© 1999 American Heart Association, Inc.
Atherosclerosis and Lipoproteins |
Role of Plasmalogens in the Enhanced Resistance of LDL to Copper-Induced Oxidation After LDL Apheresis
From the Physiologisches Institut der Universität München (D.H., B.E.) and the Institut für Klinische Chemie, Klinikum Grosshadern, Universität München (J.T.), Munich, and the Institut für Gerontologie, Universität Erlangen-Nürnberg (T.B.), Nürnberg, Germany.
Correspondence to Dr Bernd Engelmann, Physiologisches Institut der Universität München, Schillershr. 44, D-80336, Munich, Germany. E-mail Bernd.Engelmann{at}physiol.med.uni-muenchen.de
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Abstract |
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Abstract—Extracorporeal reduction of plasma low density lipoproteins (LDLs) by LDL apheresis was shown to attenuate the proatherogenic influences of LDL, such as impairment of vasodilation and increased monocyte adhesion to the endothelium. In 16 patients with familial hypercholesterolemia, we analyzed whether LDL apheresis by the heparin precipitation procedure affected the oxidative resistance of LDL. Plasma LDL cholesterol concentrations were reduced by 65% after the apheresis. The lag time of copper-mediated LDL oxidation was increased from 103 to 117 minutes (P<0.0005). The LDL contents of
-tocopherol and ß-carotene, as well as the ratio of
monounsaturated to polyunsaturated fatty acids in
LDL, were not altered. However, the LDL apheresis induced a 15%
increase in the LDL contents of plasmalogen phospholipids
(P<0.0005), a class of ether phospholipids that were
recently shown to prevent lipid oxidation. The phosphatidylcholine (PC)
to lysoPC ratio was elevated by 16% after the apheresis
(P<0.0005). The percent increase in LDL plasmalogen
phospholipids showed a close association with the increased lag time
after apheresis (P<0.0005). The LDL plasmalogen
contents of the blood samples from patients and from normolipidemic
donors were also positively related to the lag time
(P<0.005). In vitro loading of LDL with plasmalogen
phospholipids resulted in a prolongation of the lag time and an
increase in the PC/lysoPC ratio. In conclusion, the rapid rise in LDL
contents of plasmalogen phospholipids most probably causes the increase
in lag time after LDL apheresis. Plasmalogens appear to play an
important role in the oxidation resistance of LDL in vivo.
Key Words: LDL apheresis • plasmalogen phospholipids • lag times • LDL oxidation • lysophosphatidylcholine
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Introduction |
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The hypothesis that oxidized LDLs play a relevant role in the development and maintenance of atherosclerosis1 2 is based on considerable experimental evidence. In vitro–oxidized LDL reproduces many features characteristic of atherosclerotic vascular damage, such as increased adhesion of monocytes to the endothelium,3 transformation of macrophages into foam cells,4 and impaired endothelium-dependent vasorelaxation.5 Oxidized LDL was shown to be present in the atherosclerotic plaque.6 Furthermore, the LDL isolated from the plasma of patients with atherosclerosis exhibited an enhanced susceptibility toward oxidation in most studies.7 8 9
Extracorporeal LDL apheresis, the removal of the greater part of plasma LDL particles from the circulation, is currently used for the treatment of patients with severe hypercholesterolemia.10 The efficacy of this treatment in preventing fatal and nonfatal cardiovascular events has been demonstrated.11 Furthermore, LDL apheresis was shown to ameliorate the impaired endothelium-dependent vasorelaxation.12 The procedure also led to diminished expression of endothelial adhesion molecules.13 14 Thus, typical proatherogenic consequences of oxidized LDL are attenuated after therapeutic reduction of LDL. Although removal of the particles per se might partially account for these effects, changes in the oxidizability of the LDL induced by LDL apheresis could also be relevant.
In the present investigation, the oxidative resistance of
copper-oxidized LDL was found to be enhanced after apheresis, in
accordance with recent work.15 16 In view of these
findings, we attempted to gain insight into the mechanism responsible
for the increased oxidation resistance after the LDL apheresis. Several
chemical factors are known to influence the susceptibility of LDL
toward copper-mediated oxidation. These include
-tocopherol,17 18 19 the degree of
unsaturation of the LDL-based fatty acids,20
ß-carotene,21 ubiquinol-10,22 23 and
plasmalogen phospholipids.24 25 The results indicated that
the enhanced oxidation resistance after LDL apheresis was specifically
related to an increase in LDL plasmalogens.
