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Small polydisperse low density lipoproteins in familial hyperalphalipoproteinemia with complete deficiency of cholesteryl ester transfer activity
Atherosclerosis, 70 (1988) 7-12 Elsevier Scientific Publishers Ireland,
7 Ltd.
ATH 04083
Small polydisperse low density lipoproteins in familial hyperalphalipoproteinemia with complete deficiency of cholesteryl ester transfer activity Shizuya Yamashita I, Yuji Matsuzawa I, Mitsuyo Okazaki 2, Hiroyuki Kako j, Tadao Yasugi 3, Hisashi Akioka 4, Kazuko Hirano ’ and Seiichiro Tarui ’ ’Second Department of Internal Medicine, Osaka Universicv Medical School, Osaka 553 (Japan), .’Laboratoy of Chemistry, Department of General Education, Tokyo Medical and Dental University, Tokyo (Japan), -’Second Department of Internal Medicine Nthon University Medical School. Tokyo (Japan), ’ Nissei Hospital, and ’ Sakai City Health Research Institute. Sakai (Japan) (Received 12 March, 1987) (Revised, received 18 September, 1987) (Accepted 18 September. 1987)
Summary Lipoprotein abnormalities were analyzed in 3 cases of marked hyperalphalipoproteinemia caused by complete deficiency of cholesteryl ester transfer activity. The probands were all men, aged 34, 43 and 48 years, respectively. The serum high density lipoprotein (HDL)-cholesterol levels of these patients were higher than 150 mg/dl (157-254 mg/dl), while serum total cholesterol levels ranged from 227 to 360 mg/dl. Sequential flotation-ultracentrifugation analysis disclosed that low density lipoprotein (LDL)cholesterol was slightly decreased and that cholesteryl ester accumulated solely in the HDL, fraction, which was also enriched with apolipoprotein E. Cholesteryl ester transfer activity was completely absent in all of these cases. High-performance liquid chromatography showed a decrease of LDL particle size in combination with a marked enlargement of HDL particle size. Analytical ultracentrifugation disclosed heterogeneity of LDL with the presence of small LDL subpopulations. We conclude that hyperalphalipoproteinemia due to complete deficiency of cholesteryl ester transfer activity is characterized by the presence of both small polydisperse LDL and markedly large HDL enriched with cholesteryl ester and apolipoprotein E.
Key words:
Hyperalphalipoproteinemia; of cholesteryl ester transfer ultracentrifugation
Low density lipoproteins; activity; High-performance
High density lipoproteins; liquid chromatography:
Deficiency Analytical
hyperalphalipoproteinemia
(FHALP)
Introduction Correspondence to: Shizuya
Yamashita, M.D., Second Deof Internal Medicine, Osaka University Medical School. Fukushima-ku, Osaka 553. Japan.
Familial
partment
0021-9150/88/$03.50
Q 1988 Elsevier Scientific
Publishers
Ireland,
has been Ltd
considered
to be a genetic
disorder
char-
8
acterized by elevated levels of serum high density lipoprotein (HDL)-cholesterol. Although longevity with reduced morbidity and mortality due to coronary heart disease has been reported in cases of FHALP [l], the basic abnormality in this condition has not yet been elucidated. We previously reported 2 patients with FHALP showing cornea1 opacification, one of whom also suffered from angina pectoris [2,3]. In these cases, a marked increase of HDL, particle size and abnormal HDL, lipid and apolipoprotein compositions were demonstrated. There have also been several reports describing alterations in the lipid and apolipoprotein contents of increased HDL [4-61 in FHALP, although little is known about the characteristics of low density lipoproteins (LDL). Koizumi et al. [7] reported a case with FHALP showing a decrease in cholesteryl ester transfer activity. Recently, we found 3 cases of FHALP showing complete deficiency of cholesteryl ester transfer activity. In the present study, we analyzed the lipoprotein profiles in these 3 cases by both high-performance liquid chromatography (HPLC) and analytical ultracentrifugation, and revealed a decreased size and heterogeneity of LDL particles as well as a marked increase of HDL particle size. Materials and methods
The subjects of this study were 3 probands from 3 families with FHALP. They were all men, aged 34, 43 and 48 years, respectively. None of them had any clinical signs or symptoms of coronary heart disease or atherosclerosis. Blood samples were collected after an overnight fast. Total and free cholesterol and triglycerides in serum were determined by enzymatic methods [8,9]. The serum HDL-cholesterol was determined by an enzymatic method after precipitation of very low density lipoprotein (VLDL) and LDL with heparin and Ca2+ [lo]. Concentrations of apolipoprotein (apo) A-I, A-II, B, C-II, C-III and E in serum were measured by a single radial immunodiffusion method [ll]. Serum lipoproteins were separated by preparative sequential ultracentrifugation [12] using a Hitachi RP 55T rotor and a Hitachi Model 55P-7 ultracentrifuge (Hitachi Koki Co., Tokyo, Japan). Serum was
