extracted with 2 ml of pentane/ H20, 2:1 (vol/vol), and identified by flame ionization gas chromatography using a stainless steel column (183 x 0.64 cm) packed with 5% EGSS-X in Supelcoport (100-120 mesh), maintained at 170°C. Heptadecanoic acid (17:0) was used as an internal standard. Free Fatty Acid Analysis. Free fatty acids were selectively esterified with diazomethane according to the method described by Schlenk and Gellerman (13). Heptadecanoic acid (17:0) was used as an internal standard. The esterified fatty acids were identified and quantitated by flame ionization gas chromatography as described above. Phosphate and Amino Acid Analysis. Ten milligrams of lyophilized myelin was suspended in 2 ml of peroxide-free isopropanol. Aliquots were taken for phospholipid analysis (14) and for amino acid analysis on a Durrum (D-500) amino acid analyzer after hydrolysis in 5.7 M HCO for 24 hr at 1100C under vacuum. Total micromoles of amino acid was multi-
ABSTRACT Wide-angle x-ray diffraction studies revealed that the lipid-phase transition temperature of multiple sclerosis (MS) myelin was about 20°C lower than that of normal myelin, indicating differences in the physical organization of the bilayer. The transition temperature of liposomes prepared from total lipid extracts of normal myelin was 12°C lower than that for corresponding intact myelin, demonstrating that the protein of normal myelin had a substantial ordering effect on the lipid bilayer. The transition temperature for liposomes of MS myelin lipid was essentially similar to that for isolated MS myelin. Because the protein/phospholipid ratio was higher in MS myelin, and no difference in degree of fatty acid saturation was observed, the inability of MS myelin protein to organize the lipid reflects a qualitative difference in the proteins.
Multiple sclerosis (MS) is a demyelinating disease affecting primarily the white matter of the central nervous system (1, 2). Demyelination occurs in discrete plaques found throughout the white matter of the brain (3, 4). Consequently, the disease has been considered to be one with multiple foci of discrete demyelination. There remains the possibility, however, that there is a more generalized involvement of white matter in the pathology of MS. In a recent low-angle x-ray diffraction study in which long-chain fatty alcohols were incorporated into myelin, the presence of additional reflections in the MS samples implied that the alcohols formed regular structures in domains of disorganized bilayer (5). Boggs and Moscarello (6) have shown an increased protein/lipid content in MS myelin but were unable to detect any significant difference in membrane fluidity by using fatty acid spin labels or fluorescent probes. Recently, however, an x-ray diffraction study revealed that basic protein from MS myelin was much less effective in inducing lipid organization in egg phosphatidylglycerol vesicles in comparison with basic protein from normal myelin (7). As well, the formation of the characteristic multilayer arrangement has been shown to depend on the concentration of basic protein because sharp reflections were observed by x-ray diffraction only when the concentration of basic protein was 30% (wt/wt) in the basic proteinphosphatidylglycerol system (8), supporting an earlier report in which digestion by trypsin disrupted myelin structure (9). The present study focuses on alterations in biophysical and chemical changes in myelin due to the neuropathology of MS.
MATERIALS AND METHODS Myelin Isolation. The brains of six patients who died with MS and five brains from accidental deaths were obtained within 4-8 hr after death. All MS patients had the disease for 10-30 years. The cortex was removed, and the white matter The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Abbreviation: MS, multiple sclerosis.
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plied by 110 ,ug/mol to give weight of protein earlier (6).
