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A Method for the Increased Extraction of Cholesterol from Human Red Blood Cells by Modified Plasma Lipoprotein Melvin H. Gottlieb
a
a
Laboratory of Physical Biology , National Institute of Arthritis, Metabolism, and Digestive Diseases National Institutes of Health , Bethesda, Maryland, 20014 Published online: 05 Dec 2006.
To cite this article: Melvin H. Gottlieb (1978) A Method for the Increased Extraction of Cholesterol from Human Red Blood Cells by Modified Plasma Lipoprotein, Preparative Biochemistry, 8:6, 463-469, DOI: 10.1080/00327487808061663 To link to this article: http://dx.doi.org/10.1080/00327487808061663
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PREPARATIVE BIOCHEMISTRY, 8 ( 6 ) , 463-469
(1978)
A METHOD FOR THE INCREASED EXTRACTION OF CHOLESTEROL FROM HUMAN
RED BLOOD CELLS BY MODIFIED PLASMA LIPOPROTEIN
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Melvin H. G o t t l i e b L a b o r a t o r y of P h y s i c a l Biology N a t i o n a l I n s t i t u t e of A r t h r i t i s , Metabolism, and D i g e s t i v e Diseases N a t i o n a l I n s t i t u t e s of H e a l t h Bethesda, Maryland 20014 ABSTRACT The i n v i t r o e x t r a c t i o n o f c h o l e s t e r o l from e r y t h r o c y t e s by plasma l i p o p r o t e i n s of reduced c h o l e s t e r o l c o n t e n t would b e expected t o be f r e e o f c h o l e s t e r o l - u n r e l a t e d t h e c e l l membrane.
a l t e r a t i o n s of
The e a r l i e r a p p l i c a t i o n of t h i s method u t i l i -
zed whole blood plasma i n which t h e major p a r t of t h e l i p o p r o t e i n c h o l e s t e r o l w a s e s t e r i f i e d by t h e plasma enzyme l e c i t h i n - c h o l e s t e r o l a c y l t r a n s f e r a s e (LCAT) i n a p r e l i m i n a r y i n c u b a t i o n .
Be-
c a u s e o f t h e c h o l e s t e r o l remaining u n e s t e r i f i e d i n t h e plasma, o n l y 35% of t h e c e l l c h o l e s t e r o l could b e removed. r e p o r t e d h e r e u s e s HDL3,
The method
a plasma l i p o p r o t e i n which is t h e pre-
f e r r e d s u b s t r a t e f o r LCAT, i n s t e a d of whole plasma f o r t h e extraction,
M u l t i p l e e x t r a c t i o n s w i t h LCAT t r e a t e d HDL3 r e s u l t e d
i n t h e removal of up t o 77% of t h e e r y t h r o c y t e c h o l e s t e r o l w i t h o n l y minor hemolysis.
463 Copyright 0 1979 by Marcel Dekker, Inc. All kights Reserved. Neither this work nor any part may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, microfilming, and recording, or by any information storage and retrieval system, without permission in writing from the publisher.
GOTTLIEB
464 INTRODUCTION
The in vitro removal of cholesterol from erythrocytes provides
.
1 a means of studying its role in the functions of the cell membrane
Two methods have been used (besides ether extraction2 which seems a priori inadmissably drastic):
(a) extraction with phospholipid
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liposomes’, and (b) extraction with blood plasma which, in a preliminary incubation, has had the major part of its lipoprotein cholesterol esterified through the action of the plasma enzyme LCAT 4y5y6. The incubated plasma method is probably free of cholesterol-unrelated alterations in the cell membrane since the movement of cholesterol between erythrocytes and lipoproteins is a normal in vivo 8 In contrast , phospholipid liposomes add phospholipid7, remove protein ,
3 and cause hemolysis
.
Also, cholesterol removal by liposomes produces
+ changes in K -ion kinetics which removed by incubated plasma9
do not occur when cholesterol is
.
The major shortcoming of the incubated plasma method is that only about 35% of the cell cholesterol can be removed6, because of equilibration of the cells with the cholesterol remaining un-
esterified in the plasma lipoproteinslO. This present report gives a modification of the incubated plasma method by which up
Abbreviations: LCAT, lecithin-cholesterol acyl transferase; HDL2, HDL3, plasma lipoproteins with densities in the ranges 1.064-1.125 and 1.125-1.21, respectively; cp-HDL2, cp-HDL3, the respective cholesterol-poor lipoproteins; PBS, phosphate buffered saline (.15 M NaC1, .002 M NaP04, .01% Na2EDTA, pH 7.4).