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Methods |
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Subjects
Sixteen patients with familial hypercholesterolemia (FH; 10 men and 6 women; mean±SD age 49±6 years; range 42 to 61 years) who had already been treated biweekly by LDL apheresis for at least 14 months were included in the study. All patients had angiographically assessed coronary heart disease and were concomitantly treated with hydroxymethylglutaryl CoA-reductase inhibitors and aspirin. Six of the patients (5 men, 1 woman) were concomitantly supplemented with vitamin E (400 IU/d). The supplementation had been started at least 8 weeks before the study. In some experiments, the LDL particles from 10 normolipidemic donors (8 men and 2 women; mean age 49±10 years; range 39 to 64 years) were also analyzed. Fasting blood samples were drawn into EDTA-coated tubes before or after apheresis, or, in some experiments, 48 hours after apheresis. The LDLs were isolated by ultracentrifugation or heparin precipitation (as indicated), the particles being stored for <30 minutes at 4°C before the start of the analyses. Informed consent was obtained from all patients and volunteers. The studies were carried out according to the principles of the Declaration of Helsinki.
Lipoprotein Analyses
Plasma cholesterol and
triglycerides were measured using enzymatic test kits from
Boehringer Mannheim. Plasma LDL cholesterol
was quantified by a direct precipitation procedure (Immuno). HDL
cholesterol was determined enzymatically
(Boehringer Mannheim) after initial precipitation of apo
B–containing lipoproteins with
phosphotungstate/MgCl2.
Procedure of the LDL Apheresis
LDL apheresis was performed using the HELP procedure
(Heparin-induced Extracorporeal LDL Precipitation; Plasmat-Secura, B.
Braun).26 In brief, plasma obtained by filtration of whole
blood (70 mL/min) through a plasma separator was continuously mixed
with an acetate buffer containing heparin. At the resulting pH of 5.12,
LDL, lipoprotein(a), and fibrinogen were precipitated. After removal of
the precipitated heparin complex by filtration, excess heparin was
adsorbed by an anion-exchange column, and the clear, plasma filtrate
was subjected to bicarbonate dialysis to restore plasma volume as well
as the physiological pH. The plasma thus obtained
was returned together with blood cells to the patient. During a single
HELP apheresis, 2.5 to 3 L of plasma was treated. The duration of the
treatment was 2 to 3 hours.
LDL Susceptibility to Oxidation (Lag Time) and Analysis
of TBARS
LDL was prepared within 10 hours after blood collection by
sequential ultracentrifugation. Blood was drawn into
tubes containing EDTA (1 mg/mL). Plasma was separated from the blood
cells by a 10-minute centrifugation. The plasma with a
density of 1.02 g/mL was centrifuged for 4 hours at
340 000g in polycarbonate tubes in a TL-100
ultracentrifuge with a TLA-100.1 rotor (Beckman Instruments).
After recovery, the infranatant was adjusted to a density of 1.063 g/mL
with NaCl and centrifuged for 4 hours at 340 000g.
The supernatant solution containing the LDL was freed from the EDTA by
passage through desalting columns (Econo Pac IODG, Bio-Rad
Laboratories). The particles were resuspended in
O2-saturated PBS (10 mmol/L, pH 7.4) at a
concentration of 0.26 mmol cholesterol per liter.
CuCl2 (final concentration 1.67 nmol/mL) was
added from a freshly prepared stock solution. The suspension was
incubated at 30°C, and the absorbance at 234 nm was monitored at
5-minute intervals for 4 hours to follow the formation of conjugated
dienes. The lag time was determined as the intercept of the baseline
and the slope of the absorbance curve during the propagation
phase.9
Thiobarbituric acid–reactive substances (TBARS) were determined according to Wallin et al,27 with some modifications. In brief, to tubes containing 200 µL of 50% trichloroacetic acid, 75 µL of 1.3% TBA (in 0.3% NaOH, wt/vol) was added. The tubes were heated for 60 minutes at 90°C and then cooled in ice water. After centrifugation, 200 µL of the supernatant was transferred to a 96-well plate, and the differences between the absorbances at 530 nm and those at 630 nm were determined. The values thus obtained were compared with those of a standard curve of malondialdehyde equivalents, generated by hydrolysis of 1,1,3,3'-tetraethoxypropane.
