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V . .... . .....V...oo .....4....-.v... . .... ...€...r... h....- oVV.Q\..a» - . -....." .1.”.VnVVV. 1.44? Her/.ovagfi 1954’)“. w o ... .. V .t. . . V . V. . V . .V... . V V. l.- .. . ... . .. V.. .V V... V o. ..V...........oV.Vo......OVV ‘43, V. ...... ...... ..r. ... CK... ...-l V 'V ....”f...’ .uuna.. ’0. .1 ’C .V w . . a. . V V V ... V . .... .... .. o — c. I oou‘ .4 .. V4..n-. I. .V.<...oc...¢.-dv .....u.‘v/ . cu 0-. D"'@;.A‘ ,’J‘V~ ‘ V .. V . V . .. .V V . ..VV. . . . V. V'. ...... . ... r.. . ..aoo .. VVVc. v.4... .... . ...-0.9.(140'~¢ narcon’aftu V A. V .V . V. . . . . . . .V O .. o . V. .. V... .V . v V .0. V. ... ..o.. . no.4. ..f. V... a... o .J. ...V..:.’.V. c .I... .J00 .. Ira-.0 o 9.0 o. O. . V V‘ V. . V .V . V ... JV..VV.V . ... . ...§. ......VVV......4...o.......o.V.. 1.39.... .... .o.p.. V...u-V-oa..c~..V.lw”uo/Jw ”a V V V V. _ . V V . V V V . V . V V. ..V. ....V ... . ...V. ...... ... V.. V V \. V..... V .V .V V v... .....uwo.afwr ~ a!;. V .. V V V . V . ... V... ..V ...V. V. ... .V ..V .. . a. V .. ...... ....V'IV....... (.../2; 0.4.9.9. V a .VVV . . V . .. V. V. . . V . VVV V .V V V V. o V .. ... .. V . . . ......V. V .V.V .4..VV..o»..:. V ... I... V . .. "VFQ... #5.... .4 V V V . . . 9 V V .V V _ V ...V. . .V .V ....V V. .V V. .... .. . V.. ...v . . .... .VV.VV...4.J.0 ~ 4 ca. ....wro «1.0 «a . . V V . ..V . - V . .to .V A..Vo .Vo¢.4..od..a «a a.. o! .vo.¢’.f.:..n..r. . . . .V . . V. . V. V a. V... o...r....o......c.. "......ccp ..uV. . v . V V V V. . 4. V V a o .. . . V tn rivaa V. CERAMIDE The long chain base and fatty acyl-COA are converted to ceramide by means of a transferase. Degradation is achieved by hydrolysis to sphingosine in a reversible reaction by enzymes localized in the lysosome523. Ceramides have been isolated from many tissues, where they are probably intermediate forms of other sphingosine- containing lipids, or degradation products of more complex lipidsa. Cerebrosides Cerebrosides are a sub-group of the glycosphingo- lipids composedB' 77 of a ceramide to which glucose (Figure 3) or galactose (Figure 4) is bound by a beta- glycosidic linkage between C6 of the hexose and C1 of the long chain base. They glycosyl ceramides are the compounds of chief concern in this report. They are important l6 .Ampwmounmuwooosamv moflemuoo meoosam .m musmwm care o.n_c :0 5.1 m5 2319-5 :0. =0. :0. I0 I :o «:0. o (fl IV: 299.0 17 .Aopwmounmumoouomammv mpfifimumo Hamouomamu .w musmflm 3 {51 o.w 1.0 _.__ Io I2 I I0 I m N: _ _ N 5 29-5.5-5-5- 5-0 oz IONS 18 structural components of biological membranes, eSpecially myelin,and are closely associated with the sulfatides. Both galactosyl ceramide and sulfatide are present in large quantities in brain white matter, and the sum and ratio of these two compounds function as a sensitive biochemical indicator of the amount of brain myelin pre- sent. There is direct sulfation of cerebroside by brain tissue63. Sulfatide catabolism is accomplished via arylsulfatase which degrades it to galactocerebroside44' 45' 63. The sulfate ion can be removed by acid hydrolysis in an analytical procedure, and the galactosyl ceramide can then be quantified4l. The monohexosyl ceramides may be the precursor to other sphingosine containing lipids, sphingolipids or end stage degradation productsa. Extraneural tissues contain only very small amounts of cerebrosides when compared to neural tissues, but distinct differences exist from organ to organ between 41 the various classes of sphingolipids , and the make-up of individual lipids including cerebrosides varies as to hexose moiety, long chain base, and fatty acid. Cerebrosides have been recovered from a wide array of biological sources in both vertebrates and evertebrates4l. Monohexosyl ceramides have been isolated from many biological samples besides brain including plasma57' 65' 72' 30, 31, 34 14 75' leukocytes , erythrocytes , liver, kidney. lung, intestine, aorta21, placenta, len323' 41, muscle68, l9 nerve68, cerebrospinal fluid58' 71, bone marrowls, 17, and plasma membraneslB. urine All mammalian cells and many subcellular organelles are encompassed by a membrane composed of protein and lipid, and myelin is the largest plasma membrane in mammals45. Cerebrosides and sulfatides of white matter myelin constitute 20% of the dry weight41. For many years it was thought that galactosyl ceramide was the only cerebroside present in brain. Recently, however, small quantities of glucosyl ceramide have been reportedlzr 26. Dod and Gray18 found that 7% of the total lipids in rat liver plasma membrane was ceramide monohexoside and none of this was attributed to mitochondrial membranes. 72, 73 Kidney and erythrocytes contain largest amounts of the more complex hexosyl ceramides and only small amounts of the monohexosyl ceramides. Of the latter, glucosyl ceramide is found in greater amounts than the galactosyl ceramide. This is also true for placenta41. On the other hand, monohexosyl ceramides are found in largest quantities in plasma and serum with approximately ten times more glucosyl than galactosyl ceramide present. Monohexosyl ceramides represent approximately 25% of all neural glycosphingolipids and glucosyl ceramides predominate in intestine, lens, liver, lung, placenta, and spleen23. Glucosyl ceramides predominate in leuko- d31, 34, 43 t17 cytes of human bloo , urine sedimen , and aorta21. Human muscle contains only very small amounts of 20 monohexosyl ceramides, chiefly galactosyl ceramides, but analysis of femoral nerve glycolipids showed an approximate 2000-fold increase in galactosyl ceramides over muscle. Fatty acid patterns of both cerebrosides and sulfatides in muscle and nerve were identical, and it was concluded by the authors that these muscle lipids were derived from nerve tissue68. Early investigators described the cerebrosides of cerebrospinal fluid as non-phorous containing sphingo- lipids, while others using comparative mobilities on paper chromatography suggested that lipid patterns of cerebro- spinal fluid were similar to those found in plasma. Using gas chromatography-mass spectrometry and borate thin-layer 58 found no glucosyl ceramide 59 chromatography36, Samuelsson fraction. In subsequent studies comparing long chain bases, fatty acids, and carbohydrates in human brain and plasma, she found that brain and cerebrospinal fluid had practically identical cerebroside patterns which differed greatly from those of plasma. 