Transfer of cholesterol between serum lipoproteins, isolated membranes, and intact tissue

EXPJSRJ.MENTAL

AND

Transfer

MOLECULAR

PATHOLOGY

19,

293-303

of Cholesterol Between Isolated Membranes, and

(1973)

Serum Lipoproteins, Intact Tissue

FRANK P. BEL,L~ Department

of

Pathology,

of

Faculty the Clzedoke

Medicine, McMaster University, Hamilton, General Hospitals, Hamilton, Ontario

Received

February

Ontario,

and

8,1973

The transfer of unesterified cholesterol between serum lipoproteins, aortas, and erythrocyte membranes was examined under a variety of conditions, in v3ro. The availability of unesterified cholesterol of erythrocyte membranes and serum lipoproteins for transfer was unaffected by formalin fixation. By contrast however, hation of aortic tissue reduced cholesterol transfer from serum lipoproteins by approximately 50%. Transfer of serum lipoprotein unesterified cholesterol to either aortas or membranes was increased in the presence of dimethyl sulfoxide, a compound considered to be capable of weakening hydrophobic bonding between lipid molecules; this increased transfer was unaffected by fixation of either the membranes or aortic tissue. These studies indicate that cholesterol transfer between serum lipoproteins and membranes is infiuenced more by alterations in lipid-lipid interactions than by the state, fixed or unfixed, of the proteins in these moieties.

INTRODUCTION Unesterified cholesterol is known to exchange between plasma lipoproteins and erythrocytes (Hagerman and Gould, 1951; Murphy, 1962; Bell et al., 1972) while esterified cholesterol does not. It has also been reported that much of the cholesterol taken up by an aorta from plasma lipoproteins in vitro is the result of unesterified cholesterol exchange (Dayton and Hashimoto, 1966, 1970; Newman and Zilversmit, 1962). It is also clear from current evidence that cholesterol exchange to erythrocytes and aortas is a physicochemical phenomenon, not an energy dependent one (Murphy, 1962; Dayton and Hashimoto, 1966; Newman and Zilversmit, 1966). However, in spite of the numerous reports on cholesterol exchange from lipoproteins (Hagerman and Gould, 1951; Murphy, 1962; Bell et al., 1972; Dayton and Hashimoto, 1966, 1970; Newman and Zilversmit, 1962, 1966), little attempt has been made to relate the structure of membranes and lipoproteins to this phenomenon (Bruckdorfer and Green, 1967). In a previous communication from this laboratory, we reported the influence of temperature and metabolic inhibitors on cholesterol exchange between swine serum lipoproteins and erythrocytes (Bell et al., 1972). A continuation of those studies is presented here. Cholesterol transfer between isolated erythrocyte membranes, serum lipoproteins, and aorta was examined in the presence of dimethyl s&oxide, a compound considered to weaken hydrophobic bonding between lipid molecules 1 Research

Scholar

of the Canadian

Heart

Foundation. 293

1973 by Academic Press, Inc. reproduction in any form reserved.

BELL

294

(Bruckdorfer and Green, 1967), and in the presence of formalin, a protein fixative. The results are discussed in the context of current concepts of the structure of membranes and lipoproteins. In these studies, the observed movement of cholesterol between the various moieties examined has been described as cholesterol transfer. Although the movement of cholesterol in these experiments is probably not unlike that observed by ourselves (Bell et al., 1972) and others (Hagerman and Gould, 1951; Dayton and Hashimoto, 1966; Newman and Zilversmit, 1966) and attributed to cholesterol exchange, the broader term transfer is preferable since it expresses movement of cholesterol without excluding the possibility that some net movement of cholesterol occurred, particularly in incubations of aortas with serum lipoproteins. MATERIALS Animals

