/. Biochem. 102, 83-92 (1987)

Occurrence of as a Major 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 , 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 such as glucosylceramide, , GM3 , , and . 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 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. 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- 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 , 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 and 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 . 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 , 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 of the methylglycosides. fatide: 2,920 and 2,850 cm-1 due to CH, and The neutral glycosphingolipids were mostly com- CH, groups, 1,640 and 1,550 cm-1 due to amide 1 posed of glucosylceramide, galactosylceramide, linkage, 1,460 cm- due to CH3 and CH groups, 1 lactosylceramide, globotriaosylceramide, and other 1,240 cm- due to SO3H group and 1,000-1,100 minor oligoglycosylceramides such as globotetra- cm-1 due to OH group. Periodate oxidation with osylceramide (tentatively identified by TLC and 0.2 M sodium periodate (18) and solvolysis with GLC analysis of TMS derivatives of the methyl 0.1 N HC1 in methanol (19) were performed, and Downloaded from https://academic.oup.com/jb/article/102/1/83/782552 by guest on 27 September 2021 glycosides). On the other hand, relatively large the results are shown in Fig. 3. The glycosphingo- amounts of acidic glycosphingolipids were also lipid which had the same Rf value as sulfatide separated from each other by TLC (Fig. 2). One was stable to periodate oxidation, whereas it was major band which has the same Rf value as sul- converted to galactosylceramide by solvolysis. fatide was detected in all the lipoprotein fractions Galactosylceramide obtained by solvolysis had the and in whole sera of WHHL rabbit. The bands same Rf value as nonhydroxy fatty acid-containing located between sulfatide and GM3 in lanes B-3 galactosylceramide on TLC. These results con- and B-4 were negative to the anthrone reagent, firmed that the structure of the glycosphingolipid indicating nonglycolipid nature. The bands which was identical with sulfatide. Ganglioside contents migrated slightly faster than the major band in and compositions were similar in each lipoprotein each lane were methanolyzed and analyzed by fraction. In the case of LDL, GM3 (51 % of the GLC. No sugars were found in the methano- total sialic acid) and GD3 (26%) were the major lyzates, but only cholesterol. The mobility of the gangliosides in addition to small amounts of GDla band was identical to that of cholesterol sulfate. Thus, the band was tentatively identified as cho- lesterol sulfate. GLC analysis of sugar in the

B

imB^ A

Fig. 2. Thin-layer chromatograms of acidic glyco- 1 8 sphingolipids of serum, and CM, VLDL, LDL, and Fig. 3. Solvolysis and periodate oxidation of sulfatide. HDL. The plates were developed with chloroform- The plates were developed with chloroform-methanol- methanol-water (65 : 25 :4, v/v). The bands were water (65 : 25 : 4, v/v). The bands were detected as detected with cupric-phosphoric acid charring reagent in Fig. 2. I, galactosylceramide from porcine spinal (77). A-l, GM3 (control); A-2, sulfatide from porcine cord (control); 2, galactosylceramide treated with perio- spinal cord (control); A-3, CM; A-4, VLDL; A-5, date (control); 3, sulfatide treated with periodate; 4 LDL; A-6, HDL. B-l, sulfatide from porcine spinal and 5, sulfatide from LDL; 6, sulfatide from porcine cord (control); B-2, GM3 (control); B-3, normal rabbit spinal cord (control); 7, solvolysate of sulfatide; 8, serum; B-4, WHHL rabbit serum. galactosylceramide from porcine spinal cord (control).