21 from diseased and The examination of aortic tissue apparently normal vessels shows the cerebrosides present to be almost exclusively glycosyl ceramides with values ranging from 0.01 to 0.73% of the total lipid. Biosynthesis and Degradation of Cerebrosides Experimentally, ceramide synthesis has been found to occur in microsomes of the cells in which the lipids are 14 found23’ 75. Dawson and Sweeley say that plasma 21 glycosphingolipids are formed in the liver and that glucosyl ceramides are not synthesized in bone marrow, but do not speculate on location of activity. Biosynthesis of galactosyl ceramide has two known pathwayss' 41. In the first, the galactose moiety is transferred to ceramide from uridine diphosphate-galactose (UDP-gal): CERAMIDE + UDP-GAL -——-9- GAL-CER + UDP The second pathway involves the acylation of galactosyl sphingosine: SPHINGOSINE + UDP-GAL -—%> GAL-SPHINGOSINE + UDP On the other hand, only one pathway for synthesis of glucosyl ceramides has been elucidated: CERAMIDE + UDP-GAL-——€> GLC-CERAMIDE + UDP Basuz has found an enzyme in chicken embryo brain which catalyzes this reaction. This step may be important to synthesis of acidic glycosphingolipids, the gangliosides. Svennerholm67 indicates that ganglioside synthesis is accomplished in fetal brain by a step-wise addition of monosaccharide units through the action of a multi-enzyme cloud of glycosyl transferase323. The formation of glucosyl ceramide in this fashion from glucose and ceramide and, subsequently, to 22 glucosyl-galactosyl ceramide has been demonstrated in 21.222- Glycolipids undergo complete breakdown when one segment, i.e., the long chain base, fatty acid, or hexose, splits off37. Glycosyl ceramide is generally thought to be an end product in the degradation of more complex glycosphingo- 41 with hydrolysis localized in the 1ysosomes74. lipids Burton8 shows a series of catabolic reactions utilizing six hydrolases in a system in which there is a step-wise removal of the hexoses, and the end products are glucose and ceramide. Faulty or deficient enzymes specific for cleaving terminal sugars amides are responsible for most of the lipidoses so far encountered4' 5' 6' 7' 27' 30' 51. Ceramide is further degraded23: CERAMIDE ATP CERAMIDASE SPHINGOSINE + FATTY ACID w SPHINGOSINE —1-Po4 W PALMITALDEHYDE + ETHANOLAMINE-PO J’ 4 PALMITIC ACID Cerebroside Changes in Development and Aging The developing brain from an early embryonic stage to maturity undergoes a wide range of biochemical 23 activities. The earliest stage is characterized by cell proliferation (DNA replication), followed by differentia- tion (DNA transcription to RNA), then by protein synthesis (RNA translation to protein). During the latter stage, the emerging cell type takes on its own unique morphology and metabolism. With the maturation of specialized cells, i.e., those of the nervous system, myelination begins3. In man, myelination of the spinal cord tracts begins during the 22nd to the 36th week of fetal life; myelination of the brain tracts follows a short time later. Myelin is not seen in the corpus callosum until 8 weeks post- natally, while most of the myelination of the cerebral hemispheres takes place in the first two years of life23. This age is taken to be maturity and relatively small amounts of myelin are deposited after this time. The composition of human myelin does not vary to any signifi- 47, although cant degree between 10 months and 55 years data presented by another study66 show that fatty acid composition stabilizes at about 25 months and changes only to a small degree between that time and 72 years. Rapid accumulation of cerebrosides associated with myelination occurs at different ages in different species; as myelin is laid down, the cerebroside content increases proportionally. The lipid patterns, however, are similar. In the human, neutral glycosphingolipids and sulfatides are present to some degree before the onset of 24 myelination, and the patterns are very like those of extraneural tissues, especially liver. Glucosyl ceramides predominate with only small amounts of galactosyl ceramides present67. Near full term when myelination begins, the glucosyl ceramide content decreases and the amount of galactosyl ceramide and sulfatide greatly increase866. These two compounds continue to increase until myelination is complete. From then on, neither the hexosyl moiety or the sphingosine base changes. The fatty acids change only gradually by chain elongation, hydroxylation, and desaturation23' 77. This seems to be the only effect of aging. Myelin Myelin is the most elaborate and extensive of all mammalian membranes. It has been described23' 44 as a system of condensed Schwann cell or oligodendrocyte plasma membranes having alternating lamellae consisting of a protein-lipid-protein arrangement. When studied with polarized light, the lipid is seen to be oriented radially to the nerve fiberaxis, the protein, longitudinally45. Myelin is formed from the oligodendrocyte plasma membrane in the central nervous system and from the Schwann cell in the peripheral nervous system. During myelination, the nerve axon is envelOped or ensheathed between nodes of Ranvier by the plasma membrane processes, which are 25 continuous with the Schwann cell or oligodendrocytes. As many as fifty layers of myelin can be laid down around the axon. Each layer is not fused with the adjacent one, but lies in close proximity to it. By this mechanism, the myelin sheath becomes a multi-layer of the Schwann cell or oligodendrocyte membrane and encircles the axon between nodes of Ranvier23. There are differences in myelin quantity and composition between infants and adults. Fewer windings of the plasma membrane around the axon are found in infants9 and early myelin composition is therefore different from the adu1t13. The myelin of adults comprises 50% of the total dry weight of white matter44' 63. Myelin is not inert, but, for the most part, is an extremely stable structure. Davison and Dobbing9 have classified lipids as to function based on turnover time. Class 1 lipids have a half life of less than one week, function as fuel and energy reserves and for active synthesis and transport. Class 2 components have a t1/2 of one to four weeks and function as membrane components. The third class functions as structural components, and the tl/2 is greater than four weeks. Brain galactosyl ceramides show two turnover rates; a half-life of 45-46 days was demonstrated for the first, and the second, a very long half-life, indicating an extremely stable compound. A peak of activity is reached at the time of myelination which then drOps to low values41. 