/. Biochem. 102, 83-92 (1987) Occurrence of Sulfatide as a Major Glycosphingolipid in WHHL Rabbit Serum Lipoproteins1 Atsushi HARA and Tamotsu TAKETOMI Downloaded from https://academic.oup.com/jb/article/102/1/83/782552 by guest on 27 September 2021 Department of Lipid Biochemistry, Institute of Cardiovascular Disease, Shinshu University School of Medicine, Matsumoto, Nagano 390 Received for publication, February 12, 1987 Glycosphingolipids in serum and lipoproteins from Watanabe hereditable hyper- lipidemic rabbit (WHHL rabbit), which is an animal model for human familial hypercholesterolemia (FH), were analyzed for the first time in this study. Chylo- microns and very low density, low density, and high density lipoproteins contained sulfatide as a major glycosphingolipid (12 nmol//imol total phospholipids (PL) in chylomicrons, 19nmol//imol PL in VLDL, 18 nmol//imol PL in LDL, and 14 nmol/ /*mol PL in HDL) with other minor glycosphingolipids such as glucosylceramide, galactosylceramide, GM3 ganglioside, lactosylceramide, and globotriaosylceramide. The concentration of sulfatide as a major glycosphingolipid in WHHL rabbit serum (121 nmol/ml) was much higher than that in normal rabbit serum (3 nmol/ml). Fatty acids of the sulfatides comprised mainly nonhydroxy fatty acids (C22, 23, and 24) and significant amounts of hydroxy fatty acids (about 10 %), whereas long chain bases of the sulfatides comprised mostly (4E>sphingenine with a significant amount of 4D-hydroxysphinganine (about 10%). Furthermore, sulfatides in the liver and small intestine from normal and WHHL rabbits (where serum lipoproteins are produced) were determined to amount to 260nmol/g liver in WHHL rabbit, 104 nmol/g liver in control rabbit, 99.6 nmol/g small intestine in WHHL rabbit, and 31.2nmol/g small intestine in control rabbit. Ceramide portions of the sulfatides in the liver were mainly composed of (4E)-sphingenine and nonhydroxy fatty acids, while those in the small intestine were mainly composed of 4D-hydroxysphinganine and hydroxy fatty acids. These results indicated that the sulfatides of serum lipo- proteins were mostly derived from the liver (90% of the total), and that the remaining sulfatides (10% of the total) might be derived from the small intestine. These two sulfatides, which have different ceramide portions, could be useful markers for metabolic and biosynthetic studies of various lipoproteins in WHHL rabbit, and thus would be helpful to further elucidate the relationship between hypercholesterol- emia and atherosclerosis in the rabbit. 1 This work was supported in part by Grant-in-Aid for Scientific Research (No. 61480126) from the Ministry of Education, Science and Culture of Japan. Vol. 102, No. 1, 1987 83 84 A. HARA and T. TAKETOMI Glucosylceramide, galactosylceramide, lactosylcer- was determined by gas-liquid chromatography (9). amide, globotriaosylceramide, globotetraosylcer- Phosphorus contents of lipoproteins were deter- amide, and GM3-ganglioside have been found in mined by the method of Bartlett (10). As already human plasma and its lipoproteins (1-3). There reported by the authors (II), aliquots of various are some reports on increased levels of glyco- lipoproteins were negatively stained with phos- sphingolipids in serum; for example, an increase photungstic acid (pH 7.2) and dried on collodion of glucosylceramide in Gaucher patients and an carbon grids for electron-microscopic observation increase of globotriaosylceramide in Fabry patients (Hitachi, model Hu-ILA). These aliquots were (4). It was also reported that the levels of plasma also subjected to electrophoresis on Universal glycosphingolipids were elevated in patients with electrophoresis film (agarose) (Corning Medical, Downloaded from https://academic.oup.com/jb/article/102/1/83/782552 by guest on 27 September 2021 familial hypercholesterolemia (FH) (5, 6), but the U.S.A.). The lipoproteins were stained with Fat mechanism of the increase in glycosphingolipids red 7B or Coomassie brilliant blue R-250. Sodium remains to be solved. It is known that the WHHL dodecyl sulfate polyacrylamide gel electrophoresis rabbit is devoid of LDL-receptor, as is the case was performed according to Laemmli (12). The in FH. In the present study, we have analyzed procedures for extraction and isolation of glyco- glycosphingolipids in whole serum or lipoproteins sphingolipids were described in detail elsewhere in WHHL rabbit in order to further understand (9, 13). Briefly, Iipids were extracted from each the glycosphingolipid metabolism in FH. Al- lipoprotein fraction, whole serum, liver, or small though Coles and Foote have reported that plasma intestine with a chloroform-methanol mixture. lipoproteins in rabbit contained cerebroside, di- After alkaline treatment to cleave ester