adjusted to appropriate densities with NaBr. Each lipoprotein was fractionated as follows: VLDL (d < 1.006); IDL (intermediate density lipoprotein, 1.006 < d < 1.019); LDL (1.019 < d < 1.063); HDL, (1.063 < d-c 1.125) and HDL, (1.125 < d of total and free < 1.21). Concentrations cholesterol and triglycerides in each lipoprotein fraction were then determined. Analytical ultracentrifugation was performed using a Hitachi 282 analytical ultracentrifuge and an RA 60H rotor [13]. Schlieren optics and double sector cells were used for measurements of flotation rates. Measurements were performed at 44000 rpm (215 000 x g), at 4’ C, and at a density of 1.20 g/ml. NaBr was added to adjust the density. Apparent flotation rates were corrected for concentration dependence [14] and expressed as Sf,.*oo. Photographs were taken at 0, 2, 12, 18, 24 and 64 min after the centrifuge reached the designated speed. Serum (5 ~1) was also analyzed by HPLC using gel permeation columns (TSK GEL, GSWP + G50OOPW + G3000SW, Toyo Soda Co., Tokyo, Japan), and cholesterol in the final column effluent was monitored by measuring A550with the use of an enzymatic reagent kit [15]. In HPLC analysis, 10 normolipidemic male controls were used for the determination of elution time for LDL and HDL peaks. Their age was 41 & 7 years (mean + SD; range 31-50). The serum lipid levels (mean &-SD) were as follows: total cholesterol, 180 + 21 mg/dl; HDL-cholesterol, 48 f 5 mg/dl; triglycerides, 90 f 25 mg/dl. Polyacrylamide gel electrophoresis was performed as described previously [16]. Delipidated apolipoproteins of HDL, were subjected to sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis [17]. Cholesteryl ester transfer activity was measured according to the method of Albers et al. [18]. Briefly, the transfer of 14C-labelled HDLcholesteryl esters to the d-c 1.060 g/ml lipoproteins was monitored by incubation for 18 h at 37 o C with and without adding 10 ~1 of serum as a source of lipid transfer protein. The donor (HDL,) and acceptor lipoproteins were separated by heparin-MnCl 2 precipitation and the radioactivity in the supematant (HDL,) was then determined.
9
Results As shown in Table 1, the serum total cholesterol level ranged from 227 to 360 mg/dl, while the serum triglyceride level ranged from 66 to 345 mg/dl. The concentrations of serum HDLcholesterol were greater than 150 mg/dl(254, 236, 157 mg/dl, respectively) according to the heparin-Ca2+ precipitation method. To exclude the possibility that HDL in patients with deficiency of cholesteryl ester transfer activity may be partially precipitated by heparin and Ca*+, the supernatant after precipitation was subjected to both polyacrylamide gel electrophoresis (PAGE) and HPLC. It did not contain any VLDL and LDL, suggesting that the HDL of the patients were fully precipitated by heparin and Ca*+. As to apolipoproteins, serum levels of apolipoprotein A-I, A-II, C-II, C-III and E were elevated, while apolipoprotein B level was low in cases 1 and 2. Sequential flotation ultracentrifugation analysis showed that the LDL-cholesterol level was slightly decreased in cases 2 and 3, and that the concentration of HDL-cholesterol was markedly increased to the similar level determined by heparin-Ca*+ precipitation method. It was also demonstrated that the increase of HDL was accounted for solely by the increase of cholesteryl ester in the HDL,
TABLE SERUM
fraction. The apolipoproteins of HDL, of the patients were subjected to SDS-PAGE. The HDL, of the patients was slightly rich in apolipoprotein E compared with HDL, of normal controls (data not shown). The trait of hyperalphalipoproteinemia was considered to be inherited in all of the three families. In order to elucidate the underlying mechanism of hyperalphalipoproteinemia in these cases, cholesteryl ester transfer activity was measured. All of the present cases showed complete deficiency of cholesteryl ester transfer activity (O.O%/lO ~1/18 h) (control 20.0 + lO.O%/lO ~1/18 h. mean f SD, n = 20). Fig. 1 shows the elution patterns of the sera upon HPLC analysis. In normolipidemic control subjects, at least two major peaks (LDL and HDL) were resolved according to particle size. The peaks of LDL in the 3 cases were low and shifted significantly to the right (48.8-49.6 min) compared with those of normolipidemic controls (47.8 f 0.3 min, mean f SD, n = lo), while the peaks of HDL were very high and shifted markedly to the left (54.0-56.5 min) compared with those of controls (59.1 + 1.0 min, mean f SD, n = 10). In order to identify the peaks in HPLC analysis, each peak was collected, delipidated and subjected to SDSPAGE. The first peak of the patients and controls was proved to contain almost exclusively apoli-
1 LIPIDS,
LIPOPROTEIN
CHOLESTEROL
AND APOLIPOPROTEIN
LEVELS
IN THE 3 CASES
Values are in mg/dl.