26 Labelling of fatty acids indicate two pools: incorporation of precursors into C18 rose rapidly and fell rapidly, while C20, C22, and C24 acids increased in activity for two weeks and then slowly declined. Martensson41 suggests that C18 might represent a non—myelin cerebroside. After 56 days, decay curves gave indications that possibly a third pool existed which was indefinitely stable. Radin and co-workers, also using labelling techniques, reported that sulfatides are formed from cerebrosides, but do not undergo breakdown in the normal brain49. In turnover studies on subcellular fractions using 35S-sulfate, a very small fraction of sulfatide was found to undergo rapid turnover exhibiting a half-life of 2 1/2 days. This suggested a very small, very metabolically active center was present in myelin41, and this has also been considered by othersBB. Extraneural cerebrosides were found to turn over much more rapidly than does myelin, similar to that of the non-myelin structures of brain. Onset of myelination is probably triggered by the formation of enzymes during the stage of RNA translation of protein. In instances of inherited genetic defects when a deficiency or lack of essential enzymes occur, hypomyelination or dysmyelination are probably the result45 .27 Many of the lipidoses are accompanied by demyelination attributed to a defective enzyme system, and galactosyl ceramides are known to disappear from myelinso. In addition to its major role as a myelin constituent, another capability has been ascribed to galactocerebroside, that of haptenic activity. Galactosyl ceramide, isolated from brain tissue from several species, has been shown to react with great sensitivity to form immune complexes with anti-brain antibodieszg. Rapport and Graf51 demonstrated that galactocerebrosides are antigenic determinants of myelin, and that their structural localization does not exclude their availability for interaction with specific antibody. Immunological activity of glucocerebroside could not be demonstrated. Other Diseases Accompanied by Demyelination Other demyelinating diseases occur in which there is no demonstrable mechanism for demyelination. Multiple 43 in which sclerosis is characterized by demyelination galactosyl ceramide has been shown to disappear from myelin and elevated quantities appear in the cerebrospinal fluid71. Multiple sclerosis has been studied extensively and from many aspects. Reports of gross examination of brain and spinal cord from these patients describe lesions, in general, as small, well circumscribed and widely distri- buted with areas of apparently intact normal tissue. 28 Galactosyl ceramides are slightly decreased in brains of patients with multiple sclerosis and deficiencies of lipids also exist in intact white matter and myelin of apparently normal brain tissue24. Gray matter can also be involved, but usually as an extension of a white matter lesionss. Plasma cerebroside composition has been determined only on plasma pools from patients with multiple sclerosisse; no reports documenting plasma cerebroside values in individual multiple sclerosis patients are available. Millar43 has suggested that multiple sclerosis could involve a defect of the blood-brain-barrier and has outlined a sequence of events which possibly accompanies demyelination. This sequence is similar to that described for vascular disease. Whether or not this sequence is correct is less important than the likelihood that the blood-brain-barrier may be altered in this disorder which probably involves an autoimmunity to myelin basic protein. Loss of myelin has been reported in stroke following massive destruction of brain tissue which may occur following cerebrovascular accident. Stroke is a general term often used synonymously with cerebrovascular accident (CVA) to indicate infarction or hemorrhage in the brain resulting from vascular disease. Generally, infarctions are secondary to thrombosis or embolism, while hemorrhages follow rupture of microaneurysms on vessels altered by hypertension. Appendix A lists the terms used to describe diagnoses of patients used in this study. Clinically, it 29 is preferable to designate the site of occlusion or damage as specifically as possible. The terms should include the site and location of injury as well as the vascular distribution and the exact vessel involved, if that is possible. A transient ischemic attack (TIA) is symptomatic of occlusive disease since tissue hypoxia or anoxia caused by ischemia during incomplete or intermittent blockage gives transient neurological symptoms. Vascular disease is an on—going process which can begin at a fairly early age and progress for a normal lifespan without clinical manifestations. Those subjects who have occult vascular disease could be considered to be normal. In these individuals, the build-up of fatty streaks and plaques upon the intima of the vasculature, which increases with age, is unaccompanied by significant symptoms. In others, infarction and brain destruction follows thrombosis or formation of emboliss. Gross examination of the brain following stroke has shown the myelin to be interrupted. Hausheer and Bernhard27, in comparing atherosclerotic aortas with normal aortas, found that total lipids as well as cerebrosides increase as atherosclerosis increases; both glucosyl and galactosyl ceramides are present. Foote and Coles21 in their study suggest that cerebrosides found in the human aorta, especially in fatty streaks, originate in plasma. Studies documenting plasma cerebroside changes in cerebrovascular disease are not available. MATERIALS AND METHODS Blood samples were obtained from hospitalized patients. In most instances, specimens from stroke patients were obtained within 2 weeks following onset of symptoms (see Appendix A). Fifty milliliters of blood were drawn into heparin, the samples centrifuged, the plasma separated from the cells, and frozen until analyzed. The controls used were five apparently healthy individuals age-matched with stroke patients. Fifty milliliters of blood from each subject