Proc. Natl. Acad Sci. USA 81 (1984) as
described
RESULTS Wide-angle x-ray diffraction can be used to discern liquidcrystalline-phase and gel-phase lipid in membranes. Lipid in the liquid-crystalline (fluid)-phase is manifest as a broad diffuse x-ray reflection centered at a Bragg spacing 4.6 A, and gel phase gives rise to a sharp reflection centered at 4.15 A (15, 16). Densitometric tracings of wide-angle x-ray diffraction patterns of MS and normal adult myelin, recorded at 250C, both featured the broad 4.6 A reflection representing the liquid-crystalline-phase lipid and a sharp 4.15 A reflection, which appeared as a distinct shoulder on the broad 4.6 A peak (Fig. 1, tracings A and B). This 4.15 A peak was derived from an ordered crystalline (gel) phase of the lipid in which there was close hexagonal packing of the hydrocarbon chain. To confirm that detection of the gel phase in adult myelin simply reflected lipid-gel phase and was not attributable to dehydration, specimens used for diffraction studies were analyzed gravimetrically to determine their water content. Myelin containing 75-80% moisture with respect to final dry weight still gave rise to the diffraction patterns featuring the sharp reflection at 4.15 A. Wide-angle x-ray diffraction patterns of MS myelin recorded at 50'C revealed that the lipid was exclusively liquidcrystalline because only the broad diffuse x-ray reflection centered at 4.6 A was present (Fig. 2, tracing A). However, the corresponding pattern for normal myelin featured both the sharp 4.15 A reflection peak, representing gel-phase lipid, and the broad 4.6 A reflection derived from liquid-crystalline-phase lipid (Fig. 2, tracing B), indicating that this myelin contained both liquid-crystalline- and gel-phase lipid. The intensity of the 4.15 A reflection in wide-angle x-ray diffraction patterns serves as a relative measure of the amount of gql-phase lipid in membranes. However, the presence of gelsphase lipid can also be detected as a rise in transition temperature, which, in this case, is defined as the highest temperature at which gel-phase lipid can be discerned and reflects the composition of phospholipids and, in particular, fatty acids contributing to the gel phase (17, 18). Thus, above the transition temperature, membrane lipid is exclusively liquid-crystalline, and below the transition tempera-
A
4.0
5.0 Bragg spacing, A
FIG. 1. Densitometer tracings of wide-angle x-ray diffraction patterns recorded at 260C for myelin isolated from MS white matter (A) and normal adult white matter (13), both showing a broad lipid band centered at a Bragg spacing of 4.6 A, representing liquid-crystalline lipid, and a sharp lipid band centered at a Bragg spacing of 4.15 A, representing gel-phase lipid.
Bragg spacing, A
FIG. 2. Densitometer tracings of wide-angle x-ray diffraction patterns recorded at 50'C for myelin isolated from MS and normal adult white matter. A, Tracing for myelin from MS white matter showing only a broad lipid band centered at a Bragg spacing of 4.6 A, representing liquid-crystalline lipid. B, Tracing for myelin isolated from normal adult white matter showing a sharp lipid band centered at a Bragg spacing of 4.15 A, representing gel-phase lipid, and a broad lipid band centered at a Bragg spacing of 4.6 A.
ture, the membranes contain a mixture of liquid-crystallineand gel-phase lipid. In all of these experiments, the transition temperatures of MS and normal myelin were thermally reversible. Transition temperature data for normal and MS myelin are summarized in Table 1. For MS myelin, the transition temperature was 43.2 ± 1.20C, whereas for normal myelin, it was 63.0 ± 1.40C. There were no significant differences in the unsaturated/ saturated fatty acid ratios of lipid-associated fatty acid for normal and MS myelin (Table 2). In comparison with normal myelin, the MS myelin had increased levels of 16:0, 18:0, 20:4, 20:5, 22:3, and 22:6 and decreased levels of 18:1, 20:0, 18:3, 20:1, and 22:4. Because the data did not result in an altered unsaturation/saturation ratio, the different phase properties of MS and normal myelin cannot be attributed to a change in the degree of fatty acid saturation. The major free fatty acid components of normal and MS myelin were 16:0, 18:0, 18:1, 20:3, and 20:4 (Table 2). In MS myelin, the ratio of free fatty acid/total fatty acid was slightly higher than in normal myelin, but the range of variation was also relatively large and thus the difference was not staTable 1. Comparison of lipid-phase transition temperatures for MS and normal adult myelin and for liposomes of total lipid extracts Transition temperature, 0C Brain Brain number Type Myelin Liposomes 65 53 176 Normal adult 178 65 50 180 58 45 182 62 52 65 190 54 50.8 ± 1.6 Mean ± SEM 63.0 ± 1.4
MS
Mean ± SEM
A B C 179 181 193
40 43 48 45 40 43 43.2 ± 1.2
40 40 45 42 40 40 41.2 ± 0.8
Proc. Natl. Acad. Sci. USA 81 (1984)
Neurobiology: Chia et al. Table 2. Fatty acid composition of normal and MS myelin Total fatty acid, mol % MS Normal (n =4) (n =3) Fatty acid 11.72 ± 0.41 9.25 ± 0.33 16:0 21.04 ± 0.59 18.58 ± 0.98 18:0 17.51 ± 0.58 22.45 ± 1.16 18:1 0.57 ± 0.24 0.85 ± 0.02 18:2 14.74 ± 0.80 18.48 ± 0.89 20:0 1.41 ± 0.11 3.97 ± 0.25 18:3 0.52 ± 0.06 1.35 ± 0.53 20:1 4.73 ± 0.57 4.59 ± 0.60 20:3 3.54 ± 0.24 1.73 ± 0.67 20:4 3.33 ± 0.91 1.98 ± 0.17 20:5 19.43 + 1.34 15.32 + 2.04 22:3 0.52 ± 0.06 0.89 ± 0.20 22:4 2.31 ± 0.63 1.19 ± 0.14 22:6 1.17 ± 0.06*
1.13 ± 0.06*
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Free fatty acid, mol % MS Normal (n =4) (n =3) 18.02 ± 2.02 17.57 ± 0.94 14.22 ± 2.27 24.14 ± 1.14 23.68 + 3.08 20.16 ± 1.59
18.66 ± 0.62 19.72 ± 2.61
0.04 ± O.OOt 1.44 ± 0.03f
20.25 ± 8.66 23.83 ± 3.43
0.07 ± 0.Ot 2.28 ± 0.47f
Values are shown as mean ± SEM.