/. Biochem. SULFATIDE IN SERUM LIPOPROTEINS 87

(7%), GTlb (5%), GQlb (1%), and unknown the major apolipoprotein of LDL. It should be ganglioside (8%). Table I summarizes the glyco- noted that the sugar contents of glycosphingolipids sphingolipid contents in whole serum, and CM, were relatively lower than that of apolipoprotein B. VLDL, LDL, and HDL. As shown in Table I, Fatty acid compositions of glycosphingolipids WHHL rabbit serum contained much larger are shown in Table II. Hydroxy fatty acids were amounts of glycosphingolipids than normal rabbit detected in glucosylceramide, galactosylceramide, serum. It was found that sulfatides were pre- and sulfatide. Major fatty acids of these glyco- dominant among the glycosphingolipid constitu- sphingolipids were C22, C23, and C24. Lacto- ents, and the sulfatide contents were at almost the sylceramide and globotriaosylceramide contained same levels in all the lipoproteins of WHHL rabbit only nonhydroxy fatty acids such as C22, C23, Downloaded from https://academic.oup.com/jb/article/102/1/83/782552 by guest on 27 September 2021 sera when the values were expressed based on the and C24 as major fatty acids. Although GM3 phospholipid content of each lipoprotein. The also contained only nonhydroxy fatty acids, the glycosphingolipid composition in normal rabbit major fatty acids were C18 and C22. sera seemed to be similar to that of WHHL rabbit The long chain base composition of sulfatide sera except for glucosylceramide content, which from LDL was analyzed by GLC as TMS deriva- was comparable to sulfatide content in the normal tives. The sulfatide contained (4E)-sphingenine rabbit sera. (77%), sphinganine (9%), 4D-hydroxysphinganine The sugar content in the proteinaceous residue (12%), and 4D-hydroxyicosasphinganine (2%). of delipidated LDL of WHHL rabbit sera was The long chain base composition and fatty estimated by GLC of TMS derivatives of the acid composition of galactosylceramide, a metabo- methyl glycosides, and the values expressed based lite of sulfatide, were investigated. Figure 4 shows on the phospholipid contents of LDL were as the result of TLC analysis of glucosylceramide and follows: iV-acetylglucosamine (103 nmol//«nol PL galactosylceramide from LDL using 3% Borax- in LDL), mannose (86.6 nmol//umol PL in LDL), impregnated silica gel plates. The mixture of sialic acid (55.0 nmol//

TABLE I. Glycosphingolipid contents in whole serum, and CM, VLDL, LDL, and HDL of WHHL rabbit.

GlcCer GalCer LacCer GbjCer Gb4Cer(?) Sulfatide GM3

(nmol//*mol PL*) CM 8.44 nd>> nd nd nd 11.6 2.33 VLDL 2.83 1.37 0.58 0.54 0.22 19.4 2.20 (% distribution) (10) (5) (2) (2) (1) (72) (8) LDL 2.63 1.53 0.61 0.60 0.29 17.8 1.95 (% distribution) (10) (6) (2) (2) (1) (71) (8) HDL 9.05 1.17 nd nd nd 14.1 2.53

(nmol/ml serum) Serum c 25.5 8.95 6.80 3.53 1.51 1201 7.94 (% distribution) (15) (5) (4) (2) (1) (68) (5) Normal serum 3.28 0.35 0.11 0.08 0.04 2.85 0.30 (% distribution) (47) (5) (2) (1) (1) (41) (4)

Total phospholipids. " Not determined. ° WHHL rabbit serum contained 520±55 mg total phospholipids/dl. This value is equivalent to 18.0 nmol//imol PL.

Vol. 102, No. 1, 1987 88 A. HARA and T. TAKETOMI

TABLE n. Fatty acid compositions of glycosphingolipids in various lipoproteins of WHHL rabbit (%). GlcCer& GalCer Sulfatide LacCer Gb,Cer GM3

VLDL LDL CM VLD LDL HDL VLDL LDL VLDL LDL VLDL LDL

16 9 6 4 5 4 6 18 20 3 17 14 4 18 6 4 7 5 7 7 9 14 5 9 27 45 20 6 6 5 5 5 5 14 10 9 7 12 10 22 20 19 23 23 23 22 33 30 34 35 29 27 23 17 16 23 26 25 24 11 10 21 19 10 6 Downloaded from https://academic.oup.com/jb/article/102/1/83/782552 by guest on 27 September 2021 24: 1 5 5 ta t t t 3 4 13 t t t 24 16 16 23 23 23 22 12 12 15 13 8 8 25: 1 t t t 1 t t t t t t t t 25 1 2 t t t t t t t t t t

18ht> 1 1 t t t t 20 h 1 1 t t t t 22 h 5 6 3 3 3 4 23 h 7 8 7 5 6 6 24 h : 1 t 1 1 2 1 1 24 h 6 9 4 2 3 3

% of HFA b 20 26 15 12 13 14 0 0 0 0 0 0 *• Trace. b Hydroxy fatty acid.