LDL Contents of -Tocopherol and
ß-Carotene
LDL was isolated from EDTA-plasma by precipitation with
heparin-acetate buffer (0.3 mol/L sodium acetate, 100 IU/mL sodium
heparinate, pH 4.85) and resuspended in a Tris buffer containing
(in mmol/L) 154 NaCl, 3 NaN3, and 1 EDTA and
10 g/L albumin (pH 7.4). The -tocopherol
contents were measured fluorometrically by high-performance
liquid chromatography in hexane extracts of the LDL
suspensions as described previously.9 28 When the LDL
particles were isolated by ultracentrifugation, the LDL
contents of -tocopherol agreed within 5.3±1.4% with
those determined in particles prepared by precipitation with the
heparin-acetate buffer. The contents of ß-carotene were determined in
the same extracts by separation using reversed-phase
high-performance liquid chromatography and
subsequent spectrophotometric detection at 450 nm (retention time=15.8
minutes) according to procedures described in Reference 2929 .
LDL Contents of Plasmalogen and Ester
Phospholipids
LDL was recovered by precipitation with heparin-acetate buffer
as described above. LDL lipids were extracted according to Bligh and
Dyer30 by using chloroform containing 50 mg/L BHT.
Phospholipids were separated by 2-dimensional thin-layer
chromatography on DC 60 plates (Merck) by using the
solvents proposed by Broekhuyse.31 In some of the samples,
the percentages of the major LDL-associated phospholipids
(phosphatidylcholine [PC], sphingomyelin, lysoPC,
phosphatidylethanolamine [PE], and phosphatidylinositol) were
subsequently determined by phosphate analysis.32
In other samples, the spots corresponding to PC and PE were scraped
from the plate and the phospholipids eluted by addition of
chloroform/methanol (1:4, vol/vol). The extraction was repeated twice.
The phospholipids were applied to small thin-layer
chromatography plates (10x10 cm), which were exposed
for 3 minutes to HCl fumes (37% in water). After being dried, the
plates were developed in chloroform/methanol/acetic acid/water
(90:40:12:2, vol/vol/vol/vol). The phosphate contents of the spots
corresponding to lysoPC and lysoPE, which reflected the amounts of
plasmenylcholine and plasmenylethanolamine, respectively, were
measured.32 When 2 LDL samples obtained at the same time
from the same donor were analyzed separately from the start of
the analysis, the determinations of the plasmalogen
phospholipids agreed within 2.8±1.3%.
Fast Protein Liquid Chromatography
Gel filtration analysis of plasma was performed by means
of a Superose 6 column (Pharmacia). Plasma (100 µL) was injected onto
the column and eluted with a buffer containing (in mmol/L) 150
NaCl, 10 Na2HPO4, and 0.1
EDTA (pH 7.5) at a flow rate of 50 µL/min. Forty fractions of 50 µL
were collected: fractions 15 through 19, VLDL and chylomicrons;
fractions 20 through 26, IDL and LDL; and fractions 27 through 33, HDL.
Cholesterol and triglyceride determinations
were performed as described above.
Determinations of Fatty Acids
After precipitation of the LDL with the heparin-acetate buffer
and extraction of LDL lipids (as per Reference 3030 with chloroform
containing 50 mg/L BHT), fatty acid methyl esters were generated by
addition of BF3-methanol.33
Subsequently, the fatty acid methyl esters were separated using a gas
chromatograph (Hewlett-Packard HP-5880 A) equipped with a flame
ionization detector.
In Vitro Loading of LDL With Plasmalogen Phospholipids and
-Tocopherol
Fresh venous blood from normolipidemic donors was drawn into
tubes containing EDTA, and plasma was recovered by
centrifugation. For the in vitro loading of LDL with
brain plasmenylethanolamine (isolated as described previously in
Reference 3434 ), small, unilamellar vesicles were prepared. Aliquots
(0.4, 0.7, and 1 µmol) of either brain plasmenylethanolamine or
diacyl PE (16:0/18:2 PE) were dissolved together with 1.2, 2.1, and
3 µmol, respectively, of egg PC in 100 µL of ethanol ("low,
medium, and high enrichment"; cf Table 3
). The solutions were
added very slowly with stirring to 10 mL of plasma, and the suspensions
were incubated at 37°C under argon for 6 hours. The amount of
plasmenylethanolamine and diacyl PE additionally present in the
particles after this incubation period was comparable. Subsequently,
LDL was prepared by ultracentrifugation.35
The amount of LDL-associated protein was determined by using the
Bradford procedure.36 Before the start of the oxidation
with copper or 2,2'-azobis-2-amidinopropane hydrochloride
(AAPH), LDL was dialyzed at 4°C under argon against PBS buffer for 12
hours.