were drawn into heparin. Following centrifugation, the plasma was separated from the cells, and analysis immediately initiated, without freezing the plasma. Duplicate 10 milliliter samples of plasma (measured to the nearest 0.05 ml) were analyzed according to the method of Vance and Sweeley72. No hemolyzed samples were used. Redistilled solvents were used throughout. Unisil (200-325 mesh) was obtained from Clarkson Chemical Company (Williamsport, Pa.). Commercial thin layer plates coated with silica gel G (Quantum Industries, Fairfield, N. J.) were pre-run in diethyl ether to remove contaminants and reduce the amount of plasticizer. The silylating reagent, 30 31 Regisil, was obtained from the Regis Chemical Company (Chicago, Ill.). Supelco Inc. (Bellefonte, Pa.) supplied the Supelcoport column packing (3% SE 30). Folch Extraction and Column Chromatography To the serum samples were added 120 milliliters of chloroform and 60 milliliters of methanol (Figure 5). The mixture was stirred with a magnetic stirring bar for 15 minutes at room temperature, and then filtered. The precipitate on the filter paper was washed with 20 m1 of chloroform-methanol (2:1, v/v). Forty milliliters of distilled water (from glass) were mixed with the filtrates and allowed to stand overnight in the cold. The lower phase was removed from this biphasic system and evaporated to dryness on a rotary evaporator with gentle heating (50°C). This total lipid portion was taken up in 0.5 ml of chloroform and applied to a 2 gm Unisil column. The neutral lipids were removed from the column with 50 ml of chloroform and discarded; the glycosphingo— lipids, with 100 ml of acetone-methanol (9:1, v/v). Alkali-Catalyzed Methanolysis Solvents were removed from the glyc03phingolipids, the residues transferred to screw-capped vials with chloroform-methanol (2:1, v/v) and dried under a thin stream of nitroqen. Mild alkali-catalyzed methanolysis was performed at this point to remove phospholipid con- taminantslg. 32 Plasma Folch Extraction Lower Phase Silicic Acid Column Chromatography Chloroform (Discard) Acetone-Methanol (9.1 vlv) Alkali Catalyzed Methanolysis Qualitative Thin-layer Chromatography Methanolysis Trimethylsilylation Gas-Liquid Chromatography Figure 5. A summary of cerebroside isolation. Figure 6. - —o- 34 Thin-layer chromatogram of glycosyl ceramides from human plasma. The bands which were visualized after exposure to iodine vapor are monohexosyl ceramide (GL ), dihexosyl ceramide (GL2), trihexosyl ceramide (GL3), and globoside (GL4) . 35 HCl Methanolysis A stock solution of 2 umoles/ml of mannitol standard was prepared in methanol (see Appendix C). Thirty micro- liters (60 nanomoles) of mannitol and 3 ml of 0.5N methanolic HCl were added to the cerebroside residue, and incubated at 80°C for 22-24 hours. After cooling, methyl esters of fatty acids were extracted by 3 treatments of equal volumes of hexane. The remaining portion of the methanolysis mixture containing methyl glycosides and sphingolipid bases were neutralized with silver carbonate, centrifuged, and transferred to screw-capped vials. Fatty acid methyl esters and methyl glycosides were reduced to dryness under nitrogen. Trimethylsilylation Trimethylsilylation was accomplished by treating both products of methanolysis with 25 ul of bis(trimethylsilyl) fluoroacetamide plus 1% trimethylchlorosilane (Regisil) and 25 pl of dimethylformamide for 15 minutes prior to injection into the gas—liquid chromatography apparatus. The TMSi derivatives of methyl glucosides, methyl galactosides, and the mannitol standard separated readily (Figure 7) on the 3% SE 30 column at 60°C. Calculations were made according to Vance and 73 Sweeley and converted to nanomoles/ml. Figure 7. 36 mm m FA—fi I ll WU LA lumflflmlllllllllillilllllllllllllllllillMill Wllull” ‘ l ' ’ ' ' ' ' "HIV” ll" """ ‘ ‘ Mlllihlnlnmlflnmmnllm .lnllalt‘llm II In it .................... L, __ Gas-liquid chromatogram of TMSi methyl glycosides of D-glucose and D—galactose with mannitol added as an internal standard. Run on a 2mm x 3mm column of 3% SE-30 on 100/120 Supelcoport at 150°C. A Hewlett-Packard F & M model 402 gas chromatogram was used with a flash heater at 250°C and a nitrogen carrier gas flow rate of 35 ml/minute. RESULTS The values given in Table 1 indicate the levels of total monohexosyl ceramides (glycosyl cerebrosides), galactosyl ceramide, and percent galactosyl ceramide of the individuals constituting the control group. The mean galactosyl ceramide was 3.07 nanomoles/ml (nM); the total cerebrosides was 28.18 nM/ml; percent galactosyl ceramide was 11.05; nM/ml of glucosyl ceramide was 25.02. Table 2 shows the average of paired samples of the group of multiple sclerosis patients. Total cerebrosides (28.34 nM/ml), galactosyl ceramides (3.19 nM/ml), percent galactosyl ceramides (11.40), and glucosyl ceramides (25.20 nM/ml) show a striking similarity to those values determined for the control group. The range of percent galactosyl ceramides of the control group was 9—14%, for the multiple sclerosis group, 8-15%. The concentration of cerebrosides in plasma of individuals suffering from stroke is presented in Table 3. Total cerebrosides (21.99 nM/ml), glycosyl ceramides (17.75 nM/ml), and galactosyl ceramide (2.80 nM/ml) were decidedly lower than those values of the control group, while the percent galactosyl ceramides (14.32) was higher. Even so, no significant difference (p=>0.05) at the 5% 37 38 Table 1. Concentration of glycosyl ceramides*. Average of paired samples (nanomoles/ml) of controls. Controls Patient Age-Sex GAL GLC Total GL1 %GAL V.W. 50m 5.11 38.62 43.74 11.76 J.D. 61m 2.89 28.87 31.76 9.08 B.G. 47f 2.79 24.92 27.71 10.14 F.D. 50m 2.10 17.51 19.61 10.71 C.H. 66m 2.46 15.16 18.11 13.55 X 3.07 25.02 28.19 11.05 *Abbreviations: GAL indicates galactosyl ceramide; GLC, glucosyl ceramide; and GL 1’ glycosyl ceramide. 39 Table 2. Concentration of glycosyl ceramides in plasma.* Average of paired samples (nanomoles/ml) of multiple sclerosis patients. Multiple Sclerosis Patient Age—Sex GAL GLC Total GL %GAL D.P. 24f 1.94 11.35 13.34 14.95 D.W. 44f 2.88 31.64 34.52 8.34 E.M. 46m 2.79 22.00 24.80 10.51 C.V. 28m 4.81 35.77 40.53 11.86 L.V. 37f 3.54 25.25 28.49 11.37 i' 3.19 25.20 28.34 11.40 *Abbreviations: GAL indicates galactosyl ceramide; GLC, glucosyl ceramide; and GL1, glycosyl ceramides. 40 Table 3. Concentration of glycosyl ceramides in plasma.