and Labeling

AND

METHODS

Technique

Serum and erythrocytes were obtained from fasting (18 hr) blood of 8- to 12wk-old Yorkshire pigs maintained on a commercial chow (Purina Hog Chow, Ralston-Purina Co., Canada). Serum lipoproteins and erythrocytes were labeled in viva by an intravenous injection of 23 n&i [3H]-cholesterol (generally labeled, Schwarz Bio-Research, Orangeburg, NY). Cholesterol to be injected was dissolved in ethanol, mixed with Tween 20, and diluted with 0.9% saline in the volume ratios of 1: 1:30, respectively. Purity of the [3H]-cholesterol was > 99% as determined by thin-layer chromatography on silica gel G in a solvent system consisting of n-hexane, diethyl ether, and glacial acetic acid ( 146: 50:4, v/v/v). Labeled serum and erythrocytes were obtained from blood drawn via a jugular cannula 24 hr after isotope injection. In uivo labeling for 24 hr resulted in equilibration of specific activities between serum unesterified and esterified cholesterol and erythrocyte unesterified cholesterol; cholesterol specific activities were l,OOO,OOO-1,500,OOO disint/min/mg in all experiments. The incorporation of isotopic cholesterol into serum lipoproteins was checked by polyacrylamide disc gel electrophoresis (Bell et al., 1972) and it was found that 96% of the label was associated with alpha and beta-lipoprotein fractions; the remainder being in the pre-beta lipoproteins. In the studies reported here, whole serum rather than the isolated lipoprotein fractions was used throughout. Aortas Aortas from 5 to 6-mo-old pigs (175-200 lb) were obtained from a local abbattoir approximately 10 min after death and placed in oxygenated Tyrode’s buffer, pH 7.4, (containing 1 g glucose/liter) at 10°C. Experiments with the tissues were begun 30 min after death. Erythrocytes

and Erythrocyte

Membranes

Erythrocytes were isolated from blood anticoagulated with disodium EDTA (1 mg/ml) and washed three times with 3 vol of calcium-free Tyrode’s solution (pH 7.4) containing 1 g glucose/liter. Erythrocyte membranes were prepared by lysing the cells in 40 vol of 5 mA4 Tris-HCl at pH 8.0. The membranes were isolated by centrifugation at 20,OOOg

CHOLESTEROL

TRANSFER

295

for 15 min at 10°C. The membranes were resuspended and washed twice more with 5 mM Tris-HCl. Formalin Fixation Membranes, aortas, and serum lipoproteins were fixed in Tyrode’s solution pH 7.4, containing formalin. During fixation, unfixed (control) samples were kept in Tyrode’s solution without formalin but otherwise treated the same as fixed samples. Erythrocyte membranes isolated from 5 mM Tris-HCl were fixed in 10% formalin (3.7% formaldehyde solution) for various times as described in the tables and figure. After fixation, the membranes were centrifuged at 20,OOOgfor 15 min and washed three times with Tyrode’s solution to remove formalin. Aortas were opened longitudinally and the thoracic segment divided into two pieces 2 x 8 cm. One piece was fixed in 10% formalin and then thoroughly washed with Tyrode’s solution. The other piece served as a control. Precise fixation times are described in the tables and figure. Serum lipoproteins were fixed by mixing serum with 15% formalin in the volume ratio 1:2, respectively, After fixation for 2 hr, the serum was dialyzed for 24 hr against two &liter changes of Tyrode’s solution, pH 7.4 at 10°C. Incubations All incubations were performed at 37°C in capped flasks or vials in a shaking water bath. Aortas were incubated for l-3 hr in 65 ml of serum or formalin-treated serum diluted with oxygenated Tyrode’s buffer, pH 7.4, to give a final concentration of 33% serum. All other incubations were ‘carried out for l-3 hr in a total volume of 5.0 ml consisting of 1.5 ml of serum or formalin-fixed serum, 3.0 ml of Tyrode’s buffer and 0.5 ml of Tyrode’s solution containing the membrane from 0.5 ml (packed volume) of erythrocytes. After incubation, membranes were separated from the incubation mixtures by centrifugation at 20,OOOgfor 15 min at 10°C. Dimethyl sulfoxide (Fisher Scientific Co., Fairlawn, NJ) was dissolved in Tyrode’s buffer, pH 7.4, to give a final concentration of 0.8-1.2 M in the incubation mixtures. Analyses Cholesterol was extracted from serum with 20 vol of boiling acetone-ethanol ( 1: 1, v/v). Unesterified and esterifred cholesterol specific activities were determined on cholesterol isolated from the extracts by thin-layer chromatography on silica gel G plates in a solvent system consisting of n-hexane, diethyl ether, and glacial acetic acid ( 146:50:4, v/v/v). The methods used for cholesterol elution, measurement, and radioactive assay following chromatography have been described in detail (Bell et al., 1970). Cholesterol was isolated from erythrocyte membranes by hexane extraction following digestion. The membranes were digested 30 min at 65°C in 15% alcoholic KOH; after digestion, the samples were diluted with 1 vol of water and extracted three times with 3 vol of hexane. Hexane extracts were evaporated

