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Cholesterol crystal uptake and metabolism by P388D1 macrophages
Atherosclerosis, II (1989) 221-225 Elsevier Scientific Publishers Ireland,
221 Ltd.
ATH 04324
Cholesterol crystal uptake and metabolism by P388D, macrophages Walter J. McConathy,
Eugen Koren and David L. Stiers
Oklahoma Medical Research Foundation, Oklahoma City, OK 73104 (U.S.A.) (Received 12 October, 1988) (Revised, received 23 January, 1989) (Accepted 2 February, 1989)
Summary
Cholesterol monohydrate crystals are frequently detected in intermediate and advanced atherosclerotic lesions. Little is known regarding mobilization of this molecular form of cholesterol into metabolically active pools. To study a potential mechanism for mobilization of crystalline cholesterol, we examined its uptake by a mouse macrophage cell line (P388Di). Crystals were overlayered on a P388D, cell monolayer maintained in a serum-free medium. Following incubation, the monolayer was washed, and the cells were harvested and analyzed for crystal internalization. By transmission electron microscopy, crystals were found intracellularly surrounded by a bilayer membrane. Analyses of the cellular cholesterol ester content by gas-liquid chromatography and esterification of [ “C]cholesterol indicated the conversion of crystalline cholesterol to cholesterol esters. This pathway for solubilization of cholesterol crystals by macrophages could play an important role in the regression of atherosclerotic lesions.
Key words: Cholesterol crystals; Macrophage;
Cholesterol ester
Introduction Atherosclerosis is a complex degenerative disease that includes the gradual deposition of intraand extracellular lipid in the arterial wall. The development of atherosclerotic lesions consists of a combination of changes in the intima and media
Correspondence to: Walter J. McConathy, Ph.D., Lipoprotein/Atherosclerosis Research Program, Oklahoma Medical Research Foundation, 825 N.E. 13th Street, Oklahoma City, OK 73104, U.S.A. CKJ21-9150/89/$03.50
0 1989 Elsevier Scientific
Publishers
Ireland,
of arteries and is associated with focal accumulation of cholesterol, cholesterol esters, cholesterol monohydrate crystals, complex carbohydrates, fibrous material and calcium deposits. Atherosclerotic lesions can be classified into three morphologically distinct forms: the fatty streak, the fibrous plaque, and the calcified or complicated lesion [l]. All 3 lesions contain both cholesterol and cholesteryl esters. In fatty streaks, the lipid is largely intracellular. Fibrous plaques are -intermediate with respect to quantity and localization of lipid while the complicated lesions have large amounts of both intra- and extracellular lipid inLtd
222
eluding cholesterol monohydrate crystals. As recently described in studies on discrete, small regions of raised intima in human aorta, lesions intermediate between fatty streaks and fibrous plaques, also contained cholesterol monohydrate crystals which were extracellular and located near the inner limiting elastic membrane [2]. Thus, crystals may appear early in the development of atherosclerotic lesions though they have been more commonly associated with complicated lesions. As suggested by Haust [3], a mechanism for clearing damaged arterial wall is phagocytosis by mononuclear cells which appear early in the atherosclerotic process. The purpose of the present study was to explore the uptake and degradation of cholesterol monohydrate crystals using the murine tumor macrophage cell line P388Dr. Materials and methods
Cholesterol monohydrate crystals were prepared by crystallizing USP grade cholesterol from hot ethanol, 3 times. Cholesterol crystals had their characteristic appearance both by electron and light microscopy [6,11,13]. Crystals were washed with 50 mM Tris, 150 mM NaCl, pH 7.5, dispersed with a Waring blender set at medium speed for 6 min and autoclaved. To study the mobilization of cholesterol from crystals, [ l4 Clcholesterol was added to the hot ethanolic solution containing non-labeled cholesterol. Following 2 recrystallizations, the radioactive cholesterol crystals were utilized to monitor the appearance of esterified [r4C]cholesterol within the macrophage. The murine tumor macrophage cell line, P388D,, has proven useful in studies of lipoprotein and cholesterol metabolism since it maintains many relevant characteristics of the normal macrophage [4,5]. Cells were grown in a defined serum-free medium (Optimem, Gibco, Gaithersberg, MD) in T-75 flasks. Autoclaved crystals were overlayered on a semiconfluent cell monolayer. Following incubation, the cell monolayer was washed 3 times with phosphate-buffered saline (PBS, pH 7.4), to remove loosely attached and free crystals. The cells were harvested and centrifuged (1100 rpm for 5 min). The supernatant was discarded and the cell pellet resuspended in PBS. This step was repeated