A

B

Wff

1 2 3 Fig. 4. Borax-impregnated thin-layer chromatogram 12 3 4 of glucosylceramide and galactosylceramide obtained Fig. 5. Thin-layer chromatogram of sulfatides from from LDL of WHHL rabbit. The plate was developed with chloroform-methanoI-3.5% ammonia (60 : 35 : normal liver and small intestine. The plate was devel- 8, v/v). The bands were detected as in Fig. 2. 1, oped with chloroform-methanol-water (65 : 25 : 4, v/ glucosylceramide from Gaucher spleen; 2, galactosyl- v). The bands were detected as in Fig. 2. 1 and 4, eeramide from porcine spinal cord; 3, glucosylceramide sulfatide from porcine spinal cord; 2, sulfatide fraction and galactosylceramide from LDL. from liver; 3, sulfatide fraction from small intestine.

/. Bioehem. SULFATIDE IN SERUM LIPOPROTEINS 89

TABLE El. Fatty acid and long chain base compositions of glucosylceramides and in LDL of WHHL rabbit (%).

A B C D Sugar Glucose Galactose Galactose

(% of total fatty acids) 16 3 t1 5 6 18 6 1 4 3

20 9 3 5 5 Downloaded from https://academic.oup.com/jb/article/102/1/83/782552 by guest on 27 September 2021 22 32 26 19 16 23 20 27 19 8 24 : 1 7 7 8 t 24 23 26 18 8

22 h>> 4 5 16 23 h 3 6 23 24 h: 1 t 3 t 24 h 3 8 15

%ofHFAt> 0 10 22 54

(% of total long chain bases) (4E)-Sphingenine 94 93 Sphinganine 6 7 4D-Hydroxysphinganine 78 81 4D-Hydroxyicosasphinganine 22 19

Trace. •> Hydroxy fatty acid.

(which accounted for 68% of glucosylceramide) (59.0 g of wet tissue) or WHHL rabbit small contained nonhydroxy fatty acids and dihydroxy intestine (15.5 g of wet tissue) was subjected to bases. Band B (which accounted for 32% of silica gel column chromatography (Silica gel 60, glucosylceramide) contained nonhydroxy or hy- Merck, 10 x 60 mm). Free fatty acids were eluted droxy fatty acids and trihydroxy bases. Band C with chloroform-methanol (95 : 5, 40 ml) and (90 : (which accounted for 74% of galactosylceramide) 10, 10 ml), then sulfatide was eluted with chloro- contained nonhydroxy or hydroxy fatty acids and form-methanol (90 : 10, 10 ml, 85 : 15, 20 ml, and dihydroxy bases. Finally, band D (which ac- 80 : 20, 175 ml). The sulfatide fraction thus ob- counted for 26% of galactosylceramide) contained tained from normal rabbit tissues was analyzed nonhydroxy or hydroxy fatty acids and trihydroxy by TLC as shown in Fig. 5. The extract from the bases. The doublets of band C and band D are liver showed one major band, while that from the due to the differences of fatty acids. Glucosyl- small intestine showed three bands. The band ceramide and galactosylceramide contained C22, which had a lower R/ value than that of sulfatide C23, and C24 as major nonhydroxy or hydroxy in the liver was negative to the anthrone reagent. fatty acids. Thus, no further analysis of this band was done. Sulfatide in Liver and Small Intestine—Acidic The bottom band of the three major bands in fraction obtained from normal rabbit small intestine was also negative to the anthrone liver (99.5 g of wet tissue), WHHL rabbit liver reagent, indicating non-glycosphingolipid nature. (14.6 g of wet tissue), normal rabbit small intestine The top band was determined to be cholesterol

Vol. 102, No. 1, 1987 90 A. HARA and T. TAKETOMI sulfate by GLC analysis. Thus, the middle band TABLE IV. Fatty acid compositions of sulfatides in in the small intestine and one major band in the normal and WHHL rabbits (%). liver were identified as sulfatides which corre- sponded to that in LDL. The sulfatide contents Liver Small intestine were determined by GLC analysis of TMS deriva- WHHL Normal WHHL Normal tives of the methylglycosides. The normal and WHHL rabbit livers contained 104 and 260 nmol 16 5 5 4 3 sutfatide/g tissue, respectively, while the normal 17 ta and WHHL rabbit small intestines contained 31.2 18 5 5 2 1