Total cholesterol VLDL-cholesterol IDL-cholesterol LDL-cholesterol HDL,-cholesterol HDL,-cholesterol Triglycerides Apolipoprotein Apolipoprotein Apolipoprotein Apolipoprotein Apolipoprotein Apolipoprotein
A-l A-II B C-II C-III E
a Mean + SD (n = 14, male).
Case 1
Case 2
Case 3
Control
360 38 3 98 202 19 345
300 23 3 15 177 22 252
221 6 1 79 112 29 66
178 10 5 110 27 25 96
*
271 54 64 11.9 37.0 25.2
233 51 67 8.7 34.4 11.4
210 44 80 5.4 13.6 6.2
139 +_23 37 * 5 95 +22 3.8k 1.3 8.4k 2.5 3.9+ 1.0
k24 + 4 +2 +27 + 9 * 5 k28
Fig. 2. Schlieren patterns of sera from the 3 cases and a normal control. The photographs were taken at 18 min after the centrifuge reached the designated speed.
Elutlon time
(mln)
‘\. “...
\
i6.5
60 Elutlon time
-._
70
larger than normal HDL. In order to further characterize the abnormality of LDL, analytical ultracentrifugation was performed. Fig. 2 shows the Schlieren patterns of sera from the 3 cases and a typical normolipidemic male control subject. The LDL of the normolipidemic control was present as a high and sharp symmetrical peak, and the peak Sf,.,, rate of LDL from normolipidemic Japanese male controls was 25.8 + 1.5 (mean _t SD, n = 18). However, the LDL of the patients appeared as a low peak, which was composed of three distinct populations with a peak Sf rate of 17.9, ‘while such small and dense components of LDL could not be seen in control subjects. These characteristic features of small LDL with a multi-component distribution were seen in all 3 cases. Discussion
(mln)
Fig. 1. Elution profiles of sera from indicate the elution positions of control ( 9 ), respectively.
the 3 cases. Arrows LDL ( - ) and HDL
poprotein B, suggesting that this peak corresponded to LDL. The second peak of both patients and controls was proved to contain mainly apolipoproteins A-I and A-II, suggesting that this peak corresponded to HDL. These data indicated that the patients’ LDL was much smaller than normal LDL, and that their HDL was markedly
Up to now, there have been no reports dealing with the characteristics of LDL in FHALP except for that of Patsch et al. [19], who showed that VLDL and LDL of FHALP patients had normal flotation properties on zonal ultracentrifugation. In another report of a patient with deficiency of cholesteryl ester transfer activity [7], the characteristics of LDL particles were not shown because of inability to obtain good separation between LDL and HDL. However, our present data suggest that in the case of FHALP patients with deficiency of
11 cholesteryl ester transfer activity, LDL is abnormal and characterized by decreased particle size and a polydisperse distribution. Although polydispersion of LDL was reported in patients with hypertriglyceridemia [20], we consider that small LDL with polydispersion is a characteristic feature of FHALP patients with deficiency of cholesteryl ester transfer activity, since it was observed even in case 3, whose serum triglyceride level was normal. In addition, HPLC analysis revealed that the HDL particles from our patients were much larger than those from normal controls. These characteristics of the lipoprotein profiles are largely similar to those reported for rats [21]. The markedly large HDL particles seen in our patients were rich in apolipoprotein E and corresponded to HDL, in rat plasma [21]. The rat is known to be a useful animal model of cholesteryl ester transfer protein deficiency [22]. In humans, as much as 80% of LCAT-derived cholesteryl ester is transferred to LDL by the action of this protein [23]. Its absence may cause the accumulation of cholesteryl ester in HDL and a relative decrease of these molecules in other lipoproteins. The LDL of our patients was very poor in cholesteryl ester (data not shown). It has also been shown that rat LDL is slightly more dense than human LDL [21], and the lipoprotein profiles seen in our patients were not observed in normal control sera. We therefore consider that the decreased size and heterogeneity of LDL particles in our patients might have been caused by the observed deficiency of cholesteryl ester transfer activity. In conclusion, we have presented evidence for abnormality of LDL, both in particle size and components, in 3 cases of deficiency of cholesteryl ester transfer activity. Analysis of lipoproteins in these patients may lead to the further elucidation of the role of cholesteryl ester transfer protein in cholesterol transport. Acknowledgements This work was supported by a grant No. 60480268 from the Ministry of Education, Grantin-Aid for Special Project Research: “Metabolic Researches of Blood Vessels”, from the Ministry of Education, Science and Culture, Japan, and a