under Na and redissolved in isopropanol. One portion of the isopropanol solution was used for the automated determination of cholesterol (Block et al., 1966) and another portion for radioactive measurements by liquid scintillation counting (Bell et al., 1970). Three tissue discs of a combined surface area of 4.6 cm2 were punched from the pieces of aortas with a No. 8 cork borer after incubation. The adventitia-outer media was stripped from the discs, and discarded. This method of obtaining tissue for analysis minimized the contribution of cholesterol uptake via the adventitial surface or cut edges of the aorta. Cholesterol was recovered from the tissue discs by hexane extraction after digestion of the tissue with 15% alcoholic KOH as described above and radioactivity measured (Bell et al., 1970). Cholesterol radioactivity transferred from labeled membranes to serum lipoprotein was assayed by solubilizing ‘0.2 ml of the incubation supernatant in 1.0 ml of a commercial tissue solubilizer (NCS reagent, Amersham/ Searle, Des Plaines, Ill.; the ‘digests were then diluted with scintillation fluid and the radioactivity counted (Bell and Schwartz, 1971). Results obtained by this method were found to be as accurate as those ‘obtained for cholesterol radioactivity determined on samples in which cholesterol was isolated by solvent extraction and thin-layer chromatography. Calculation

of Cholesterol

Transfer

In membrane-serum incubations, [3H] -cholesterol radioactivity recovered in the initially unlabeled fraction of experimental incubations was compared to radioactivity (range 50,00O-S0,000 dpm) recovered in the same fraction of control incubations taken as 100%. This method of expression (relative percent transfer) permits comparisons of data from different experiments. In aorta-serum incubations, uptake of lipoprotein [3H]-cholesterol by aortas was measured in tissue discs punched from the arteries after incubation as described above. Radioactivity taken up per cm3 intimal surface (range 100400 dpm/cm2 suface) in experimental incubations was expressed relative to radioactivity taken up per cm? intimal surface in their respective control incubations taken as 100%. For the experiments shown in Fig. 1, transfer of [ 3H]-cholesterol from serum lipoproteins to aortas was calculated as ng of cholesterol transferred per cm” of intimal surface (rig/cm*). These calculations were made by dividing [3H]cholesterol radioactivity of the tissue by the initial specific activity of the serum unesterified cholesterol. The use of initial specific activities rather than mean cholesterol specific activities to estimate cholesterol transfer in these experiments is justified since changes in serum unesterified cholesterol specific activities were less than 1.5% even in incubations of up to 3 hr duration. In all these incubations, radioactivity. recovered in serum or aortas after incubation with labeled membranes or serum, respectively, must be considered to represent minimal values; the calculations do not take into account the recycling of labeled cholesterol between serum lipoproteins and the membranes or tissues or the changing specific activity of the labeled donor. Cholesterol speciiic activities did not equilibrate between the various components of these incubations. After a 3-hr incubation, aortic cholesterol specific activity attained only 0.4% that of labeled serum while serum unesterified cholesterol