once. The final pellet coming from one T-75 flask was resuspended in 1 ml of PBS and used for electron microscopy (0.3 ml) and gas liquid chromatography (0.7 ml) analyses. In addition, cells were extracted [7] and analyzed for the net accumulation of cholesterol esters by gas liquid chromatography [8] or esterification of [ “C]cholesterol by thin-layer chromatography [9]. Protein concentration was determined by the method of Bradford [lo]. Results
Following 24 h incubation of the P388D, macrophages with an overlay of crystals, numerous inclusions were clearly detectable in sectioned cells (Fig. lA,B). Relatively large crystals (> 1 x 1 pm) having an opaque appearance were seen in the macrophages exposed to cholesterol monohydrate crystals, while there was no similar material in control macrophages. Examination of the boundaries of the crystals at higher resolution demonstrated the presence of a surrounding bilayer (Fig. 1C). The bilayer surrounding the crystal had the appearance typical of a membrane [ll]. As illustrated, the large crystal was in contact with a vacuole which contained smaller crystals also surrounded by membranes (Fig. 1D). Such an observation is consistent with phagocytosis of both large and small cholesterol monohydrate crystals. To clarify whether the phagocytosized crystals undergo solubilization and chemical modification, cells incubated with cholesterol crystals were analyzed for the appearance of cholesterol esters by gas-liquid chromatography and the appearance of [r4C]cholesterol esters. In 3 separate experiments, the cholesterol ester increased significantly over control levels (Table 1) yielding a net accumulation of cholesterol esters. Macrophages also contained high levels of unesterified cholesterol which was consistent with the uptake of crystalline cholesterol and indicated that only a fraction of internalized cholesterol is esterified. Since EM and phase-contrast microscopy showed virtually no extracellular cholesterol crystals in the washed and centrifuged cell fraction, most of the free cholesterol determined by GLC is considered to originate from internalized cholesterol crystals. Direct conversion of crystalline cholesterol into
223
50 nm Fig. 1. Transmission electron micrographs depicting cholesterol monohydrate crystals wttmn mouse macrophage cell line (P388D,). (A) Cross-section of macrophage with large crystal and numerous vacuoles with smaller crystal remnants present. (B) High magnification of crystal and vacuole with remnants of cholesterol crystals. (C) Presence of surrounding bilayer at boundary of crystal. (D) High magnification of vacuole with remnants of cholesterol crystals, some with double membrane still present. Cells were incubated with cholesterol crystals for 48 h prior to analysis.
esterified cholesterol was confirmed by the appearance of [‘4C]cholesterol esters in cells incubated with crystalline [ “C]cholesterol (Table 2). Discussion The rate of cholesterol accumulation in the artery is a function of 3 processes: the transfer of
lipid from plasma, the binding and sequestering of lipids, and the solubilization and removal of lipid from the artery [12]. Katz and Small [13] suggested in their studies on atherosclerotic plaques that the “cholesterol monohydrate crystalline phase will be metabolically inert and constitute a formidable obstacle to plaque regression”. Our
224 TABLE 1 CHOLESTEROL AND CHOLESTEROL ESTER ACCUMULATION IN MURINE P338Dt MACROPHAGES Unesterified cholesterol (C) and cholesterol esters (CE) determined by gas-liquid chromatography (n = 3) after incubation of cells with cholesterol crystals for 48 h. Values are mean (SD) pg/mg cell protein C
Cells Cells + crystals
CE 20.9
1561
(0.4) (608) *
4.6 (0.9) 26.6 (4.5) *
* P < 0.005.
TABLE 2
ated during the degradation of triglyceride-rich lipoproteins [15]. Such crystalline structures were observed in plasma from postprandial fat-loaded subjects infused with heparin. These included type I and type III hyperlipoproteinemic subjects and those with Zieve’s syndrome. Thus, microcrystalline cholesterol could also arise in the postprandial state and exist transiently in normolipidemic subjects consuming a high fat-cholesterol meal. The processing of such postprandial cholesterol crystals could be mediated by macrophages and their removal might also be important in impeding the progression of arteriosclerosis.