Research Grant for Studies on Adult Diseases. We thank Dr. E.L. Bierman, University of Washington, Seattle, for his valuable advice and Dr. J.J. Albers and T. Nishide, Northwest Lipid Research Clinic, Harborview Medical Center, Seattle, for the determination of cholesteryl ester transfer activity. We are also grateful to Miss Mie Katsuda and Mrs. Shizuno Yasuda for their technical assistance and to Miss Yasuyo Moriyama, Miss Mayumi Ohbori and Miss Mariko Yoshida for the preparation of the manuscript. References 1 Glueck. C.J., Fallat, R.W., Millett, F.. Gartside, P., Elston. R.C. and Go, R.C.P.. Familial hyperalphalipoproteinemia: studies in 18 kindreds. Metabolism, 24 (1975) 1243. 2 Matsuzawa, Y., Yamashita, S., Kameda, K., Kubo, M.. Tarui. S. and Hara, I., Marked hyper-HDLz-cholesterolemia associated with premature cornea1 opacity - A case report, Atherosclerosis, 53 (1984) 207. 3 Yamashita, S.. Ueyama, Y., Kawamoto, T., No&i, S., Kameda, K., Kubo, M., Matsuzawa. Y. and Tarui, S.. Lipoprotein abnormalities in familial hyperalphalipoproteinemia associated with cornea1 opacification and coronary heart disease, Jpn. Circ. J.. 50 (1986) 757. 4 Avogaro, P.. Cassolata, G., Kostner. G. and Holasek. D.R.A.. Familial hyperalphalipoproteinemia - further studies on serum lipoproteins and some serum enzymes, Clin. Chim. Acta. 77 (1977) 139. P., Fallat. R.W., Sielski, J. and 5 Glueck, C.J., Gartside, Steiner, P.M., Longevity syndromes: familial hypobeta- and familial hyperalphalipoproteinemia. J. Lab. Clin. Med., 88 (1976) 941. 6 Mendoza, S., Lutmer, R.F.. Glueck, C.J., Chen, C.. Steiner. P.M.. Fallat, R.W. and Kashyap. M.L., Composition of HDL-2 and HDL-3 in familial hyperalphalipoproteinemia. Atherosclerosis, 25 (1976) 131. 7 Koizumi. J.. Mabuchi, H., Yoshimura, A., Michishita, I.. Takeda. M., Itoh, H., Sakai. Y., Sakai. T.. Ueda. K. and Takeda. R.. Deficiency of serum cholesteryl-ester transfer activity in patients with familial hyperalphalipoproteinemia, Atherosclerosis, 58 (1985) 175. 8 Allain, C.C.. Poon, L.S., Chan, C.S.G., Richmond. W. and Fu, P.C., Enzymatic determination of total serum cholesterol, Clin. Chem., 20 (1974) 470. 9 Tiffany. K.O.. Morton. J.M., Hall, E.M. and Garrette. A.S., Clinical evaluation of kinetic enzymatic fixed time and integral analysis of serum triglyceride, Clin. Chem., 20 (1974) 476. 10 Burstein. M., Scholnick. H.R. and Morfin, R., Lipoproteinpolyanion-metal interactions, Adv. Lipid Res., 11 (1973) 67. 11 Goto, Y., Akanuma. Y., Harano, Y., Hata. Y., Itakura. H., Kajiyama, G., Kawade, M., Koga, S., Kuzuya, F., Maruhama, Y.. Matsuzawa. Y., Murai. A.. Murase. T..
12
12
13
14
15
16
Naito, C., Nakai, T., Noma, A., Saitoh, Y., Sasaki, J., Takeuchi, N., Tamachi, H., Ozawa, H., Yamamoto, A., Yamazaki, S., Yasugi, T. and Yukawa, S., Determination by the SRID method of normal values of serum apolipoproteins (A-I, A-II, B, C-II, C-III and E) in normolipidemic healthy Japanese subjects, J. Clin. Biochem. Nutr., 1 (1986) 73. Havel, R.J., Eder, H.A. and Bragdon, J.H., The distribution and chemical composition of ultracentrifugally separated lipoproteins in human serum, J. Clin. Invest., 34 (1955) 1345. Gofman, J.W., Jones, H.B., Lindgren, F.T., Lyon, T.P., Elliott, H.A. and Strisower, B., Blood lipids and human atherosclerosis, Circulation, 2 (1950) 161. Lindgren, F.T., Jensen, L.C. and Hatch, F.T., The isolation and quantitative analysis of serum lipoproteins. In: G.J. Nelson (Ed.), Blood Lipids and Lipoproteins: Quantitation, Composition, and Metabolism, Wiley-Interscience, New York, 1972, pp. 181-274. Okazaki, M., Shiraishi, K., Ohno, Y. and Hara; I., Rapid method for the quantitation of cholesterol in human serum lipoproteins by high performance liquid chromatography, J. B&hem. (Tokyo), 89 (1981) 879. Kameda, K., Matsuzawa, Y., Kubo, M., Ishikawa, K., Maejima, I., Yamamura, T., Yamamoto, A. and Tarui, S., Increased frequency of lipoprotein disorders similar to type