CHOLESTEROL

TRANSFER

1 INCUBATION

FIG. 1. The terol transfer [sH]-cholesterol and absence experiments f sulfoxide; (A

2 TIME

297

3 (hours)

effect of formalin fixation and dimethyl sulfoxide on the kinetics of between serum lipoproteins and aorta. Serum lifioproteins labeled were incubated with either un6xed or formalin fixed aortas in of 1.2 M dimethyl &oxide. Each point represents the mean standard error of the mean. ( 0 ), Unfixed aorta; ( l ), unfixed aorta ), fixed aorta; ( A ), fixed aorta + dimethyl sulfoxide.

PHI-cholesin vi00 with the presence of 8 to 10 + dimethyl

specific activity attained only 15% that of labeled membrane cholesterol specific activity. RESULTS The effects of formalin fixation on transfer of unesterified cholesterol between serum lipoproteins and erythrocyte membranes, measured after 2 hr of incubation, are shown in Table I. [3H]-cholesterol of fixed erythrocyte membranes was available for transfer to the same extent as that from u&xed membranes. The duration of formalin treatment of membranes did not appear to have any marked effect on the availability of cholesterol for transfer. In addition, fixed serum lipoproteins were not significantly different from unfixed serum lipoproteins in ability to accept cholesterol transferred from membranes. In a similar experiment shown in Table II, [3H]-cholesterol-labeled membranes were incubated for 3 hr with untreated serum. The rate of increase of serum unesterified cholesterol specific activity was similar in incubations with fixed and unfixed membranes indicating the kinetics of transfer was unaffected by membrane fixation. Table III shows the effect of fixation on the transfer of unesterified [SH]-cholesterol, measured after a 1-hr incubation, between serum lipoproteins and aortas. Fixation of either the aorta or the serum lipoproteins did not prevent cholesterol transfer, but, in contrast to experiments with fixed membranes (Tables I and II), fixation of tissue with formalin significantly (P < 0.001) decreased transfer of cholesterol from serum lipoproteins by approximately 50%. This decrease was independent of aorta fixation time beyond 15 min and independent of the state

TABLE THE EFFECT OF FORM.ILIN FIXATION (UNESTERIFIED) FROM ERYTHROCYTS Membrane time

I

ON THE TRANSFER OF [3H]-C~~~~~~~~~~ hkMnn~Ni?s TO SERUM LIPOPROTEINS &Serum lipoproteins

fixation (min)” Unfixed

Fixed

0 5 20

100 f 95 f 105 f

0 (8) 3 (5)* 3 (5)*

Cholesterol

kansfer

(rel. 104 f -

yL)b 40 (4)*

60

101 f

8 (8)*

110 f

10 (4)*

* Erythrocyte membranes were labeled with [3H]-cholesterol in ai~o, fixed with formalin and incubated 2 hr with fixed or unfixed serum lipoproteins as described in the text. b Relative percent cholesterol transfer was measured, as described in the text; all values in the table are calculated relative to the value 100 f 0 shown for the incubation, of unfixed membranes with unfixed serum lipoproteins. o Values are means f standard error of the means. The number of experiments is shown in parentheses. * Not significantly different (P > 0.05) from unfixed serum lipoproteins (Student’s t-test).

of the labeled serum lipoproteins since there was no significant difference in transfer to the aorta from either fixed or unfixed serum lipoproteins. The influence of dimethyl sulfoxide on the transfer of unesterified cholesterol between serum lipoproteins and fixed and unfixed erythrocyte membranes or aortas is shown in Tables IV and V, respectively. At a concentration of 0.8-1.2 M in the incubation medium, dimethyl sulfoxide significantly (P < .OOl) increased cholesterol transfer from membranes to serum (Table IV) and from serum to aortas (Tables V); the magnitude of the increase was approximately 30 and 90%, respectively. This stimulation of cholesterol transfer was not affected by fixation of the membranes or aortas. Figure 1 shows the kinetics of cholesterol transfer between untreated serum lipoproteins and aortic tissue during 3 hr incubation. The rate of cholesterol transfer to fixed aortic tissue was significantly lower (P < 0.001) than the rate of cholesterol transfer to unfixed tissue after 1 hr of incubation, as in Table III, and after 2 and 3 hr of incubation (Fig. 1). TABLE THE