EXTRACTION OF CHOLESTEROL
465
to 77% of erythrocyte cholesterol has been removed with only minor (
1.125 fraction, which contains HDL
3
and
LCAT along with other plasma proteins, was then dialyzed against PBS and (after addition of antibiotic) was incubated at 37'C
24 hrs.
for
The resulting cp-HDL3 was isolated by ultracentrifugal
flotation at p
=
1.21, washed at
bumin, and dialyzed against PBS.
p =
1.21 to remove residual al-
Collection and washing of cells,
and determination of their cholesterol content, were as described6; unesterified lipoprotein cholesterol was determined with cholesterol
.
13 oxidase
RESULTS AND DISCUSSION Figure 1 shows the percentage of cell cholesterol extracted by LCAT treated HDL3 as a function of time, where extractant solution was replaced at each data point.
Hemolysis (as determined
from the 540 nm absorption of the extractant supernatant) was
.5% during the first, second and fourth extractions and 2% during the longer third extraction; this hemolysis is similar to that occurring in control experiments, in the absence of HDL3.
In
other, similar experiments 61%, 68% and 70% of cell cholesterol
GOTTLIEB
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466
0
10
20
3 l
EXTRACTION TIME Ihoursl
FIGURE 1 Percentage of cholesterol removed from erythrocytes after extraction by cholesterol-poor HDL3, with extractant solution replaced at each data point. 0.2 ml erythrocytes extracted with 1 5 ml portions of cp-HDLg at 3 7 ° C in PBS containing also .5% glucose, .l% adenosine and 1% Garamycin antibiotic (Schering). Lipoprotein solution concentration was 1 mg proteinlml as determined by the Lowryls method. HDL3 originally contained .065 mg cholesterollmg protein; after incubation the cp-HDL3 contained .009 mg cholesterol/mg protein.
was extracted. Greater extractions can be obtained with larger excesses of cholesterol-poor (cp) HDL3; with three times the ratio of cp-HDL3 to cells used in Fig. 1, used in each of three extrac-
.
14
tions, 77% of the cholesterol was extracted
The use of multiple extractions requires some comment. Assuming that the cholesterol binding capacity of the cp-HDL
3 is
equal to the amount of cholesterol esterified during the LCAT incubation, the quantity of cp-HDL3 used in a single extraction in the experiment of Fig. 1 would be able to bind three times the cholesterol content of the cells.
Thus if distribution equi-
librium were attained (and taking into account the residual cholesterol of the cp-HDLg), 70% of the cell cholesterol would be
EXTRACTION OF CHOLESTEROL extracted.
467
However, i n a s i n g l e 22 hour e x t r a c t i o n under condi-
t i o n s s i m i l a r t o t h a t of Fig. 1, o n l y 45% of t h e e r y t h r o c y t e chol e s t e r o l was removed.
This low s i n g l e e x t r a c t i o n i n d i c a t e s t h a t
t h e c h o l e s t e r o l b i n d i n g c a p a c i t y of t h e cp-HDL3 i s smaller t h a n t h e c h o l e s t e r o l c o n t e n t of u n t r e a t e d HDL3.
T h i s may b e a r e s u l t
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of t h e g r e a t e r c h o l e s t e r y l ester c o n t e n t of t h e cp-HDL I n c u b a t e d HDL
2
w a s a l s o examined as e x t r a c t a n t .
i n c u b a t i o n similar t o t h a t d e s c r i b e d f o r HDL t e r o l was esterified.
3’
3’ On LCAT
67% of t h e choles-
However, t h i s cp-HDL2 e x t r a c t e d o n l y 1 2 %
of t h e e r y t h r o c y t e c h o l e s t e r o l i n a s i n g l e 2 2 hour e x t r a c t i o n . I n p a r a l l e l experiments w i t h HDL3 from t h e same blood s o u r c e , 83% of t h e c h o l e s t e r o l w a s e s t e r i f i e d and 49% of t h e c h o l e s t e r o l
w a s removed i n t h e s i n g l e e x t r a c t i o n . With g r e a t e r cp-HDL
3
t o e r y t h r o c y t e r a t i o s , p r o b a b l y more
t h a n 77% of t h e e r y t h r o c y t e c h o l e s t e r o l c o u l d be removed.
The
p r o c e d u r e s d e s c r i b e d should a l s o be a p p l i c a b l e t o p l a t e l e t s and lymphocytes, which a l s o c o n t a i n exchangeable c h o l e s t e r o l
16,17
While t h e a p p l i c a t i o n of t h i s method t o t h e s t u d y of c h o l e s t e r o l -
d e p e n d e n t , p h y s i c a l p r o p e r t i e s of t h e c e l l membrane, s u c h as v i s c o s i t y 1 7 , would b e of i n t e r e s t , t h e p o t e n t i a l l y most u s e f u l app l i c a t i o n of e x t e n s i v e l y c h o l e s t e r o l - d e p l e t e d c e l l s i s i n r e g a r d t o t h e enzymatic and t r a n s p o r t p r o c e s s e s i n which a chemical s p e c i f i c i t y f o r c h o l e s t e r o l i s involved
.
18
GOTTLIEB
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