17
18
19
20
21
22
23
III hyperlipoproteinemia in survivors of myocardial infarction in Japan, Atherosclerosis, 51 (1984) 241. Shapiro, A.L., Vinuela, E. and Maizel, J.V., Jr., Molecular weight estimation of polypeptide chains by electrophoresis in SDS-polyacrylamide gels, B&hem. Biophys. Res. Commun., 28 (1967) 815. Albers, J.J., Tollefson, J.H., Chen, C. and Steimnetz, A., Isolation and characterization of human plasma lipid transfer proteins, Arteriosclerosis, 4 (1984) 49. Patsch, W., Kuisk, I., Glueck, C. and Schonfeld, G., Lipoproteins in familial hyperalphalipoproteinernia, Arteriosclerosis, 1 (1981) 156. Hammond, M.G., Mengel, M.C., Warmke, G.L. and Fisher, W.R., Macromolecular dispersion of human plasma lowdensity lipoproteins in hyperlipoproteinemia, Metabolism, 26 (1977) 1231. Oschry, Y. and Eisenberg, S., Rat plasma lipoproteins: reevaluation of a lipoprotein system in an animal devoid of cholesteryl ester transfer activity, J. Lipid Res., 23 (1982) 1099. Barter, P.J. and Lally, J.I., The activity of an esterified cholesterol transferring factor in human and rat serum, B&him. Biophys. Acta, 531 (1978) 233. Nestel, P.J., Reardon, M. and Billington, T., In vivo transfer of cholesteryl esters from high density lipoproteins to very low density lipoproteins in man, Biochim. Biophys. Acta, 573 (1979) 403.
7 Ltd.
ATH 04083
Small polydisperse low density lipoproteins in familial hyperalphalipoproteinemia with complete deficiency of cholesteryl ester transfer activity Shizuya Yamashita I, Yuji Matsuzawa I, Mitsuyo Okazaki 2, Hiroyuki Kako j, Tadao Yasugi 3, Hisashi Akioka 4, Kazuko Hirano ’ and Seiichiro Tarui ’ ’Second Department of Internal Medicine, Osaka Universicv Medical School, Osaka 553 (Japan), .’Laboratoy of Chemistry, Department of General Education, Tokyo Medical and Dental University, Tokyo (Japan), -’Second Department of Internal Medicine Nthon University Medical School. Tokyo (Japan), ’ Nissei Hospital, and ’ Sakai City Health Research Institute. Sakai (Japan) (Received 12 March, 1987) (Revised, received 18 September, 1987) (Accepted 18 September. 1987)
Summary Lipoprotein abnormalities were analyzed in 3 cases of marked hyperalphalipoproteinemia caused by complete deficiency of cholesteryl ester transfer activity. The probands were all men, aged 34, 43 and 48 years, respectively. The serum high density lipoprotein (HDL)-cholesterol levels of these patients were higher than 150 mg/dl (157-254 mg/dl), while serum total cholesterol levels ranged from 227 to 360 mg/dl. Sequential flotation-ultracentrifugation analysis disclosed that low density lipoprotein (LDL)cholesterol was slightly decreased and that cholesteryl ester accumulated solely in the HDL, fraction, which was also enriched with apolipoprotein E. Cholesteryl ester transfer activity was completely absent in all of these cases. High-performance liquid chromatography showed a decrease of LDL particle size in combination with a marked enlargement of HDL particle size. Analytical ultracentrifugation disclosed heterogeneity of LDL with the presence of small LDL subpopulations. We conclude that hyperalphalipoproteinemia due to complete deficiency of cholesteryl ester transfer activity is characterized by the presence of both small polydisperse LDL and markedly large HDL enriched with cholesteryl ester and apolipoprotein E.
Key words:
Hyperalphalipoproteinemia; of cholesteryl ester transfer ultracentrifugation
Low density lipoproteins; activity; High-performance
High density lipoproteins; liquid chromatography:
Deficiency Analytical
hyperalphalipoproteinemia
(FHALP)
Introduction Correspondence to: Shizuya
Yamashita, M.D., Second Deof Internal Medicine, Osaka University Medical School. Fukushima-ku, Osaka 553. Japan.