References
APPEARANCE OF [‘4C]CHOLESTEROL ESTERS IN MURINE P388Dr MACROPHAGE FOLLOWING THE ADDITION OF [‘4C]CHOLESTEROL MONOHYDRATE CRYSTALS Homogenized crystalline cholesterol (spec. act. 19 990 f 570 cpm/mg) was added to macrophage cell culture, cells were harvested at indicated times, extracted, lipids separated by TLC (n = 3), counted, and values converted to nmol esterified cholesterol (CE). Values are mean (SD). Incubation
nmol CE
24 h 48 h
13.6 (1.2) 30.5 (0.6)
studies show that cholesterol crystals can be taken up and partially solubilized by macrophages. Such a mechanism could be important in the removal of cholesterol crystals from focal cholesterol deposits and therefore important in the regression of atherosclerotic lesions. Since cholesterol crystals may appear early in the development of atherosclerotic lesions [2] and have been demonstrated in foam cells in experimental atheroma [14], their removal could also be important in delaying or preventing the progression to more advanced lesions. Another less recognized pathway where metabolic processing of cholesterol crystals could be physiologically important is the postprandial state. Cholesterol monohydrate crystals were demonstrated by EM in the plasma compartment in lipoprotein fractions (d < 1.063 g/ml) which were suggested to stem from excess surface lipid gener-
1 Gem, G.S., Vesselinovitch, D. and Wissler, R.W., A dynamic pathology of atherosclerosis, Am. J. Med., 46 (1969) 657. 2 Bocan, T.M.A. and Guyton, J.R., Human aortic fibrolipid lesions. Progenitor lesions for fibrous plaques, exhibiting early formation of the cholesterol-rich core, Am. J. Pathol., 120 (1985) 193. 3 Haust, M.D., The morphogenesis and fate of potential and early atherosclerosis lesions in man, Hum. Pathol. 2 (1971) 1. 4 Via, D.P., Plant, A.L., Craig, I.F., Gotto, A.M., Jr. and Smith, L.C., Metabolism of normal and modified low-density lipoproteins by macrophage cell lines of murine and human origin, B&him. Biophys. Acta, 833 (1985) 417. 5 Gianturco, S.H., Brown, S.A., Via, D.P. and Bradley, W.A., The /3-VLDL receptor pathway of murine P388Dt macrophages, J. Lipid Res., 27 (1986) 412. 6 Jones, A.L. and Price, J.M., Some methods of electron microscopic visualixation of lipoproteins in plasma and chyle, J. Histochem. Cytochem., 16 (1968) 366. 7 Lipid Research Clinics Program, DHEW Pub. No. (NIH) 76-628, Vol. 1, U.S. Govt. Printing Office, Washington, DC, 1974, pp. 74-81. 8 Wang, C.-S., We&r, D., Alaupovic, P. and McConathy, W.J., Studies on the degradation of human very low density lipoproteins by human milk lipoprotein lipase, Arch. Biothem. Biophys., 214 (1982) 26. 9 Alaupovic, P., Sanbar, S.S., Furman, R.H., Sullivan, M.L. and Walraven, S.L., Studies of the composition and structure of serum lipoproteins: isolation and characterization of very high density lipoproteins of human serum, Biochemistry, 5 (1966) 4044. 10 Bradford, M., A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. B&hem., 72 (1976) 284. 11 Singer, S.J. and Nicholsen, G.L., The fluid mosaic model of the structure of cell membranes, Science, 175 (1972) 720.
225 12 Zilversmit, D.B., Mechanisms of cholesterol accumulation in the arterial wall, Am. J. Cardiol., 35 (1975) 559. 13 Katz, S.S. and Small, D.M., Isolation and partial characterization of the lipid phases of human atherosclerotic plaques, J. Biol. Chem., 255 (1980) 9753. 14 Luper, F., Danaricu, I. and Simionescu, N., Development
of intracellular lipid deposits in the lipid-laden cells of atherosclerotic lesions. A cytochemical and ultrastructural study, Atherosclerosis, 67 (1987) 127. 15 Bode, G., Kloer, H.U. and Ditschuneit, H., Lipoprotein morphology in some LCAT-deficient states, Stand. J. Clin. Lab. Invest., 38, Suppl. 150 (1978) 199.