Membrane

Unfixed Fixed

II

TRANSFER OF [3H]-C~~~~~~~~~~ (UNESTERIFIED) ERYTHROCYTE h~EMBX4NES TO SERUM LIPOPROTEINS Serum

23,350 21,680

unesterified 1 hr & 100b ZJZ 40

cholesterol

32,540 35,320

specific 2 hr f f

250 2600

activity

FKOY

(disint/min/mg) 3hr 39,870 40,040

f f

1000 60

“Erythrocyte membranes were labeled with [aHI-cholesterol in viva as described in the text. Membranes, which were either unfixed or fixed for 60 min in formalin, were incubated untreated serum as described in the text. Serum unesterified cholesterol specific activity measured on extracts of aliquots of the incubation medium after I, 2, and 3 hr of incubation. b Values are the mean of duplicate incubations f the standard error of the mean.

with was

CHOLESTEROL TABLE THE

EFFECT

OF FORMALIN (UNESTERIFIED)

299

TRANSFER III

FIXATION ON THE TRANSFER FROM SERUM LIPOPROTEINS

OF [3H]-C~~~~~~~~~~ TO AORTA

Serum lipoprotein&

Aorta fixations time (min)

Fixed

Unfixed

Cholesterol transfer (rel. %)c 105 f 6d (8)** 62 f 6 (9)* (8)* 57 f 7 (9)* 67zt7 43f5 (8)* 52 f 6 (8)*

0 15 30 60

100 f: 0 (8) 53 f 4 (lo)*

8 Aortas from 5- to 6mo-old pigs were fixed in formalin as described in the text. b Serum lipoproteins were labeled in viva with [3H]-cholesterol, fixed as described in the text, and incubated for 1 hr with aortas. 0 Relative percent cholesterol transfer was measured as described in the text; all values in the table are calculated relative to the value 100 f 0 shown for the incubation of unfixed aortic tissue with unfixed serum lipoproteins. d Values are the mean f standard error of the mean. The number of experiments is shown in parentheses. * Significantly different (P < 0.001) from values obtained with unfixed aorta (Student’s t test). ** Not significantly different (P > 0.05) from values obtained with unfixed aorta (Student’s t test).

Similarly, the stimulatory effect of dimethyl sulfoxide on cholesterol transfer between untreated serum lipoproteins and aortic tissue observed in Table V after 1 hr of incubation persisted during 2 ‘and 3 hr incubations (Fig. 1). The rate of cholesterol transfer to aortic tissue, either fixed or unfixed, was significantly (P < 0.001) greater at 1, 2, and 3 hr in the presence of dimethyl sulfoxide; the addition of the dimethyl suIfoxide to fixed or unfixed aortic tissue resulted in an TABLE THE EFFECT

IV

OF DIMETHYL SULFOXIDE ON THE TRANSFER OF [3H]-CHOLF:STEROL FROM ERYTHROCYTE MEMBRANES TO SERUM LIPOPROTEINS

Dimethyl

Membranes

Unfixed Fixed

sulfoxideb

+

Cholesterol transfer (rel. %)o 100 f 0 (8) 133 f 4d (8)* 100 f 0 (6) 132 f 2 (6)*

a Erythrocyte membranes were labeled in vivo with [3H]-cholesterol. The membranes, either unfixed or fixed for 60 min in formalin, were incubated for 2 hr with untreated serum lipoproteins. b Dimethyl sulfoxide was added to the incubation medium to a final concentration of 0.8 M. 0 Relative y. cholesterol transfer was measured as described in the text. Values in the table are calculated relative to the value 100 f 0 shown for the incubations with unfixed membranes in the absence of diiethyl sulfoxide. d Values are means f standard errors of the means. The number of experiments is shown in parentheses. * Significantly different (P < 0.001) from values obtained with unfixed membranes in the absence of diiethyl sulfoxide (Student’s t test).