Familial
partment
0021-9150/88/$03.50
Q 1988 Elsevier Scientific
Publishers
Ireland,
has been Ltd
considered
to be a genetic
disorder
char-
8
acterized by elevated levels of serum high density lipoprotein (HDL)-cholesterol. Although longevity with reduced morbidity and mortality due to coronary heart disease has been reported in cases of FHALP [l], the basic abnormality in this condition has not yet been elucidated. We previously reported 2 patients with FHALP showing cornea1 opacification, one of whom also suffered from angina pectoris [2,3]. In these cases, a marked increase of HDL, particle size and abnormal HDL, lipid and apolipoprotein compositions were demonstrated. There have also been several reports describing alterations in the lipid and apolipoprotein contents of increased HDL [4-61 in FHALP, although little is known about the characteristics of low density lipoproteins (LDL). Koizumi et al. [7] reported a case with FHALP showing a decrease in cholesteryl ester transfer activity. Recently, we found 3 cases of FHALP showing complete deficiency of cholesteryl ester transfer activity. In the present study, we analyzed the lipoprotein profiles in these 3 cases by both high-performance liquid chromatography (HPLC) and analytical ultracentrifugation, and revealed a decreased size and heterogeneity of LDL particles as well as a marked increase of HDL particle size. Materials and methods
The subjects of this study were 3 probands from 3 families with FHALP. They were all men, aged 34, 43 and 48 years, respectively. None of them had any clinical signs or symptoms of coronary heart disease or atherosclerosis. Blood samples were collected after an overnight fast. Total and free cholesterol and triglycerides in serum were determined by enzymatic methods [8,9]. The serum HDL-cholesterol was determined by an enzymatic method after precipitation of very low density lipoprotein (VLDL) and LDL with heparin and Ca2+ [lo]. Concentrations of apolipoprotein (apo) A-I, A-II, B, C-II, C-III and E in serum were measured by a single radial immunodiffusion method [ll]. Serum lipoproteins were separated by preparative sequential ultracentrifugation [12] using a Hitachi RP 55T rotor and a Hitachi Model 55P-7 ultracentrifuge (Hitachi Koki Co., Tokyo, Japan). Serum was
adjusted to appropriate densities with NaBr. Each lipoprotein was fractionated as follows: VLDL (d < 1.006); IDL (intermediate density lipoprotein, 1.006 < d < 1.019); LDL (1.019 < d < 1.063); HDL, (1.063 < d-c 1.125) and HDL, (1.125 < d of total and free < 1.21). Concentrations cholesterol and triglycerides in each lipoprotein fraction were then determined. Analytical ultracentrifugation was performed using a Hitachi 282 analytical ultracentrifuge and an RA 60H rotor [13]. Schlieren optics and double sector cells were used for measurements of flotation rates. Measurements were performed at 44000 rpm (215 000 x g), at 4’ C, and at a density of 1.20 g/ml. NaBr was added to adjust the density. Apparent flotation rates were corrected for concentration dependence [14] and expressed as Sf,.*oo. Photographs were taken at 0, 2, 12, 18, 24 and 64 min after the centrifuge reached the designated speed. Serum (5 ~1) was also analyzed by HPLC using gel permeation columns (TSK GEL, GSWP + G50OOPW + G3000SW, Toyo Soda Co., Tokyo, Japan), and cholesterol in the final column effluent was monitored by measuring A550with the use of an enzymatic reagent kit [15]. In HPLC analysis, 10 normolipidemic male controls were used for the determination of elution time for LDL and HDL peaks. Their age was 41 & 7 years (mean + SD; range 31-50). The serum lipid levels (mean &-SD) were as follows: total cholesterol, 180 + 21 mg/dl; HDL-cholesterol, 48 f 5 mg/dl; triglycerides, 90 f 25 mg/dl. Polyacrylamide gel electrophoresis was performed as described previously [16]. Delipidated apolipoproteins of HDL, were subjected to sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis [17]. Cholesteryl ester transfer activity was measured according to the method of Albers et al. [18]. Briefly, the transfer of 14C-labelled HDLcholesteryl esters to the d-c 1.060 g/ml lipoproteins was monitored by incubation for 18 h at 37 o C with and without adding 10 ~1 of serum as a source of lipid transfer protein. The donor (HDL,) and acceptor lipoproteins were separated by heparin-MnCl 2 precipitation and the radioactivity in the supematant (HDL,) was then determined.