Iipids, glycosylceramide, triglycosylceramide, and globo- sphingolipids were separated into neutral sphin- side (7), more detailed information about glyco- golipids and acidic sphingolipids with DEAE- sphingolipids of plasma lipoproteins is needed to Sephadex A-25 (acetate form) as described by elucidate the glycosphingolipid metabolism in lipo- Ledeen et al. (14). The neutral glycosphingolipids proteins. In this paper, it is reported for the first were acetylated and separated into glycosphingo- time that the sulfatide content is highest in the lipids and sphingomyelin by silica gel column glycosphingolipid constituents in the serum lipo- chromatography and the glycosphingolipids were protein of WHHL rabbit, although the sulfatide finally deacetylated. The neutral glycosphingo- is seldom a major glycosphingolipid in other tis- lipids thus obtained were separated from each sues or fluids. other by preparative thin-layer chromatography (TLC) on plates precoated with Silica gel 60 (Merck, B.R.D.), developed with chloroform- MATERIALS AND METHODS methanol-water (65 : 25 : 4, v/v). The acidic Iipids were purified by silica gel column chromatography Materials—WHHL rabbits (Japanese white as follows; stepwise elution with chloroform- rabbit) were kindly supplied by Dr. Yoshio Wata- methanol mixtures (95 : 5, 90 : 10, 85 : 15, 80 : 20, nabe, Kobe University School of Medicine, Kobe. and 50 : 50) gave free fatty acids, sulfatide, and Normal rabbits (Japanese white rabbit) were used gangliosides. Sulfatide was further purified by as controls. Blood samples were drawn from the preparative TLC as above. Analytical procedures animals after overnight fasting and separated into used for the present study were described in detail sera and erythrocytes by centrifugation. The elsewhere (9, 13). Briefly, after methanolysis of pooled sera were used for experiments as well as glycosphingolipid, the resulting fatty acid methyl fresh tissues of rabbit liver and small intestine, esters were analyzed by gas liquid chromatography including mucous membrane. (GLQ using an OV-101 silicone capillary column Methods—The. pooled WHHL rabbit sera (0.2 mm x 25 m) at 25O°C. The methyl glycosides were fractionated into chylomicrons (CM, rf<0.93 from the methanolyzate were re-N-acetylated and g/ml), very low density lipoprotein (VLDL, 1.01 > analyzed as trimethylsilyl (TMS) derivatives by </>0.93), low density lipoprotein (LDL, 1.06>d> GLC on an OV-101 silicone capillary column. 1.01), and high density lipoprotein (HDL, 1.15 > The column temperature was programmed from tf>1.08) by the method of Hatch and Lees (8). 190 to 260°C at 2°C/min. Mannitol was used as Cholesterol content in each lipoprotein fraction J. Biochem. SULFAT1DE IN SERUM LIPOPROTE1NS 85 an internal standard. Long chain bases were lipoproteins were determined by electronmicro- prepared as before (13), and analyzed as TMS- scopic examination. As shown in Fig. 1, the derivatives by GLC on an OV-101 silicone capillary diameters of LDL and VLDL were 17-20 nm and column at 25O°C. The amount of ganglioside was 33-58 nm, respectively. These lipoproteins were determined with resorcinol reagent (75) or quan- also confirmed by electrophoresis on agarose film titated by TLC-densitometry at 570 nm (16). (data not shown). Simple lipid compositions of VLDL and LDL were determined by GLC anal- ysis of cholesterol or fatty acid methyl ester. RESULTS VLDL contained 54% cholesterol ester, 35% tri- Fractionation of Various Lipoproteins—The glyceride, and 11 % free cholesterol, while LDL Downloaded from https://academic.oup.com/jb/article/102/1/83/782552 by guest on 27 September 2021 pooled sera of fasting WHHL rabbits were frac- contained 62% cholesterol ester, 23% triglyceride, tionated into CM, VLDL, LDL, and HDL by the and 15% free cholesterol. procedure of Hatch and Lees (8). The lipoprotein Glycosphingolipids in Serum, and CM, VLDL, composition of the WHHL rabbit sera was deter- LDL, and HDL—Crude sphingolipids were sepa- mined on the basis of cholesterol content as fol- rated into neutral and acidic sphingolipids on lows: CM (1.9%), VLDL (24.5%), LDL (71.4%), DEAE-Sephadex A-25. After sphingomyelin was and HDL (2.2%). The size and shape of these removed from the neutral sphingolipid, relatively Vi.Di LDL Fig. I. Electron micrographs of VLDL and LDL. Each lipoprotein was negatively stained with phosphotungstic acid and dried on a collodion carbon grid. Vol. 102, No. I, 1987 86 A. HARA and T. TAKETOMI small amounts of neutral glycosphingolipids were major glycosphingolipid revealed that galactose obtained. They were separated from each other was the only component. The IR spectrum of by preparative TLC and identified by GLC anal- the glycosphingolipid was similar to that of sul- ysis of TMS derivatives