* Average of paired samples (nanomoles/ml) of stroke patients. Stroke Patient Age-Sex GAL GLC Total GL1 %GAL A.K. 65m 1.56 11.20 12.76 12.21 E.S. 84f 4.57 14.90 19.47 25.66 E.M. 76m 4.69 25.57 30.36 14.52 G.M. 58m 2.28 22.65 24.93 9.26 P.S. 55m 1.20 8.55 9.75 12.32 C.D. 56m 1.96 14.49 16.45 11.94 D.S. 76m 3.36 36.89 40.23 8.36 36 2.80 17.75 21.99 14.32 *Abbreviations: GAL indicates galactosyl ceramide; GLC, glycosyl ceramide; and GL1, glycosyl ceramide. 41 level was observed between the total cerebrosides of the stroke and multiple sclerosis groups when compared with the control group (Table 4). Table 5 gives a comparison of nanomoles/m1 and percent of galactosyl ceramides of the three groups. Using analysis of variance, there was no significant difference at the 5% level (p=>0.05). Patient E.S. showed 25.66% of galactosyl ceramide (mean = 14.32%). The range of percent for the other patients in the group was 8-14%. The diagnosis of this patient was cerebral thrombosis (stroke), and she had been a known diabetic for fifty years. Her case history (see Appendix A) gives no indication, however, of any diabetic neuropathy. The question of the effect of age upon the galactosphingolipid content of plasma certainly arises in this instance. Analysis of the glycosyl ceramides and percent galactosyl ceramides, excluding patient E.S., showed (Table 6) the total cerebrosides (22.41 nanomoles/ml) and percent galactosyl ceramides (11.44) to be more closely related to the control group. The range of the percent galactosyl ceramides between the individuals constituting the stroke group is the same (8-15%) as for the multiple sclerosis group (Table 7). Galactosyl ceramides are decreased to 2.51 nanomoles/ml. There was no significant difference (p=>0.05) between these two groups as deter- mined by nested analysis of variance. 42 Table 4. Total glycosyl ceramides, galactosyl ceramides, and glucosyl ceramides of all groups (nanomoles/m1). GL1 GAL GLC Controls 28.19 3.07 . 25.02 Multiple Sclerosis 28.34 3.19 25.20 Stroke 21.99 2.80 17.75 p=>0.05 43 Table 5. Galactosyl ceramides in plasma (nanomoles/ml). GAL %GAL Controls 3.07 11.05 Multiple Sclerosis 3.19 11.40 Stroke 2.80 14.32 p=>0.05 44 Table 6. Concentration of glycosyl ceramides, galactosyl ceramides, and percent galactosyl ceramide* of the stroke group. Patient E.S. is excluded. Average of paired samples (nanomoles/m1). Stroke Patient Age-Sex GAL Total GL1 %GAL A.K. 65m 1.56 12.76 12.21 E.M. 76m 4.69 30.36 14.52 G.M. 58m 2.28 24.93 9.26 P.S. 55m 1.20 9.75 12.32 C.D. 56m 1.96 16.45 11.94 D.S. 76m 3.36 40.23 8.36 X 2.51 22.41 11.44 *Abbreviations: GAL indicates galactosyl ceramide and GL1, glycosyl ceramide. 45 Table 7. Concentration of galactosyl ceramides and glycosyl ceramides (nanomoles/ml) and percent galactosyl ceramides of the stroke, multiple sclerosis, and control groups when patient E.S. is excluded from the stroke group. GAL %GAL GL1 Control 3.07 11.06 28.19 Multiple Sclerosis 3.19 11.40 28.34 Stroke 2.51 11.44 22.42 p=>0.05 DISCUSSION The values for controls in this report are higher than previously given for normals, but the individuals who compose this group were older than control groups previously studied. The percent of galactosyl ceramides is slightly lower (9-14%) than reported by Rathke and JonesS3. The control group was age-matched with stroke patients. The range of percent galactosyl ceramides found in multiple sclerosis patients was also slightly lower (8-15%), but the mean percent galactosyl ceramides of these patients (11.40) compared very closely with that of the controls (11.05). The values for galactosyl ceramides (2.80) and total cerebrosides (21.99) were lower than the controls, but were not significantly lower; percent galactosyl ceramides were higher (14.32), but still within the range found in the controls, and not significantly different. Patient E.S. had 25.66% galactosyl ceramides. This patient was an 84 year old female, and a known diabetic for fifty years, although her case history gave no indi- cation of any diabetic neuropathy (see Appendix A). The question of the effect of age on glycosphingolipid content of plasma certainly arises in this patient. In fact this 46 47 patient could probably be included in three different categories: aging, stroke, and other neurological disorders. For this reason, it was decided to re-evaluate the data excluding this patient. The galactosyl ceramide mean dropped from 2.80 nN/ml to 2.51 nM/ml; total cerebrosides rose only a small amount, to 22.41 nM/ml as compared to 21.99 nM/ml in the original calculation. The mean percent galactosyl ceramides was found to be 11.44%. This was very similar to the mean percent galactosyl ceramides found in the control group (11.05%) and the group of multiple sclerosis patients (11.40%). There are several points in the analytical procedure where errors could have been incorporated. Gangliosides, that is, glyc05phingolipids containing neuraminic acid, and sphingolipids having five or more molecules of carbohydrate, are soluble in both chloroform- methanol mixtures and in water; cerebrosides having one to four carbohydrate moieties are notlg. This is the basis for the Folch extraction procedurezo. Gangliosides, moreover, are strongly influenced in biphasic systems77 by K+ and Ca++. Some workers have used KCl in the aqueous phase to facilitate quantitative separation of gangliosides from cerebrosides. The work described in this paper was performed using distilled water. It is conceivable that some gangliosides remained with the cerebrosides. This problem should have been resolved, however, by the thin- layer chromatographic procedure. GLl bands co-migrated 48 with known standards; bands were well separated and there was no overlap. Products of the methanolysis procedure were identified as methyl glycosides of hexoses by gas- liquid chromatography. Any work done with plasma bears the risk of some contamination from erythrocytes despite careful centrifu- gation and removal of plasma from the red cell layer. Glycosyl ceramides occur in small quantities in erythrocyte 72: 73 and in leukocyt8834- The possibility does stromata exist that some very small quantity of glucosyl ceramides from blood cells was co-analyzed. There was, however, no visible hemolysis in any sample, and any red or white cells escaping visual detection would have contributed an insignificant amount of cerebrosides. 