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Statistics
Statistical differences induced by the LDL apheresis were
evaluated by repeated-measures ANOVA. Where appropriate, the
Wilcoxon signed-rank test was employed. In other cases, the
differences were analyzed by 1-way ANOVA (comparisons between
patients with and without vitamin E supplementation; effects of in
vitro loading of LDL with plasmalogens). All values are given as
mean±SD.
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Results |
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Increased Lag Time After Apheresis
After a single LDL apheresis, total plasma and LDL cholesterol contents were reduced by 47% and 65%, respectively, in the 16 patients with FH (Table 1
). Plasma HDL cholesterol
contents remained unaffected by the procedure. The lag time of
copper-mediated conjugated-diene formation was determined in LDL
particles isolated before and after the apheresis. In 15 of the 16
patients analyzed, the lag time was prolonged after the
apheresis, while in 1 patient it was unchanged (Figure 1, upper panel). The mean lag time value
as determined before apheresis was 52% higher in the patients
supplemented with vitamin E compared with that in patients without
vitamin E supplementation (lower panel of Figure 1). After the
apheresis, the lag time was increased by 16% and 15% in patients with
and without vitamin E supplementation, respectively (Figure 1, lower panel). When all 16 patients were included in the
analysis, the median of the lag time values was found to be
increased by 14% (from 103.1 before to 117.2 minutes after,
P<0.0005). The LDL particles isolated before and after the
procedure exhibited no change in their electrophoretic mobility.
Furthermore, no TBARS could be detected in the lipoprotein samples at
the 2 time points. When the particles were analyzed by fast
protein liquid chromatography, the size of the LDL was
found to be unaltered after apheresis.
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patients without vitamin E supplementation;
• patients receiving vitamin E (400 IU/d). Lower panel, Mean values
of the data shown in the upper panel. pre indicates before apheresis;
post, after apheresis. Columns with horizontal bars, patients without
vitamin E supplementation; columns with vertical bars, patients
receiving vitamin E. *P<0.05,
**P<0.0005 vs pre.Two days after the apheresis, the duration of the lag time reapproached the preapheresis values (98.5±12.3 minutes before versus 114.8±9.7 minutes after; P<0.05 after versus before; 48 hours after apheresis: 106.7±11.6 minutes; mean values of 6 patients with FH). In patients with FH treated by a different procedure of LDL apheresis (immunoabsorption with the use of apo B antibodies37 ), we also observed an increase in the lag time after a single apheresis (performed by the heparin precipitation procedure; not shown). The possibility was considered that the anticoagulation with heparin during the extracorporeal procedure might have contributed to the increased lag time after the apheresis. Accordingly, 5000 IU of heparin was given intravenously to 3 normolipidemic donors as well as to 3 patients with FH (without vitamin E supplementation). The lag time of the isolated LDL oxidized with copper (1.67 nmol/mL) was not altered by the heparin administration (normolipidemic individuals: 99.8±18.1 minutes before versus 100.7±12.7 minutes 1 hour after heparin; patients with FH: 82.5±12.3 minutes before versus 79.6±10.2 minutes after heparin).
Elevation of LDL Plasmalogen Phospholipids After Apheresis
The oxidative resistance of LDL is governed by several factors,
among which the LDL-associated antioxidants are of particular
importance.19 -Tocopherol is considered to
be 1 of the main LDL-based antioxidants. The -tocopherol
contents of the LDL from patients supplemented with vitamin E were
2.3-fold higher than those of the patients without vitamin E
supplementation (Table 2). The LDL
contents of -tocopherol were not altered by the
apheresis in the 2 groups of patients with FH (Table 2).
Furthermore, the LDL contents of ß-carotene as well as the ratio of
monounsaturated to polyunsaturated fatty acids of
the particles were also not significantly modified.
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In vitro oxidation of LDL also induces changes in LDL phospholipids, in
particular a reduction in LDL PC and a concomitant increase of lysoPC,
its degradation product.38 39 The PC/lysoPC ratio of
the particles was increased by 15% (patients without vitamin E
supplementation) and 16% (with vitamin E supplementation) subsequent
to the procedure (Table 2
). After inclusion of all 16 patients
into the analysis, the mean PC/lysoPC ratio was found to be
elevated by 16% (from 20.9±3.3 before to 24.2±3.9 after apheresis,
P<0.0001).