221 Ltd.
ATH 04324
Cholesterol crystal uptake and metabolism by P388D, macrophages Walter J. McConathy,
Eugen Koren and David L. Stiers
Oklahoma Medical Research Foundation, Oklahoma City, OK 73104 (U.S.A.) (Received 12 October, 1988) (Revised, received 23 January, 1989) (Accepted 2 February, 1989)
Summary
Cholesterol monohydrate crystals are frequently detected in intermediate and advanced atherosclerotic lesions. Little is known regarding mobilization of this molecular form of cholesterol into metabolically active pools. To study a potential mechanism for mobilization of crystalline cholesterol, we examined its uptake by a mouse macrophage cell line (P388Di). Crystals were overlayered on a P388D, cell monolayer maintained in a serum-free medium. Following incubation, the monolayer was washed, and the cells were harvested and analyzed for crystal internalization. By transmission electron microscopy, crystals were found intracellularly surrounded by a bilayer membrane. Analyses of the cellular cholesterol ester content by gas-liquid chromatography and esterification of [ “C]cholesterol indicated the conversion of crystalline cholesterol to cholesterol esters. This pathway for solubilization of cholesterol crystals by macrophages could play an important role in the regression of atherosclerotic lesions.
Key words: Cholesterol crystals; Macrophage;
Cholesterol ester
Introduction Atherosclerosis is a complex degenerative disease that includes the gradual deposition of intraand extracellular lipid in the arterial wall. The development of atherosclerotic lesions consists of a combination of changes in the intima and media
Correspondence to: Walter J. McConathy, Ph.D., Lipoprotein/Atherosclerosis Research Program, Oklahoma Medical Research Foundation, 825 N.E. 13th Street, Oklahoma City, OK 73104, U.S.A. CKJ21-9150/89/$03.50
0 1989 Elsevier Scientific
Publishers
Ireland,
of arteries and is associated with focal accumulation of cholesterol, cholesterol esters, cholesterol monohydrate crystals, complex carbohydrates, fibrous material and calcium deposits. Atherosclerotic lesions can be classified into three morphologically distinct forms: the fatty streak, the fibrous plaque, and the calcified or complicated lesion [l]. All 3 lesions contain both cholesterol and cholesteryl esters. In fatty streaks, the lipid is largely intracellular. Fibrous plaques are -intermediate with respect to quantity and localization of lipid while the complicated lesions have large amounts of both intra- and extracellular lipid inLtd
222
eluding cholesterol monohydrate crystals. As recently described in studies on discrete, small regions of raised intima in human aorta, lesions intermediate between fatty streaks and fibrous plaques, also contained cholesterol monohydrate crystals which were extracellular and located near the inner limiting elastic membrane [2]. Thus, crystals may appear early in the development of atherosclerotic lesions though they have been more commonly associated with complicated lesions. As suggested by Haust [3], a mechanism for clearing damaged arterial wall is phagocytosis by mononuclear cells which appear early in the atherosclerotic process. The purpose of the present study was to explore the uptake and degradation of cholesterol monohydrate crystals using the murine tumor macrophage cell line P388Dr. Materials and methods
Cholesterol monohydrate crystals were prepared by crystallizing USP grade cholesterol from hot ethanol, 3 times. Cholesterol crystals had their characteristic appearance both by electron and light microscopy [6,11,13]. Crystals were washed with 50 mM Tris, 150 mM NaCl, pH 7.5, dispersed with a Waring blender set at medium speed for 6 min and autoclaved. To study the mobilization of cholesterol from crystals, [ l4 Clcholesterol was added to the hot ethanolic solution containing non-labeled cholesterol. Following 2 recrystallizations, the radioactive cholesterol crystals were utilized to monitor the appearance of esterified [r4C]cholesterol within the macrophage. The murine tumor macrophage cell line, P388D,, has proven useful in studies of lipoprotein and cholesterol metabolism since it maintains many relevant characteristics of the normal macrophage [4,5]. Cells were grown in a defined serum-free medium (Optimem, Gibco, Gaithersberg, MD) in T-75 flasks. Autoclaved crystals were overlayered on a semiconfluent cell monolayer. Following incubation, the cell monolayer was washed 3 times with phosphate-buffered saline (PBS, pH 7.4), to remove loosely attached and free crystals. The cells were harvested and centrifuged (1100 rpm for 5 min). The supernatant was discarded and the cell pellet resuspended in PBS. This step was repeated