BELL

300

TABLE

V

THY E:FFECT OF DIMETHYL SULFOXIDE ON THE TIMNSFKR OF [“HICHOLESTEROL FROM SERUM LIPOPROTEINS TO AOKT.I Dimethyl -

Aortas

Unfixed Fixed

sulfoxideb +

Cholesterol transfer (rel. 7;)” loo f 0 (10) 186 f 6d (lo)* 57f5 (6)* 112 f 7 (6)

* Aortas from 5- to 6-mo-old pigs, either fixed for 30 min in formalin or unfixed, were incubated for 1 hr with untreated serum lipoproteins labeled in viva with [3H-]cholesterol. b Dimethyl sulfoxide was added to the incubation medium to a final concentration of 0.8 M. 0 Relative y0 cholesterol transfer was measured as described in the text. Values in the table are calculated relative to the value 100 f 0 shown for the incubation of unfixed aortas in the absence of dimethyl sulfoxide. d Values are means f standard errors of the means. The number of experiments is shown in parentheses. * Significantly different (P < 0.001) from values obtained with unfixed aortas in the absence of dimethyl sulfoxide (Student’s t test).

doubling in the rate of transfer to the tissues at all time intervals examined ( Fig. 1).

approximate

DISCUSSION The effects of formalin fixation and dimethyl sulfoxide on cholesterol transfer between serum lipoproteins and erythrocyte membranes or aorta have been examined for the purpose of defining more clearly the factors influencing the process of cholesterol transfer. The precise nature of the alterations in protein structure induced by formaldehyde in the aorta, erythrocyte membranes, and serum lipoproteins in these experiments is not known. However, protein denaturation by formaldehyde results, in part, from the disruption of secondary structure of the proteins as a consequence of formaldehyde reactivity with amino groups of the proteins to form hydroxymethyl derivatives. The amino methyl01 groups thus formed modify the protein structure even further by forming methylene bridges with other functional groups in the protein such as phenol, indole, and imidiazole (Hayat, 1970; Fraenkel-Conrat et al., 1945). The fixation of erythrocyte membranes or serum lipoproteins with formalin does not prevent cholesterol transfer between these fractions, does not diminish the availability of cholesterol of either of these fractions for transfer (Tables I, II, and III), nor does it alter the kinetics of cholesterol transfer (Table II). From the data one may conclude that, under the conditions of these experiments, the transfer of cholesterol between membrane and lipoproteins is a process independent of the state (fixed or unfixed) of the proteins in these moieties; a conclusion compatible with current concepts for the structure of membranes and lipoproteins. It has been suggested (Glaser et al., 1970; Lenard and Singer, 1966; Vanderkooi and Sundralingam, 1970) that lipid and protein molecules of the membrane are

CHOLESTEROL

TRANSFER

301

organized in a mosaic pattern in which globular protein molecules are interspersed in a matrix of lipid. Fixation of the protein in such a model would likely have little effect on the availability of cholesterol for transfer, particularly since cholesterol of most membranes is thought to be bound to the hydrophobic positions of phospholipids rather than to proteins (Parpart and Ballentine, 1952; VanDeenen, 1965). Similarly, the proposed molecular structure of serum high density lipoproteins (HDL) and low density lipoproteins (LDL) based on the repeating “lipotide” unit (Day and Levy, 1969) ,allows for the presence of both lipid and protein at the surface of the molecules; an arrangement whereby lipid would be available for transfer despite protein fixation. As with membranes, lipoprotein cholesterol is also thought to be bound to apolar groups of phospholipids and not directly bound to proteins (Day
BELL