Recent work indicates that plasmalogen phospholipids may play a
relevant role in the oxidation resistance of LDL in
vitro.24 25 The preapheresis values for the LDL-associated
plasmalogens were 2.6-fold higher in patients with vitamin E
supplementation compared with patients not supplemented with vitamin E
(Table 2). After LDL apheresis, the plasmalogen contents were
increased by 14% and 13% in patients without and with vitamin E
supplementation, respectively (Table 2). When all patients were
included in the analysis, the median of the LDL plasmalogen
contents was elevated by 15% after the apheresis (from 20.9 before to
24.0 µmol/mmol of LDL cholesterol after apheresis,
P<0.005).
Relations Between Lag Time and LDL Plasmalogen Contents
In view of these results, we analyzed whether the increase
in LDL plasmalogens after apheresis was related to the prolongation in
the lag time. As shown in Figure 2, there
was a positive correlation between the percent elevation of the LDL
plasmalogen contents induced by the apheresis and the degree of
prolongation of the lag time. The positive associations were also noted
when the 2 groups of patients without and with vitamin E
supplementation were analyzed separately (without vitamin E:
r=0.66, P<0.05; with vitamin E:
r=0.92, P<0.005). The extent of increase in the
lag time after apheresis was not related to the percent elevation of
the PC/lysoPC ratio (r=0.06, NS).
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The LDL contents of -tocopherol and of plasmalogen
phospholipids as measured before apheresis in patients with FH and
those of normolipidemic donors were compared with their lag time
values. In the total group of 20 individuals thus evaluated, the LDL
-tocopherol contents exhibited a weak, positive
association with the lag time values (Figure 3, upper panel). The plasmalogen contents
showed a stronger relation to the lag time values in the same
individuals (Figure 3, lower panel). The PC/lysoPC ratios as
determined at the same time point in the same donors did not exhibit a
significant relation to the duration of the lag time
(r=0.18). When the preapheresis values of all patients with
FH and of the normolipidemic individuals were compared with the
duration of the lag time, positive correlations were obtained
(-tocopherol versus lag time: r=0.79,
P<0.0001; plasmalogen phospholipids versus lag time:
r=0.83, P<0.0001; n=26).
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, normolipidemic donors.
In Vitro Enrichment of LDL With Plasmalogen Phospholipids
To evaluate whether changes in the LDL contents of the plasmalogen
phospholipids could be responsible for the variations in LDL oxidation
resistance, LDL particles were enriched in vitro with 3 different
quantities of plasmenylethanolamine and, as a control, with similar
amounts of diacyl PE (low, medium, and high enrichment). Thereby the
total plasmalogen contents (plasmenylcholine plus
plasmenylethanolamine) were increased by 17%, 31%, and 46% compared
with the respective diacyl PE-loaded particles (Table 3). The procedure did not alter the
-tocopherol contents of the particles (not shown).
The duration of the lag time as well as the changes in the PC/lysoPC
ratios induced by copper oxidation in the diacyl PE-enriched particles
were comparable to those observed in control LDL (without any
enrichment; Table 3). The lag time of conjugated-diene formation
in copper-oxidized particles was prolonged by 10%, 13%, and 26%,
respectively, in LDL particles in vitro-loaded with the 3 different
amounts of plasmenylethanolamine compared with the respective
lipoproteins enriched with diacyl PE (Table 3). The LDL
particles from 2 patients with FH (not receiving vitamin E) were in
vitro–loaded with either plasmenylethanolamine or diacyl PE under
conditions corresponding to the low plasmalogen enrichment of Table 3. The total LDL plasmalogen contents thus obtained were 21.1
(enrichment with diacyl PE) and 24.9 µmol/mmol of LDL
cholesterol (enrichment with plasmenylethanolamine).
Concomitantly, the lag time of LDL oxidized with copper (1.67 nmol/mL)
was increased by 11%, from 85.4±7.2 (diacyl PE loading) to 94.6±2.3
(plasmenylethanolamine loading; means of 4 determinations,
P<0.05).