and 99.6 nmol sulfatide/g tissue, respectively. It Downloaded from https://academic.oup.com/jb/article/102/1/83/782552 by guest on 27 September 2021 1 1 should be noted that the liver and the small intes- 19 tine of WHHL rabbit contained larger amounts 20 6 4 1 1 of sulfatide than normal. The sulfatide contents 21 2 2 in the rabbit livers were very high as compared 22 25 17 3 4 with that in human liver, which contained 1 nmol sulfatide/g tissue (20). The fatty acid compositions 23 25 24 2 t of the sulfatides from the livers and the small 24: 1 3 6 t intestines of normal and WHHL rabbits are shown 24 23 27 1 1 in Table IV. The chain lengths of the major fatty 25 : 1 1 1 acids of the sulfatides were C22, C23, and C24 in both organs, but the sulfatide in both livers 25 1 2 contained mostly nonhydroxy fatty acids, while 26: 1 t that in the small intestines contained mostly hy- 26 t t droxy fatty acids. There was no marked differ- ence in fatty acid composition of sulfatides be- 18h» 1 2 tween normal and WHHL rabbits. 20 h 5 6 Table V shows the long chain base compo- 21 h 3 2 sitions of the sulfatides in the livers and the small intestines. Normal and WHHL rabbits showed 22h 1 1 31 30 a similarity in their long chain base compositions. 23 h 1 2 27 27 The sulfatides in the livers contained mostly di- 24 h : 1 t t 2 t hydroxy bases, while those in the small intestines 24 h 1 3 16 22 contained mostly trihydroxy bases. It was thus noted that the sulfatides in the livers contained 25 h t 2 1 ceramide which was mainly composed of nonhy- droxy fatty acids and dihydroxy bases, whereas %ofHFA<> 3 6 87 90 the sulfatides in the small intestines contained a ceramide which was mostly composed of hydroxy Trace. *• Hydroxy fatty acids.

TABLE V. Long chain base compositions of sulfatides in normal and WHHL rabbit (%).

Liver Small intestine LDL (WHHL) WHHL Normal WHHL Normal

(4E>Sphingenine 86 88 25 14 77 Sphinganine 7 7 5 3 9 4D-Hydroxysphinganine 6 4 55 68 12 4D-Hydroxyicosasphinganine 1 1 15 15 2