71 conducted an exhaustive Tourtellotte and Haerer study on lipids in cerebrospinal fluid in 156 patients diagnosed as having multiple sclerosis or retrobulbar neuritis. Patients were categorized according to number of exacerbations, stage of remission, age, length of time the patient had had the disease, amount of disability, levels of cerebrospinal protein, and acute or chronic stage of the disease. Cerebrosides were elevated in all patients, and more so in patients showing the greatest elevations in cerebrospinal fluid protein and those patients over 40 years of age. The assumption is that patients over 40 have had multiple sclerosis for longer periods of time and have greater areas of demyelination 49 resulting in breakdown of the blood-brain-barrier with an increase in CSF protein. Except for patient E.M. who is described as having had multiple sclerosis for 14 years, case histories of patients do not contain this information. There are no data available as to whether the patient is undergoing an initial bout of the disease or an excerba- tion. Neither is any information given regarding the number of exacerbations the patient has had, nor the possible extent of his disease. This kind of data might help to explain the variation between patients in this study, and the experiment should have been designed so that the information was included. The higher amounts of galactosyl ceramides in the plasma of patients C.V. and L.V. may indicate more extensive demyelination. -On the other hand, even though an increase in cerebrospinal fluid galactosyl ceramide has been reported in multiple sclerosis patients, the total body quantity of cerebrOSpinal fluid is small compared to that of plasma. Consequently, assuming that products of demyelination do appear in plasma, these products would suffer a several-fold dilution which may not be sufficient to elevate the concentration of galactosyl ceramide above a rather wide normal range. This assumes that the blood-brain-barrier permits entrance of substances from the central nervous system into plasma. Communication with the brain via the blood has been shown to be highly selective, and the 50 possibility exists that unless this barrier is damaged directly in the process of demyelination, galactosyl ceramides cannot gain access to plasma. The mean values of glycosyl and galactosyl ceramides for stroke patients are reduced from those of control and multiple sclerosis subjects, but not to a significant degree. The percent galactosyl ceramides was found to be higher. Although considerable variation of glycosyl and galactosyl ceramides was observed, this variation was also present in the other two groups. As in the case of the multiple sclerosis patients, more complete case histories might have proved helpful. For example, more Specific documentation of the onset of symptoms, the stage in the course of the disease when plasma was withdrawn from the patient, and an estimation of the severity of the disease might have proved helpful in determining if a time interval elapsed before products of destroyed brain were dumped into plasma. Massive destruction of brain is known to disrupt the blood—brain-barrier, and the prediction that galactosyl ceramides would appear in blood plasma is a natural one. This conclusion, however, is not consistent with the data. The variation in concentration of galactosyl ceramide encompassed a wide range. Two explanations are possible: 1) The time of sampling could be very significant; a time interval may be required before molecules from destroyed brain tissue appear in plasma, and earlier values 51 may be normal or subnormal. It may be that cerebrosides are removed from plasma and deposited upon the intima prior to stroke and contribute to thrombus formation. Following stroke, cerebrosides from damaged brain are returned to plasma, and 2) the extent of the lesion may be a contributing factor. Smaller lesions contribute smaller amounts and, as postulated for cerebrospinal fluid, dilution of these products of brain destruction, may not produce a significant increase above the fairly wide range of normal values. The source of plasma cerebrosides has not been established although Dawson and Sweeleyl4 have postulated that glycosphingolipids from senescent erythrocytes establish an equilibrium with plasma, and Kattlove 33 21. present formulae for calculating quantities of cerebro- sides catabolyzed daily from leukocytes. Except for blood cells, the equilibrium of the various extraneural organs with plasma in health and disease has not been verified. Plasma is known to be a carrier of and a depot for other metabolic products from these organs. While long chain bases and fatty acids are known to be organ Specific, whether these molecules form a composite in plasma following degradation is uncertain. It is of interest to note that the fatty acids found in plasma were predominantly of the shorter chain lengths, especially palmitic acid (C16), and that it is this particular fatty acid which is thought to be the basic 52 fatty acid molecule passing from blood to brain and ultimately undergoing elongation, hydroxylation, and 75 in the formation of brain glycosphingo- desaturation lipids. Communication between plasma and brain constituents is a highly selective process. Elevation of glycosphingo- lipid levels in both plasma and urine have been found in some lipid storage diseases, even those accompanied by demyelination. These lipids have been attributed to underdestruction, not overproduction, due to enzyme defects and are generally not considered to be products of demyelination. Studies on the mechanism of aging have brought to light few biochemical changes occurring in the aging process in normal subjects. No information is available to describe if, or at what point, organ structures such as kidney or liver begin to degenerate on a small scale throwing off structural moelcules, such as cerebrosides, into plasma. This could explain the elevation of plasma cerebrosides found in the older control subjects used in this study. SUMMARY Duplicate plasma samples from seven stroke patients, five multiple sclerosis patients, and five control subjects age—matched with stroke patients were analyzed to deter- mine the levels of concentration of cerebrosides. The analysis involved the use of the Folch extraction procedure to separate the