once. The final pellet coming from one T-75 flask was resuspended in 1 ml of PBS and used for electron microscopy (0.3 ml) and gas liquid chromatography (0.7 ml) analyses. In addition, cells were extracted [7] and analyzed for the net accumulation of cholesterol esters by gas liquid chromatography [8] or esterification of [ “C]cholesterol by thin-layer chromatography [9]. Protein concentration was determined by the method of Bradford [lo]. Results
Following 24 h incubation of the P388D, macrophages with an overlay of crystals, numerous inclusions were clearly detectable in sectioned cells (Fig. lA,B). Relatively large crystals (> 1 x 1 pm) having an opaque appearance were seen in the macrophages exposed to cholesterol monohydrate crystals, while there was no similar material in control macrophages. Examination of the boundaries of the crystals at higher resolution demonstrated the presence of a surrounding bilayer (Fig. 1C). The bilayer surrounding the crystal had the appearance typical of a membrane [ll]. As illustrated, the large crystal was in contact with a vacuole which contained smaller crystals also surrounded by membranes (Fig. 1D). Such an observation is consistent with phagocytosis of both large and small cholesterol monohydrate crystals. To clarify whether the phagocytosized crystals undergo solubilization and chemical modification, cells incubated with cholesterol crystals were analyzed for the appearance of cholesterol esters by gas-liquid chromatography and the appearance of [r4C]cholesterol esters. In 3 separate experiments, the cholesterol ester increased significantly over control levels (Table 1) yielding a net accumulation of cholesterol esters. Macrophages also contained high levels of unesterified cholesterol which was consistent with the uptake of crystalline cholesterol and indicated that only a fraction of internalized cholesterol is esterified. Since EM and phase-contrast microscopy showed virtually no extracellular cholesterol crystals in the washed and centrifuged cell fraction, most of the free cholesterol determined by GLC is considered to originate from internalized cholesterol crystals. Direct conversion of crystalline cholesterol into
223
50 nm Fig. 1. Transmission electron micrographs depicting cholesterol monohydrate crystals wttmn mouse macrophage cell line (P388D,). (A) Cross-section of macrophage with large crystal and numerous vacuoles with smaller crystal remnants present. (B) High magnification of crystal and vacuole with remnants of cholesterol crystals. (C) Presence of surrounding bilayer at boundary of crystal. (D) High magnification of vacuole with remnants of cholesterol crystals, some with double membrane still present. Cells were incubated with cholesterol crystals for 48 h prior to analysis.
esterified cholesterol was confirmed by the appearance of [‘4C]cholesterol esters in cells incubated with crystalline [ “C]cholesterol (Table 2). Discussion The rate of cholesterol accumulation in the artery is a function of 3 processes: the transfer of
lipid from plasma, the binding and sequestering of lipids, and the solubilization and removal of lipid from the artery [12]. Katz and Small [13] suggested in their studies on atherosclerotic plaques that the “cholesterol monohydrate crystalline phase will be metabolically inert and constitute a formidable obstacle to plaque regression”. Our
224 TABLE 1 CHOLESTEROL AND CHOLESTEROL ESTER ACCUMULATION IN MURINE P338Dt MACROPHAGES Unesterified cholesterol (C) and cholesterol esters (CE) determined by gas-liquid chromatography (n = 3) after incubation of cells with cholesterol crystals for 48 h. Values are mean (SD) pg/mg cell protein C
Cells Cells + crystals
CE 20.9
1561
(0.4) (608) *
4.6 (0.9) 26.6 (4.5) *
* P < 0.005.
TABLE 2
ated during the degradation of triglyceride-rich lipoproteins [15]. Such crystalline structures were observed in plasma from postprandial fat-loaded subjects infused with heparin. These included type I and type III hyperlipoproteinemic subjects and those with Zieve’s syndrome. Thus, microcrystalline cholesterol could also arise in the postprandial state and exist transiently in normolipidemic subjects consuming a high fat-cholesterol meal. The processing of such postprandial cholesterol crystals could be mediated by macrophages and their removal might also be important in impeding the progression of arteriosclerosis.