*Unsaturated/saturated fatty acid. tFree fatty acid/total fatty acid.
fUnsaturated/saturated free fatty acid.
tistically significant. The ratios of unsaturated/saturated from fatty acid for MS and normal myelin were also not statistically different (Table 2). The transition temperature for liposomes of total lipid extracts from MS myelin was similar to that for the intact MS myelin (Table 1). The liquid-crystalline to gel-phase transition occurred at 41.2 0.8°C for total lipid and at 43.2 1.2°C for intact myelin. In contrast, the transition temperature of liposomes from normal myelin was dramatically different from that of intact normal myelin. The liquid-crystalline to gel-phase transition occurred at 63.0 1.4°C for intact myelin and at only 50.8 1.6°C for corresponding total lipid extracts (Table 1). ±
±
±
±
DISCUSSION Wide-angle x-ray diffraction has revealed significant differin the physical organization of the lipid bilayer between MS myelin and normal myelin. This technique has the advantage of not perturbing the bilayer in the way that probe techniques (such as ESR) do. Moreover, by x-ray diffraction, it has proved possible to detect gel-phase lipid in membranes that was not discernible by ESR, presumably because spin probes tend to become localized in the liquid-crystalline or fluid regions of the bilayer (18). Possibly for this reason, differences in fluidity between MS and normal myelin were not detected in previous studies (6) employing fatty acid spin labels, even though the ratio of protein/lipid was found to be higher in MS myelin. The absence of gel-phase lipid in wide-angle x-ray diffraction patterns of MS myelin at 50°C and its presence in corresponding patterns for normal myelin suggest that the lipid bilayer was more disordered in MS myelin than in normal myelin. The bands of primary interest in this study were the lipid reflections at Bragg spacings of 4.6 and 4.15 A. The broad 4.6 A band was derived from membrane lipid that was in a liquid-crystalline state and was essentially disordered (19-22). The appearance of the sharp band at 4.15 A designated the presence of ordered gel-crystalline-phase lipid (19-21, 23). Thus, both types of membrane contain a mixture of liquid-crystalline and gel phases at physiological temperature, but the transition temperature, which is taken to be the highest temperature at which gel-phase lipid can be detected, was -200C lower for MS myelin than for normal
ences
myelin. To determine whether this change in phase behavior resulted from alteration of the myelin lipid itself or from its interaction with protein, transition temperatures of liposomes prepared from total lipid extracts of myelin were compared. For MS liposomes, the transition to exclusively liquid-crystalline lipid was essentially the same as that for myelin, indicating that the protein in MS myelin did not influence the transition temperature. On the other hand, the transition temperature for liposomes from lipids derived from normal myelin were 13'C lower than that for normal myelin membrane, implying a role for the protein in the normal membrane. The fatty acid composition of total lipid from MS myelin used in this study was very similar to that reported previously (6, 22) and was not markedly different from normal. Thus, the altered phase properties of MS myelin lipid cannot be attributed to changes in fatty acid composition or in the degree of fatty acid saturation. Also, it is unlikely that the altered phase properties of MS myelin lipid can be attributed to changes in the cholesterol content because a previous study found no difference between the mean cholesterol/ phospholipid ratio of MS and normal myeliti (6). Demyelination in MS involves the breakdown of relatively large quantities of phospholipids (24-26), presumably due to hydrolysis by phospholipases releasing free fatty acids (2426), and any overt differences between normal and MS myelin in the degree to which free fatty acids were retained in the bilayer could give rise to corresponding alterations in lipid-phase properties (27). Histochemical and