9
Results As shown in Table 1, the serum total cholesterol level ranged from 227 to 360 mg/dl, while the serum triglyceride level ranged from 66 to 345 mg/dl. The concentrations of serum HDLcholesterol were greater than 150 mg/dl(254, 236, 157 mg/dl, respectively) according to the heparin-Ca2+ precipitation method. To exclude the possibility that HDL in patients with deficiency of cholesteryl ester transfer activity may be partially precipitated by heparin and Ca*+, the supernatant after precipitation was subjected to both polyacrylamide gel electrophoresis (PAGE) and HPLC. It did not contain any VLDL and LDL, suggesting that the HDL of the patients were fully precipitated by heparin and Ca*+. As to apolipoproteins, serum levels of apolipoprotein A-I, A-II, C-II, C-III and E were elevated, while apolipoprotein B level was low in cases 1 and 2. Sequential flotation ultracentrifugation analysis showed that the LDL-cholesterol level was slightly decreased in cases 2 and 3, and that the concentration of HDL-cholesterol was markedly increased to the similar level determined by heparin-Ca*+ precipitation method. It was also demonstrated that the increase of HDL was accounted for solely by the increase of cholesteryl ester in the HDL,
TABLE SERUM
fraction. The apolipoproteins of HDL, of the patients were subjected to SDS-PAGE. The HDL, of the patients was slightly rich in apolipoprotein E compared with HDL, of normal controls (data not shown). The trait of hyperalphalipoproteinemia was considered to be inherited in all of the three families. In order to elucidate the underlying mechanism of hyperalphalipoproteinemia in these cases, cholesteryl ester transfer activity was measured. All of the present cases showed complete deficiency of cholesteryl ester transfer activity (O.O%/lO ~1/18 h) (control 20.0 + lO.O%/lO ~1/18 h. mean f SD, n = 20). Fig. 1 shows the elution patterns of the sera upon HPLC analysis. In normolipidemic control subjects, at least two major peaks (LDL and HDL) were resolved according to particle size. The peaks of LDL in the 3 cases were low and shifted significantly to the right (48.8-49.6 min) compared with those of normolipidemic controls (47.8 f 0.3 min, mean f SD, n = lo), while the peaks of HDL were very high and shifted markedly to the left (54.0-56.5 min) compared with those of controls (59.1 + 1.0 min, mean f SD, n = 10). In order to identify the peaks in HPLC analysis, each peak was collected, delipidated and subjected to SDSPAGE. The first peak of the patients and controls was proved to contain almost exclusively apoli-
1 LIPIDS,
LIPOPROTEIN
CHOLESTEROL
AND APOLIPOPROTEIN
LEVELS
IN THE 3 CASES
Values are in mg/dl.
Total cholesterol VLDL-cholesterol IDL-cholesterol LDL-cholesterol HDL,-cholesterol HDL,-cholesterol Triglycerides Apolipoprotein Apolipoprotein Apolipoprotein Apolipoprotein Apolipoprotein Apolipoprotein
A-l A-II B C-II C-III E
a Mean + SD (n = 14, male).
Case 1
Case 2
Case 3
Control
360 38 3 98 202 19 345
300 23 3 15 177 22 252
221 6 1 79 112 29 66
178 10 5 110 27 25 96
*
271 54 64 11.9 37.0 25.2
233 51 67 8.7 34.4 11.4
210 44 80 5.4 13.6 6.2
139 +_23 37 * 5 95 +22 3.8k 1.3 8.4k 2.5 3.9+ 1.0
k24 + 4 +2 +27 + 9 * 5 k28
Fig. 2. Schlieren patterns of sera from the 3 cases and a normal control. The photographs were taken at 18 min after the centrifuge reached the designated speed.
Elutlon time
(mln)
‘\. “...
\
i6.5
60 Elutlon time
-._
70
larger than normal HDL. In order to further characterize the abnormality of LDL, analytical ultracentrifugation was performed. Fig. 2 shows the Schlieren patterns of sera from the 3 cases and a typical normolipidemic male control subject. The LDL of the normolipidemic control was present as a high and sharp symmetrical peak, and the peak Sf,.,, rate of LDL from normolipidemic Japanese male controls was 25.8 + 1.5 (mean _t SD, n = 18). However, the LDL of the patients appeared as a low peak, which was composed of three distinct populations with a peak Sf rate of 17.9, ‘while such small and dense components of LDL could not be seen in control subjects. These characteristic features of small LDL with a multi-component distribution were seen in all 3 cases. Discussion
(mln)
Fig. 1. Elution profiles of sera from indicate the elution positions of control ( 9 ), respectively.