total lipids, Silica gel column and thin-layer chromatography to separate monohexosyl ceramides, HCl methanolysis to rupture the glycosidic and amide bonds of the cerebroside molecule, and quantifica- tion of the hexose moiety by gas-liquid chromatography. While the normal values in this study were elevated above those previously reported, the percent of galactosyl ceramides was similar to that determined on younger patients. The concentration of plasma cerebrosides in control subjects and multiple sclerosis patients was strikingly Similar. Cerebroside values for the stroke patients were lower and the percent galactosyl ceramides were higher than for the control subjects. No values, however, varied to a significant degree (p=>0.05). Recalculation of data from the stroke group following elimination of the patient who appeared to be afflicted with more than one disease entity and who showed an 53 54 approximate two-fold elevation of galactosyl ceramides above the other patients in the stroke group gave the following results: the total cerebrosides were slightly elevated, galactosyl ceramides decreased considerably, but not to a significant degree, and the percent galactosyl ceramides was in very close agreement with the other two groups. The pathology of stroke and multiple sclerosis is described and possible reasons for the wide variation in patient cerebroside values given. The influence of aging on plasma cerebrosides is discussed. BIBLIOGRAPHY 10. BIBLIOGRAPHY Austin, J. H. and Maxwell, W. E., 1962. Significance of plasma glycolipid levels in normals and in 3 disorders of brain glycolipids. Soc. exp. Biol. and Med. (Proc) 121:197-200. Basu, S., Kaufmann, B. and Roseman, S., 1968. Enzymatic synthesis of ceramide-glucose and ceramide-lactose by glycosyltransferases from embryonic chicken brain. J. Biol. Chem. 243:5802-5804. Benjamin, J. and McKhann, G., 1972. Neurochemistry of development. In Basic Neurochemistry, Ch. 14. Edited by R. W. Albers gt 31. Boston: Little, Brown & Co. Brady, R., 1966. The Sphingolipidoses. New Eng. J. Med. 215:312-318. Brady, R., 1970. Metabolic disorders of sphingolipid metabolism in man. Chem.Phys. Lipids 5:261-269. Brady, R., 1971. Cerebral lipidoses.~ Ann. Rev. Med. 21:317-324. Brady, R., 1972. Sphingolipidoses. In Basic Neuro- chemistry, Ch. 23. Albers 23H31., eds. New York: Little, Brown & Co. Burton, R., 1967. Biochemistry of sphingosine con- taining lipids. In Lipids and Lipidoses, G. Schettler, ed. New York: Springer-Verlag. Burton, R., 1970. Factors affecting incorporation of precursors into body constituents: A review of common sense considerations with glycolipids as examples. Lipids 5:475-484. Cherayil, G. and Cyrus, A., Jr., 1966. The quantitative estimation of glycolipids in Alzheimer's disease. J. Neurochem. 13:579-590. 55 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 56 Christensen Lou, H. and Matzke, J., 1965. Cerebro- side and other polar lipids of the cerebrOSpinal fluid in neurological diseases. Acta Neurol. Scand. 41:445-447. Cumings, J., Thompson, E. and Goodwin, H., 1968. Sphingolipids and phospholipids in microsomes and myelin in normal and path brains. J. Neurochem. 15:243-248. Dalal, K. and Einstein, E., 1969. Biochemical maturation of the central nervous system. I. Lipid changes. Brain Res. 16:441-451.. Dawson, G. and Sweeley, C., 1970. $2 vivo studies on glycosphingolipid metabolism in porCine blood. J. Biol. Chem. 245:410—416. Dawson, G., 1972. Detection of glycosphingolipids in small samples of human tissue. Ann. Clin. Lab. Sci. 33274-284. Dawson, G., 1972. GlyCOSphingolipid levels in an unusual neurovisceral storage disease characterized by lactosylceramide galactosyl hydrolase deficiency: Lactosylceramidosis. J. Lipid Res. 13:207—219. Desnick, R., Sweeley, C. and Krivit, W., 1970. A ,method for the quantitative determination of neutral glycosphingolipids in urine sediment. J. Lipid Res.‘11:31—37. Dod, B. and Gray, G., 1968. The lipid composition of rat liver membranes. Biochim. BiOphys. Acta 150: 397-404. Esselman, W., Laine, R. and Sweeley, C., 1972. Isolation and characterization of glycosphingo- lipids. In Methods in Enz ol , Vol. 18, V. Ginsberg, ed. Nefi’YorE: AcaHemic Press, pp. 140-156. Folch, J., Lees, M. and Sloane Stanley, G., 1957. A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226:497-509. Foote, J. and Coles, E., 1968. Cerebrosides of human aorta: Isolation and identification of the hexose and fatty acid distribution. J. Lip. Res. 2: 482-486. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 57 Fredrickson, D., 1968. Classification and features of the lipidoses affecting the nervous system. In Cerebral Lipidoses II, A. Vincente et al., eds. Presses Academique Efiropeenes Bruxelles. Fredrickson, D. and Sloan, H., 1972. Glucosyl ceramide lipidoses: Gaucher's disease. In The Metabolic Basis of Inherited Disease, Ch. 33. Editeduby Stafibury, Wyngaafden and Fredrickson, 3rd ed. New York: McGraw-Hill (Blakiston). Gerstl, B., Tavaststjerna, M., Hayman, R., Eng, L. and Smith, J., 1965. Alterations in myelin fatty acids and plasmalogens in multiple sclerosis. Ann. N.Y. Acad. Sci. 122:405-416. Gerstl, 8., Eng, L., Tvaststjerna, M., Smith, J. and Krause, S., 1970. Lipids and proteins in multiple sclerosis white matter. J. Neurochem. 11:677-689. Hammarstrom, S., 1971. Brain glucosyl ceramides containing 2-hydroxy acids. Eur. J. Biochem. 21: 388-392. Hausheer, L. and Bernhard, K., 1963. Cerebrosides aus menschlichen aorten (Eng. summ.) Zeitschrift fur Physiol. Chem. 331:41-44. Hers, H. and Van Hoof, F., 1970. The genetic pathology of lysosomes. In Pr ress in Liver Disease, Vol. III. H. POppert and F. Schaffner, eds. New York: Grune & Stratton. Joffee, S. and Rapport, M., 1963. Identification of an organ specific lipid hapten in brain. Nature 197:60-62. Kampine, J., Brady, R., Yankee, R., Kanfer, J., Shapiro, D. and Gal, A., 1967. Sphingolipid metabolism in leukemic leukocytes. Cancer Res. 21:1312-1315. Kampine, J., Martensson, E., Yankee, R. and Kanfer, J., 1968. Sphingolipid metabolism in legkocytes. I. Incorporation of C glucose and C galactose into glycosphingolipids by intact human leukocytes. Lipids‘3:151-156. Karlsson, K.A. and Martensson, E., 1968. Studies on sphingosine. XIV. On the phytosphingosine content of the major human kidney glycolipids. Biochim. Biophys. Acta 152:230-233. 33. 34. 35. 36. 37. 38. 39. 4o. 41. 42. 