/. Biochem. SULFATTOE IN SERUM LIPOPROTEINS 91 fatty acids and trihydroxy bases in both normal obtained from the scanning sodium dodecyl sulfate and WHHL rabbits. polyacrylamide gel electrophoresis of LDL apo- lipoprotein at 570 nm at the gel concentration of DISCUSSION 5% after staining with Coomassie brilliant blue R-250 (data not sown). The content of apo- There have been no reports on the presence of lipoprotein B-100, which might be derived from sulfatides in human and other animal sera or the liver, was approximately 90%, while that of lipoproteins. It was found for the first time in apolipoprotein B-48, which might be derived from the present study that sulfatides are the major small intestine, was about 10%. High contents glycosphingolipid in all the lipoprotein fractions of sulfatides in lipoproteins as well as different Downloaded from https://academic.oup.com/jb/article/102/1/83/782552 by guest on 27 September 2021 such as CM, VLDL, LDL, and HDL in WHHL. ceramide portions of sulfatides derived from the rabbit serum. Other minor glycosphingolipids such two different sources suggest that sulfatides may as glucosylceramide, galactosylceramide, GM3 serve as useful tools in studying lipoprotein metab- ganglioside, lactosylceramide, globotriaosylcer- olism and biosynthesis in WHHL rabbits, and thus amide, and other oligohexosylceramides were also be helpful to further investigate the relationship detected in the lipoproteins, as has already been between hypercholesterolemia and atherosclerosis reported in human sera (1-3). The content and in WHHL rabbits. Wago (22) has already re- fatty acid composition of sulfatides were rather ported that intravenous administration of sulfatide similar in each lipoprotein fraction. These find- to cholesterol-fed rabbits inhibited the elevation of ings not only support the metabolic conversion of serum total lipid and total cholesterol, and miti- VLDL to LDL via IDL, but also indicate the gated the atheromatous involvements in the aorta. possibility that sulfatides are freely exchangeable Also, Roberts et al. (23) have recently re- among lipoproteins like neutral glycosphingolipids ported very interesting data concerning highly (21). It is well known that lipoproteins are bio- specific interactions between sulfatides and cell synthesized independently in both liver and small adhesion proteins such as laminin, thrombospondin intestine. Thus, in order to examine whether the and von Willebrand factor. The presence of sul- liver and the small intestine contain sulfatides fatides in serum lipoproteins suggests that the which are found in the lipoproteins, these tissues sulfatide-containing lipoproteins may play a role were subjected to analyses of glycosphingolipids. in regulating the physiological function of the cell If the sulfatides in these organs can be distinguished adhesion proteins. However, it remains to be in terms of fatty acid and long chain base com- investigated whether sulfatides exist in the sera position, the origins of the lipoproteins may be or serum lipoproteins of all mammalian species. deduced from the sulfatides compositions. From the contents of hydroxy fatty acids of the sulfatides in the WHHL rabbit liver (HFA content: 3% of REFERENCES total fatty acids) and in the WHHL rabbit small 1. Svennerholm, E. & Svennerholm, L. (1963) Nature intestine (HFA content: 87% of total fatty acids), 198, 688-689 88% of the sulfatides in LDL (HFA content: 13% 2. Vance, D.E. & Sweeley, C.C. (1967) /. Lipid Res. of total fatty acids) were assumed to be derived 8, 621-630 from the liver, and the remaining sulfatides (12%) 3. Yu, R.K. & Lcdeen, R.W. (1972) /. Lipid Res. 13, from the small intestine. On the other hand, 680-686 4. Vance, D.E., Krivit, W., & Sweeley, C.C. (1969) when trihydroxy bases of the sulfatides in WHHL J. Lipid Res. 10, 188-192 rabbit were used for calculation in the same way 5. Chatterjce, S. & Kwiterovich, P.O. (1976) Lipids (Table V), 89% of the sulfatides in LDL were 11, 462-166 assumed to be derived from the liver, and 11% 6. Dawson, C, Kruski, A.W., & Scanu, AM. (1976) from the small intestine. Thus, it was suggested J. Lipid Res. 17, 125-131 that the sulfatides in serum lipoprotein of WHHL 7. Coles, E. & Foote, J.L. (1974) /. Lipid Res. 15, rabbit were mostly derived from the liver (90%) 192-199 and, to a lesser extent, from the small intestine 8. Hatch, F.T. & Lees, R.S. (1968) in Advances in (10%). This ratio was also supported by the data Lipid Research (Paoletti, R. & Kritchevsky, D., eds.) Vol. 6, pp. 1-68, Academic Press, New York

Vol. 102, No. 1, 1987 92 A. HARA and T. TAKETOMI

9. Hara, A. & Taketomi, T. (1982) Llpids 17, 515-518 J. Chromatogr. 43, 120-126 10. Bartlett, G.R. (1959) /. Blol. Chem. 234, 466-468 18. Carter, H.E., Rothfus, J.A., & Gigg, R. (1961) /. 11. Taketomi, T., Kawamura, N., Hara, A., & Mura- LipidRes. 2, 228-234 kami, S. (1975) Jpn. J. Exp. Med. 45, 55-62 19. Stoffyn, P. & Stofiyn, A. (1963) Btochim. Biophys. 12. Laemmli, U.K. (1970) Nature 227, 680-685 Ada 70, 107-108 13. Hara, A., Kitazawa, N., & Taketomi, T. (1984) /. 20. Nilsson, O. & Svennerholm, L. (1982) /. Lipid Res. Lipid Res. 25, 175-184 23, 327-334 14. Ledcen, R.W., Yu, R.K., & Eng, L.F. (1973) /. 21. Loeb, J.A. & Dawson, G. (1982) /. Biol. Chem. Neurochem. 21, 829-839 257, 11982-11987 15. Svennerholm, L. (1957) Biochim. Biophys. Ada 24, 22. Wago, K. (1961) Jpn. Heart J. 2, 354-367 604-611 23. Roberts, D.D., Rao, N.C., Liotta, L.A., Gralnick, Downloaded from https://academic.oup.com/jb/article/102/1/83/782552 by guest on 27 September 2021 16. Ando, S., Chang, N.-C, & Yu, R.K. (1978) Anal. H.R., & Ginsburg, V. (1986) /. Biol. Chem. 261, Biochem. 89, 437-450 6872-6877 17. Fewster, M.E., Burns, B.J., & Mead, J.F. (1969)

/. Biochem.