After a 2- and 4-hour oxidation of diacyl PE-enriched LDL with copper,
the PC/lysoPC ratios of LDL were reduced by 31% to 38% and by 63% to
68%, respectively (Table 3). The copper-induced decrease of the
PC/lysoPC ratio was attenuated by 36% (2-hour incubation) and 20%
(4-hour incubation) in particles with low plasmalogen enrichment
compared with the respective particles loaded with diacyl PE. The same
plasmalogen-enriched LDL particles were also subjected to
oxidation with the peroxyl radical generator AAPH (2 µmol/mL). The
decrease in the PC/lysoPC ratio elicited by AAPH after 2 and 4 hours of
incubation was comparable to that induced by copper (Table 3).
The AAPH-mediated reductions of the ratio were lowered by 35% (2
hours) and by 23% (4 hours) as a consequence of the plasmalogen
enrichment (compared with the LDL loading with diacyl PE).
In LDLs with medium plasmalogen enrichment, the copper-mediated
reduction of the PC/lysoPC ratio was prevented by 28% (2-hour
incubation) and 32% (4-hour incubation) in relation to the respective
control particles containing additional diacyl PE. After enrichment
with the highest amount of plasmenylethanolamine, the oxidative
degradation of PC was reduced by 43% and 39% after 2 and 4 hours of
incubation of LDL with copper compared with the respective controls
(Table 3).
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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In the present investigation, we observed rapid changes in the oxidizability of the LDL particles after LDL apheresis in patients with FH. After the extracorporeal reduction of LDL, the resistance of LDL to copper-induced oxidation was enhanced, as indicated by a prolongation in the lag time of conjugated-diene formation. Concomitantly, the LDL contents of plasmalogen phospholipids were increased after apheresis, their elevation being most likely responsible for the strengthening of the oxidation resistance of the particles.
The reduced susceptibility of LDL toward copper oxidation observed in the present study after apheresis by the heparin precipitation method is in line with results of recent investigations performed with other procedures.15 16 Although relatively limited, the increase in lag time elicited by the apheresis might well be of physiopathologic relevance. Indeed, the previously observed differences in the lag time between healthy individuals and patients with coronary atherosclerosis and hyperlipidemia are mostly in a comparable percent range as those induced by the apheresis.7 8 9
The oxidation resistance of LDL was determined in the present study
by the use of a relatively high concentration of copper (1.67 nmol/mL).
Previous work shows that at this and higher concentrations of the metal
ion, the length of the lag time is inversely related to the copper
concentration.19 40 Although several findings indicate a
relevant role for copper as a major oxidant under in vivo conditions
(see, for example, References 41 through 4341 42 43 ), the importance of the
metal ion is still a matter of debate.44 Nevertheless, the
increase in LDL plasmalogen contents after LDL apheresis is expected to
also enhance the resistance of LDL to other oxidants. This can be
deduced from earlier results indicating a comparable reduction of
LDL-associated plasmalogens after copper- and peroxyl radical–mediated
oxidation.24 Furthermore, plasmalogens are able to protect
polyunsaturated fatty acids against oxidation by peroxyl
radicals.34 In addition, experimental increases in LDL
plasmalogen contents comparable to those elicited by the LDL apheresis
prevented the degradation of LDL PC induced by peroxyl radicals (Table 3).
To identify the factor(s) responsible for the augmented lag time after
apheresis, several possibilities were considered.
-Tocopherol is a major determinant of the oxidation
resistance of LDL.17 18 19 However, the LDL contents of
-tocopherol were unchanged after the procedure (Table 2). Also, the level of other factors potentially influencing the
susceptibility of LDL toward copper oxidation, such as the ratio of
monounsaturated to polyunsaturated fatty
acids22 or the ß-carotene contents,23 were
not significantly altered by the apheresis.
Plasmalogen phospholipids were recently shown to contribute to the oxidation resistance of LDL.24 25 Further work indicates that plasmalogens reduce the oxidation of lipids in cells,45 liposomes, and micelles,34 46 as elicited by various oxidants, including the metals copper and iron, peroxyl radicals, and photooxidation. Their inhibitory effects on lipid oxidation are apparently related to a direct reaction of the plasmalogen-specific enol-ether double bond with the oxidants. It was recently shown that 1 enol-ether double bond is able to scavenge 2 peroxyl radicals.34 In addition, the enol-ether appears to possess a high affinity toward interaction with copper ions, as determined by the use of nuclear magnetic resonance techniques in detergent micelles (D.H. et al, unpublished observations, 1999).