43. 44. 58 Karlsson, K. A., 1970. On the chemistry and occurrence of sphingolipid long-chain bases. In Chemistry and Metabolism g£_Sphingolipids. C. C. Sweeley, ed. Amsterdam: North-Holland. Kattlove, H., Williams, J., Gaynor, E., Spivak, M., Bradley, R. and Brady, R., 1969. Gaucher cells in chronic myelocytic leukemia: An acquired abnormality. Blood 33:379-390. Katzman, R., 1972. Blood-brain-CSF barriers. In Basic Neurochemistry, Ch. 16. Albers 32 al., eds. New York: Little, Brown & Co. Kean, E., 1966. Separation of gluco- and galacto- cerebrosides by means of thin-layer chromatography. J. Lip. Res. 1:449-452. Kishimoto, Y., Davis, E. and Radin, N., 1965. Turn- over of rat brain gangliosides, glycerophosphatides, cerebrosides, and sulfatides as a function of age. J. Lipid Res. 6:525-531. Kuske, T., 1972. Plasma glycosphingolipids in lipid disorders. Ann. Clin. Lab. Sci. 2:268-273. LeBaron, F., 1970. Metabolism of myelin constituents. Handbook pf Neurochemistry 3. Metabolic Reactions in the Nervous System, Ch. 21. PIenum Press. Mapes, C., Anderson, R., Sweeley, C., Desnick, R. and Krivit, R., 1970. Enzyme replacement in Fabry's disease, an inborn error of metabolism. Sci. 162:987-989. Martensson, E., 1969. Glycolipids of animal tissue. In Pro ress in the Chemistrygf Fats and Other Lipids, R. HdIman, ed} Pergamon Press, 15:367-407. Millar, J., 1971. Multiple Sclerosis. A Disease Ac uired in_Childhood. Springfield, 111.: C as. T omas. Miras, C., Mantzoz, J. and Levis, G., 1966. The isolation and partial characterization of glyco- lipids of normal human leukocytes. Biochem. J. 28:782-786. Moser, H., 1972. Sulfatide lipidoses: Metachromatic leukodystrophy. In Metabolic Basis of Inherited Disease, Ch. 32. Stanbury, wyngaarden and FrEdrickson, eds. 3rd ed. New York: McGraw-Hill. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 59 Norton, W., 1972. Myelin. In Basic Neurochemistry. Albers et al., eds. New York: Little, Brown & Co. O'Brien, J. and Sampson, L., 1965. Lipid composition of the normal human brain: gray matter, white matter, and myelin. J. Lipid Res. 6:537-544. O'Brien, J. and Sampson, E., 1965. Fatty acid and fatty aldehyde composition of the major brain lipids in normal human gray matter, white matter, and myelin. J. Lip. Res. 6:545-551. Prostenik, M., 1970. Chemistry of sphingolipids - some historical aspects. In Chemistry and Metabolism g£_Sphingolipids, C. C. SweeIdy, ed. Amsterdam: North-Holland. Radin, N., Martin, F. and Brown, J., 1957. Galacto- lipide metabolism. J. Biol. Chem. 224:499-507. Radin, N., 1970. The galactosyl moiety of brain cerebrosides. Chem. Phys. Lipids 5:178-192. Radin, N., 1970. Cerebrosides and sulfatides. In Handbook ngNeurochemistry 3, Ch. 13. A. Lajtha, ed. Plenum Press. Rapport, M. and Graf, L., 1965. Immunological reactions of myelin ig_vitro. Ann. N. Y. Acad. Sci. 122:277-279. Rathke, E. and Jones, M., 1973. Serum cerebrosides in multiple sclerosis. J. Neurochem., in press. Renkonen, 0., 1970. Presence of sphingadienene and trans-monoenoic fatty acids in ceramide mono- hexosides of human plasma. Biochim. Biophys. Acta 219:190-192. Robbins, S., 1967. Pathol , 3rd Ed. Philadelphia: Saunders, pp. 1395-1398. Samuelsson, K., 1969. On the occurrence and nature of free ceramides in human plasma. Biochim. Biophys. Acta 176:211-213. Samuelsson, K., 1971. Identification and quantitative determination of ceramides in human plasma. Scand. J. Clin. Lab. Invest. 21:371-380. 58. 59. 60. 61. 62. 63. 64. 65. 66. 67. 68. 6O Samuelsson, K., 1971. Separation and identification of cerebrosides in cerebrospinal fluid by gas chromatography-mass spectrometry. Scand. J. Clin. Lab. Invest. 27:381-391. Samuelsson, K., 1971. Studies on ceramides and mono- hexosyl ceramides. Dissertation, Department of Chemistry I. Karolinska Institute of Stockhohm, Sweden. Sokoloff, L., 1972. Circulation and energy metabolism of the brain. In Basic Neurochemistry, Ch. 15. Edited by R. W. AlEers 22 E1. Boston: Little, Brown 8 Co. Stanbury, wyngaarden and Fredrickson, 1972. The Metabolic Basis of Inherited Disease, 3rd ed. New York: McGraw-HiII’IBlakiston). Stoffel, W., 1967. The chemistry of mammalian lipids. Lipids and Lipidoses, G. Schettler, ed. New York: Springer-Verlag. Suzuki, K., 1972. Chemistry and metabolism of brain lipids. In Basic Neurochemistr , Ch. 11. Albers et al., eds. Boston: LittIe, Brown & Co. Suzuki, K. and Suzuki, Y., 1973. Galactosyl ceramide lipidosis: globoid cell leukodystroPhy (Krabbe's disease). In The Metabolic Basis of Inherited Disease, Ch. 34. Edited by Stanbufy} wyngaarden and Fredrickson. New York: McGraw-Hill. Svennerholm, E. and Svennerholm, L., 1963. Neutral glycolipids in human blood serum, spleen and liver. Nature 198:688-689. Svennerholm, L., 1964. The distribution of lipids in the human nervous system. I. Analytical procedure. Lipids of fetal and newborn brain. J. Neurochem. 11:839-853. Svennerholm, L. and Stallberg-Stenhagen, S., 1968. Changes in fatty acid composition of cerebrosides and sulfatides of human nervous tissue with age. J. Lip. Res. 1:215-225. Svennerholm, L., Bruce, A., Mansson, J., Rynmart, B. and Vanier, M., 1972. Sphingolipids of human skeletal muscle. Biochim. Biophys. Acta 280:626- 636. 69. 7o. 71. 72. 73. 74. 75. 76. 77. 61 Sweeley, C. and Moscatelli, E., 1959. Quantitative microanalysis and estimation of sphingolipid bases. J. Lip. Res. 1:40-47. Tourtellotte, W., 1970. Cerebrospinal fluid in multiple sclerosis. In Handbook g£_Clinical Neurology, Vol. IX, Ch. 11. Edited by Vinken and Bruyn. Amsterdam: North-Holland. Tourtellotte, W. and Haerer, A., 1969. Lipids in cerebrospinal fluid. XII. In multiple sclerosis and retobulbar neuritis. Arch. Neurol. 32:605-615. Vance, D. and Sweeley, C., 1967. Quantitative determination of the neutral glycosyl ceramides in human blood. J. Lip. Res. 8:621-630. Vance, D., Krivit, W. and Sweeley, C., 1969. Concen- trations of glycosyl ceramides in plasma and red cells in Fabry's disease, a glycolipid lipidosis. J. Lipid Res. 12:188-192. Weissmann, G., 1972. Lysosomal mechanisms of tissue injury in arthritis. New Eng. J. Med. 286:141-146. Wells, H. and Jones, M., 1973. Galactosyl-ceramide levels in human plasma. In press. White, Handler and Smith, 1968. Principles 2: Biochemistry, 4th ed. McGraw-Hill. Wiegandt, H., 1971. Glycosphingolipids. Adv. Lip. Res. 9:249-289. 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