302

membranes and lipoproteins can be modified without affecting the process of cholesterol transfer and/or exchange between these entities. Although the nature of lipoprotein-membrane or membrane-membrane interactions which result in cholesterol transfer or exchange is still unknown, it has been proposed that cholesterol exchange involves the formation of a collision complex (Gurd, 1960), held together by van der Waals-London dispersion forces, in which lipid containing sites fuse for sufficient time to allow the diffusion of cholesterol (Bruckdorfer and Green, 1967). Exchange or transfer of lipid molecules between lipoproteins and membranes must by-pass direct contact with an aqueous environment; the coalescence of lipid “sites” would achieve that condition. This being the case, any agent of factor (s ) influencing or modifying lipidlipid interactions or the physical state of the lipids (e.g., fluidity) may be expected to modify exchange or transfer rates; dimethyl sulfoxide is such an agent and temperature one of the factors (Bell et al., 1972; Chapman, 1969). The biological significance of cholesterol exchange is unknown but of interest, particularly, to those workers in the field of arterial metabolism and atherosclerosis. Most studies of arterial cholesterol flux have been hampered by the overwhelming proportion of “flux” attributable to exchange without net movement of the sterol (Dayton and Hashimoto, 1966, 1970). Although cholesterol exchange to the artery has been regarded as unimportant, the possibility exists that cholesterol exchange to arteries may be converted to net transfer of cholesterol under certain conditions. ACKNOWLEDGMENTS This researchwassupportedby the CanadianHeart Foundationand the Medical Research Councilof CanadaGrant MT-3067 (held by Dr. C. J. Schwartz), and the Wilson Trust Fund. ADAMS, C. W. M., &RAG, S. V., MORGAX, R. S., and ORTON, C. C. (1968). Dissociationof EssexPackersfor supplyingaortic tissue. REFERENCES C. W. M., VIRAG, S. V., 3H-cholesteroland 1251-labeled

MORGAN, R. S., and ORTON, C. C. (1968). Dissociationof plasma protein influx in normal atheromatous rabbit aorta: A quantitative histochemical study. J. Atheroscler. Res. 8, 679-696. BELL, F. P., LOFLAND, H. B., and STOKES, N. A. (1970). Cholesterol flux in vitro in aortas of cholesterol-fed and non-cholesterol-fed pigeons. AtheroscZero.k 11, 235-246. BELL, F. P., and SCHWARTZ, C. J. (1971). Exchangeability of cholesterol between swine serum lipoproteins and erythrocytes, in vitro, Biochem. Biophys. Acta 231, 553-557. BELL, F. P., SOMER, J. B., CRAIG, I. H., and SCHWARTZ, C. J. (1972). Membrane-lipid exchange: Exchange of cholesterol between porcine serum lipoproteins and erythrocytes. Pathology 4, 205-214. BLOCK, W. D., JARRETT, K. J., and LEVINE, J. B. (1966). An improved automated determination of serum total cholesterol with a single color reagent. Clin. Chem. 12, 681-689. BRUCKLWRFER, K. R., and GREEN, C. ( 1967). The exchange of unesterified cholesterol between human low-density lipoproteins and rat erythrocyte ghosts. Biochem. J. 104, 270-277. CHAPMAN, D. (1969). Physical studies of lipid-lipid and lipid-protein interactions. Lipids 4,251-260. DAYTON, S., and HASHIMOTO, S. (1966). Movement of labeled cholesterol between plasma lipoprotein and normal arterial wall across the initimal surface. Circ. Res. 19, 1041-1049. DAYTON, S., and HASHIMOTO, S. (1970). Recent advances in molecular pathology: A review. Cholesterol flux and metabolism in arterial tissue and in atheromata. Exp. Mol. Pathol. ADAMS,

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