In the patients with FH analyzed in the present study, the
percent increase in the LDL contents of total plasmalogen phospholipids
(plasmenylethanolamine plus plasmenylcholine) exhibited a strong,
positive relation to the prolongation of the lag time (Figure 2). When the preapheresis values for the LDL plasmalogen
contents of the patients and those of normolipidemic donors were
compared with the duration of the lag time, a close positive
association between both parameters was again noted (Figure 3). The correlation between the -tocopherol
contents of LDL and the lag time was less pronounced in individuals not
supplemented with vitamin E but became stronger after inclusion of the
patients with vitamin E supplementation, in accordance with earlier
data.19 47 Future studies are needed to evaluate whether
the association between the plasmalogen contents and the oxidation
resistance of LDL is also evident in larger populations of individuals.
When LDL particles were loaded in vitro with plasmenylethanolamine
("low plasmalogen enrichment"; Table 3), comparable percent
changes in the lag time of copper-oxidized LDL were obtained as by the
increase in plasmalogen contents induced by the LDL apheresis. Thus,
the elevated LDL plasmalogen contents are most likely responsible for
the prolongation in the lag time values observed after LDL
apheresis.
The reasons for the elevation in LDL plasmalogen contents after the apheresis are unknown at present. Plasmalogens were shown to be rapidly exchanged between lipid donors and erythrocytes.48 The plasmalogen pool of red blood cells, which is >10 times higher than that of the LDL particles, is augmented after LDL apheresis.49 The enhanced delivery of erythrocyte-derived plasmalogen phospholipids to the LDL could contribute to the increase in LDL plasmalogen contents after apheresis. The steeper plasmalogen gradient between red blood cells and LDL induced by the rapid fall in LDL concentrations might favor such an enhancement of plasmalogen transfer.
LysoPC was previously reported to mediate the atherogenic effects of in
vitro–oxidized LDL, including the impairment of
endothelium-dependent vasodilation,50 the
enhanced adhesion of monocytes to the
endothelium,38 and the increased
expression of endothelial growth
factors.51 After LDL apheresis, the
endothelium-dependent vasodilation was found to be
ameliorated,12 52 53 and the adhesion of monocytes to the
endothelium was observed to be
diminished.13 14 On the basis of the results of the
present study, these earlier findings might be partially explained
by the diminished lysoPC contents of the particles (Table 2).
The reasons for the increased PC/lysoPC ratios after apheresis are
unknown. It is unlikely that the enhanced oxidative resistance of LDL
after the apheresis might play a prominent causal role, because there
was no correlation between this ratio and the duration of the lag time
(see Results). Therefore, other factors may be relevant, such as
alterations in the activities of enzymes known to remodel
LDL-associated PC (eg, lecithin:cholesterol
acyltransferase).
In summary, the close associations between the LDL contents of plasmalogen phospholipids and the duration of the lag time observed in the present investigation indicate that plasmalogens are of considerable relevance for the resistance of LDL to copper oxidation. The diminished oxidizability of the LDL after LDL apheresis is suggested to contribute to the beneficial effects of the procedure on endothelial dysfunction and the prevention of coronary artery disease.
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Acknowledgments |
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This study was supported by grants from the Wilhelm Sander-Stiftung and of the Friedrich-Baur-Stiftung to B.E. The skillful technical assistance of Gudrun Haas and Achim Brunner is gratefully acknowledged. Thanks are also due to Prof Dr R. Lorenz for the determinations of ß-carotene.
Received October 5, 1998; accepted March 11, 1999.
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References |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
1. Steinberg D. Conner memorial lecture: oxidative modifications of LDL and atherogenesis. Circulation. 1997;95:1062–1071.
2. Holvoet P, Collen D. Thrombosis and atherosclerosis. Curr Opin Lipidol. 1997;8:320–328.[Medline] [Order article via Infotrieve]
3.
Quinn MT, Parthasarathy S, Fong LG, Steinberg D.
Oxidatively modified lipoproteins: a potential role in recruitment and
retention of monocyte/macrophages during atherogenesis.
Proc Natl Acad Sci U S A.. 1987;84:2995–2998.
4.
Fogelman AM, Shechter J, Seager J, Hokom M, Child JS,
Edwards PA. Malondialdehyde alteration of low density lipoprotein leads
to cholesteryl ester accumulation in human
monocyte-macrophages. Proc Natl Acad Sci U S A.. 1980;77:2214–2218.