biochemical evideitce showed that free fatty acids do accumulate in apparently unaffected areas of MS brain (28, 29). Our present study showed that there was a slightly higher level of free fatty acid in MS myelin as compared to normal myelin, but, because of the large variation, the difference was not statistically significant. Nevertheless, s'-ch an increase in short acyl chains could decrease binding of the polar head groups to proteins (30, 31) and alter the molecular organization of the bilayer. The total amino acid content of MS myelin suggests a relative increase in proteolipid and a relative decrease in basic protein (6). Human myelin proteolipid does not appear to exert a strong influence on the phase-transition temperature of lipid (32). Basic protein promotes organization of the phospholipid bilayer into multilayer structures characteristic
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of normal myelin (7). However, the basic protein of MS myelin is much less effective in inducing lipid organization than normal basic protein (7). This qualitative difference of MS basic protein, coupled with the possible quantitative variation, may contribute to the altered phase properties of MS myelin. This work was supported by the Medical Research Council of Canada, Grant MT-4844. 1. McKhann, G. M. (1982) Annu. Rev. Neurosci. 5, 219-239. 2. Suzuki, K., Kamoshita, S., Eto, Y., Tourtellotte, W. W. & Ganatas, J. (1973) Arch. Neurol. (Chicago) 28, 293. 3. Itoyama, Y., Sternberger, H. H., Webster, H. de F., Quarles, R. H., Cohen, S. R. & Richardson, E. P. (1980) Ann. Neurol. 7, 167-177. 4. Hallpike, J. F. (1972) Prog. Histochem. Cytochem. 3, 1-39. 5. Weiss, A., Neumann, A. W. & Moscarello, M. A. (1982) in Protides of the Biological Fluids, ed. Peeters, H. (Pergamon, New York), Vol. 30, pp. 167-170. 6. Boggs, J. M. & Moscarello, M. A. (1980) Neurochem. Res. 5,
319-335. 7. Brady, G. W., Fein, D. B., Wood, D. D. & Moscarello, M. A. (1981) FEBS Lett. 125, 159-160. 8. Brady, G. W., Fein, D. B., Wood, D. D. & Moscarello, M. A. (1980) Biophys. J. 34, 345-350. 9. Adams, C. W. M. & Tugan, N. A. (1961) J. Neurochem. 6, 334-341. 10. Chia, L. S., Thompson, J. E. & Moscarello, M. A. (1983) FEBS Lett. 157, 155-158. 11. Nichols, B. W. (1964) in New Biochemical Separations, eds. James, A. J. & Morris, L. S. (Van Nostrand, Toronto), pp. 321-337.
Proc. NatL Acad Sci USA 81 (1984) 12. Jacobsen, K., Nir, S., Isaac, T. & Papahadjopoulos, D. (1973) Biochim. Biophys. Acta 311, 330-348. 13. Schlenk, H. & Gellerman, J. L. (1960) Anal. Chem. 32, 14121414. 14. Bartlett, G. R. (1959) J. Biol. Chem. 234, 466-468. 15. McKersie, B. D., Thompson, J. E. & Brandon, J. K. (1976) Can. J. Bot. 54, 1074-1078. 16. Chia, L. S. (1981) Dissertation (Univ. of Waterloo, Waterloo, Ontario, Canada). 17. McKersie, B. 0. & Thompson, J. E. (1978) Plant Physiol. 61, 635-643. 18. Chia, L. S., Thompson, J. G. & Dumbroff, E. B. (1981) Plant Physiol. 67, 415-420. 19. Esfahani, M., Limbrick, A. R., Knutton, S., Oka, T. & Wakil, S. J. (1971) Proc. Nati. Acad. Sci. USA 68, 3180-3184. 20. Engelman, D. M. (1970) J. Mol. Biol. 58, 115-117. 21. Engelman, D. M. (1971) J. Mol. Biol. 58, 153-165. 22. Muller, A. (1932) Proc. R. Soc. London Ser. A 138, 514-530. 23. Schechter, E., Letellier, L. & Gulikkrzywicki, T. (1974) Eur. J. Biochem. 49, 61-76. 24. Davison, A. N. (1977) Proc. R. Soc. Med. 70, 349-351. 25. Bowen, D. M., Davison, A. N. & Ramsey, R. B. (1974) in Biochemistry of Lipids, ed. Goodwin, T. W. (Butterworth, Toronto), pp. 141-179. 26. Suzuki, K. (1978) in Pathobiology of Axons, ed. Waxman, S. G. (Raven, New York), pp. 357-368. 27. Muranushi, N., Takagi, N., Muranishi, S. & Sezaki, H. (1981) Chem. Phys. Lipids 28, 269-279. 28. Hartog-Jager, W. E. den (1978) Histochemistry 58, 273-280. 29. Craelius, W., Rosenheck, D., Schacter, D. & Shaer, A. (1979) Neurosci. Abstr. 5, 399. 30. Salem, L. (1962) Can. J. Biochem. 40, 1287-1298. 31. Vandernheuval, T. A. (1963) J. Am. Oil Chem. Soc. 40, 455. 32. Boggs, J. M., Clement, I. R. & Moscarello, M. A. (1980) Biochim. Biophys. Acta 601, 134-151.