References
APPEARANCE OF [‘4C]CHOLESTEROL ESTERS IN MURINE P388Dr MACROPHAGE FOLLOWING THE ADDITION OF [‘4C]CHOLESTEROL MONOHYDRATE CRYSTALS Homogenized crystalline cholesterol (spec. act. 19 990 f 570 cpm/mg) was added to macrophage cell culture, cells were harvested at indicated times, extracted, lipids separated by TLC (n = 3), counted, and values converted to nmol esterified cholesterol (CE). Values are mean (SD). Incubation
nmol CE
24 h 48 h
13.6 (1.2) 30.5 (0.6)
studies show that cholesterol crystals can be taken up and partially solubilized by macrophages. Such a mechanism could be important in the removal of cholesterol crystals from focal cholesterol deposits and therefore important in the regression of atherosclerotic lesions. Since cholesterol crystals may appear early in the development of atherosclerotic lesions [2] and have been demonstrated in foam cells in experimental atheroma [14], their removal could also be important in delaying or preventing the progression to more advanced lesions. Another less recognized pathway where metabolic processing of cholesterol crystals could be physiologically important is the postprandial state. Cholesterol monohydrate crystals were demonstrated by EM in the plasma compartment in lipoprotein fractions (d < 1.063 g/ml) which were suggested to stem from excess surface lipid gener-
1 Gem, G.S., Vesselinovitch, D. and Wissler, R.W., A dynamic pathology of atherosclerosis, Am. J. Med., 46 (1969) 657. 2 Bocan, T.M.A. and Guyton, J.R., Human aortic fibrolipid lesions. Progenitor lesions for fibrous plaques, exhibiting early formation of the cholesterol-rich core, Am. J. Pathol., 120 (1985) 193. 3 Haust, M.D., The morphogenesis and fate of potential and early atherosclerosis lesions in man, Hum. Pathol. 2 (1971) 1. 4 Via, D.P., Plant, A.L., Craig, I.F., Gotto, A.M., Jr. and Smith, L.C., Metabolism of normal and modified low-density lipoproteins by macrophage cell lines of murine and human origin, B&him. Biophys. Acta, 833 (1985) 417. 5 Gianturco, S.H., Brown, S.A., Via, D.P. and Bradley, W.A., The /3-VLDL receptor pathway of murine P388Dt macrophages, J. Lipid Res., 27 (1986) 412. 6 Jones, A.L. and Price, J.M., Some methods of electron microscopic visualixation of lipoproteins in plasma and chyle, J. Histochem. Cytochem., 16 (1968) 366. 7 Lipid Research Clinics Program, DHEW Pub. No. (NIH) 76-628, Vol. 1, U.S. Govt. Printing Office, Washington, DC, 1974, pp. 74-81. 8 Wang, C.-S., We&r, D., Alaupovic, P. and McConathy, W.J., Studies on the degradation of human very low density lipoproteins by human milk lipoprotein lipase, Arch. Biothem. Biophys., 214 (1982) 26. 9 Alaupovic, P., Sanbar, S.S., Furman, R.H., Sullivan, M.L. and Walraven, S.L., Studies of the composition and structure of serum lipoproteins: isolation and characterization of very high density lipoproteins of human serum, Biochemistry, 5 (1966) 4044. 10 Bradford, M., A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. B&hem., 72 (1976) 284. 11 Singer, S.J. and Nicholsen, G.L., The fluid mosaic model of the structure of cell membranes, Science, 175 (1972) 720.
225 12 Zilversmit, D.B., Mechanisms of cholesterol accumulation in the arterial wall, Am. J. Cardiol., 35 (1975) 559. 13 Katz, S.S. and Small, D.M., Isolation and partial characterization of the lipid phases of human atherosclerotic plaques, J. Biol. Chem., 255 (1980) 9753. 14 Luper, F., Danaricu, I. and Simionescu, N., Development
of intracellular lipid deposits in the lipid-laden cells of atherosclerotic lesions. A cytochemical and ultrastructural study, Atherosclerosis, 67 (1987) 127. 15 Bode, G., Kloer, H.U. and Ditschuneit, H., Lipoprotein morphology in some LCAT-deficient states, Stand. J. Clin. Lab. Invest., 38, Suppl. 150 (1978) 199.