the 3 cases. Arrows LDL ( - ) and HDL
poprotein B, suggesting that this peak corresponded to LDL. The second peak of both patients and controls was proved to contain mainly apolipoproteins A-I and A-II, suggesting that this peak corresponded to HDL. These data indicated that the patients’ LDL was much smaller than normal LDL, and that their HDL was markedly
Up to now, there have been no reports dealing with the characteristics of LDL in FHALP except for that of Patsch et al. [19], who showed that VLDL and LDL of FHALP patients had normal flotation properties on zonal ultracentrifugation. In another report of a patient with deficiency of cholesteryl ester transfer activity [7], the characteristics of LDL particles were not shown because of inability to obtain good separation between LDL and HDL. However, our present data suggest that in the case of FHALP patients with deficiency of
11 cholesteryl ester transfer activity, LDL is abnormal and characterized by decreased particle size and a polydisperse distribution. Although polydispersion of LDL was reported in patients with hypertriglyceridemia [20], we consider that small LDL with polydispersion is a characteristic feature of FHALP patients with deficiency of cholesteryl ester transfer activity, since it was observed even in case 3, whose serum triglyceride level was normal. In addition, HPLC analysis revealed that the HDL particles from our patients were much larger than those from normal controls. These characteristics of the lipoprotein profiles are largely similar to those reported for rats [21]. The markedly large HDL particles seen in our patients were rich in apolipoprotein E and corresponded to HDL, in rat plasma [21]. The rat is known to be a useful animal model of cholesteryl ester transfer protein deficiency [22]. In humans, as much as 80% of LCAT-derived cholesteryl ester is transferred to LDL by the action of this protein [23]. Its absence may cause the accumulation of cholesteryl ester in HDL and a relative decrease of these molecules in other lipoproteins. The LDL of our patients was very poor in cholesteryl ester (data not shown). It has also been shown that rat LDL is slightly more dense than human LDL [21], and the lipoprotein profiles seen in our patients were not observed in normal control sera. We therefore consider that the decreased size and heterogeneity of LDL particles in our patients might have been caused by the observed deficiency of cholesteryl ester transfer activity. In conclusion, we have presented evidence for abnormality of LDL, both in particle size and components, in 3 cases of deficiency of cholesteryl ester transfer activity. Analysis of lipoproteins in these patients may lead to the further elucidation of the role of cholesteryl ester transfer protein in cholesterol transport. Acknowledgements This work was supported by a grant No. 60480268 from the Ministry of Education, Grantin-Aid for Special Project Research: “Metabolic Researches of Blood Vessels”, from the Ministry of Education, Science and Culture, Japan, and a
Research Grant for Studies on Adult Diseases. We thank Dr. E.L. Bierman, University of Washington, Seattle, for his valuable advice and Dr. J.J. Albers and T. Nishide, Northwest Lipid Research Clinic, Harborview Medical Center, Seattle, for the determination of cholesteryl ester transfer activity. We are also grateful to Miss Mie Katsuda and Mrs. Shizuno Yasuda for their technical assistance and to Miss Yasuyo Moriyama, Miss Mayumi Ohbori and Miss Mariko Yoshida for the preparation of the manuscript. References 1 Glueck. C.J., Fallat, R.W., Millett, F.. Gartside, P., Elston. R.C. and Go, R.C.P.. Familial hyperalphalipoproteinemia: studies in 18 kindreds. Metabolism, 24 (1975) 1243. 2 Matsuzawa, Y., Yamashita, S., Kameda, K., Kubo, M.. Tarui. S. and Hara, I., Marked hyper-HDLz-cholesterolemia associated with premature cornea1 opacity - A case report, Atherosclerosis, 53 (1984) 207. 3 Yamashita, S.. Ueyama, Y., Kawamoto, T., No&i, S., Kameda, K., Kubo, M., Matsuzawa. Y. and Tarui, S.. Lipoprotein abnormalities in familial hyperalphalipoproteinemia associated with cornea1 opacification and coronary heart disease, Jpn. Circ. J.. 50 (1986) 757. 4 Avogaro, P.. Cassolata, G., Kostner. G. and Holasek. D.R.A.. Familial hyperalphalipoproteinemia - further studies on serum lipoproteins and some serum enzymes, Clin. Chim. Acta. 77 (1977) 139. P., Fallat. R.W., Sielski, J. and 5 Glueck, C.J., Gartside, Steiner, P.M., Longevity syndromes: familial hypobeta- and familial hyperalphalipoproteinemia. J. Lab. Clin. Med., 88 (1976) 941. 6 Mendoza, S., Lutmer, R.F.. Glueck, C.J., Chen, C.. Steiner. P.M.. Fallat, R.W. and Kashyap. M.L., Composition of HDL-2 and HDL-3 in familial hyperalphalipoproteinemia. Atherosclerosis, 25 (1976) 131. 7 Koizumi. J.. Mabuchi, H., Yoshimura, A., Michishita, I.. Takeda. M., Itoh, H., Sakai. Y., Sakai. T.. Ueda. K. and Takeda. R.. Deficiency of serum cholesteryl-ester transfer activity in patients with familial hyperalphalipoproteinemia, Atherosclerosis, 58 (1985) 175. 8 Allain, C.C.. Poon, L.S., Chan, C.S.G., Richmond. W. and Fu, P.C., Enzymatic determination of total serum cholesterol, Clin. Chem., 20 (1974) 470. 9 Tiffany. K.O.. Morton. J.M., Hall, E.M. and Garrette. A.S., Clinical evaluation of kinetic enzymatic fixed time and integral analysis of serum triglyceride, Clin. Chem., 20 (1974) 476. 10 Burstein. M., Scholnick. H.R. and Morfin, R., Lipoproteinpolyanion-metal interactions, Adv. Lipid Res., 11 (1973) 67. 11 Goto, Y., Akanuma. Y., Harano, Y., Hata. Y., Itakura. H., Kajiyama, G., Kawade, M., Koga, S., Kuzuya, F., Maruhama, Y.. Matsuzawa. Y., Murai. A.. Murase. T..
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