5.
Galle J, Bassenge R, Busse R. Oxidized low density
lipoproteins potentiate vasoconstrictions to various agonists by direct
interaction with vascular smooth muscle. Circ Res. 1990;66:1287–1293.
6.
Palinski W, Rosenfeld ME, Ylä-Herttuala S,
Gurtner GC, Socher SS, Butler SW, Parthasarathy S, Carew TE, Steinberg
D, Witztum JL. Low density lipoprotein undergoes oxidative modification
in vivo. Proc Natl Acad Sci U S A.. 1989;86:1372–1376.
7. Regnström J, Nilsson J, Per Tornvall CL, Hamsten A. Susceptibility to low-density lipoprotein oxidation and coronary atherosclerosis in man. Lancet. 1992;339:1183–1186.[Medline] [Order article via Infotrieve]
8. Chiu HC, Jeng JR, Shieh SM. Increased oxidizability of plasma low density lipoprotein from patients with coronary artery disease. Biochim Biophys Acta. 1994;1225:200–208.[Medline] [Order article via Infotrieve]
9.
Halevy D, Thiery J, Nagel D, Arnold S, Erdmann E,
Höfling B, Cremer P, Seidel D. Increased oxidation of LDL in
patients with coronary artery disease is independent from
dietary vitamins E and C. Arterioscler Thromb Vasc Biol. 1997;17:1432–1437.
10. Gordon BR, Saal SD. Current status of low density lipoprotein apheresis for the therapy of severe hyperlipidemia. Curr Opin Lipidol. 1996;7:381–384.[Medline] [Order article via Infotrieve]
11. Gordon BR, Kelsey SF, Dau PC, Gotto AM Jr, Graham K, Illingworth DR, Isaacsohn J, Jones PH, Leitman SF, Saal SD, Stern TN, Troendle A, Zwiener RJ. Long-term effects of low-density lipoprotein apheresis using an automated dextran sulfate cellulose adsorption system: Liposorber Study Group. Am J Cardiol.. 1998;81:407–411.[Medline] [Order article via Infotrieve]
12.
Tamai O, Matsuoka H, Itabe H, Wada Y, Kohno K, Imaizumi
T. Single LDL apheresis improves endothelium-dependent
vasodilatation in hypercholesterolemic humans.
Circulation. 1997;95:76–82.
13. Cattin L, Petrucco A, Cazzolato G, Bon GB, Borelli V, Nardon E, Zabucchi G, Fonda M, Bordin P. Low density lipoprotein apheresis decreases oxidized low density lipoproteins and monocyte adhesion to endothelial cells. ASAIO J. 1997;43:209–213.[Medline] [Order article via Infotrieve]
14.
Sampietro T, Tuoni M, Ferdeghini M, Ciardi A,
Marraccini P, Prontera C, Sassi G, Taddei M, Bionda A. Plasma
cholesterol regulates soluble cell adhesion molecule
expression in familial hypercholesterolemia.
Circulation. 1997;96:1381–1385.
15. Leitinger N, Pirich C, Blazek I, Endler G, Sinzinger H. Decreased susceptibility of low-density lipoproteins to in vitro-oxidation after dextran-sulfate LDL apheresis treatment. Atherosclerosis. 1996;25:305–312.
16. Napoli C, Ambrosio G, Scarpato N, Corso G, Palumbo G, D'Armiento FP, Mancini FP, Malorini A, Formisano S, Ruocco A, Cali A, Chiariello M. Decreased low-density lipoprotein oxidation after repeated selective apheresis in homozygous familial hypercholesterolemia. Am Heart J. 1997;133:585–595.[Medline] [Order article via Infotrieve]
17. Bowry VW, Ingold KU, Stocker R. Vitamin E in human low-density lipoprotein: when and how this antioxidant becomes a pro-oxidant. Biochem J. 1992;288:341–344.
18.
Kontush A, Finckh B, Karten B, Kohlschütter A,
Beisiegel U. Antioxidant and prooxidant activity of
-tocopherol in human plasma and low density lipoprotein.
J Lipid Res. 1996;37:1436–1448.[Abstract]
19. Ziouzenkova O, Gieseg SP, Ramos P, Esterbauer H. Factors affecting resistance of low density lipoproteins to oxidation. Lipids. 1996;31(suppl):S-71–S-76.
20.
Bonanome A., Pagnan A, Biffanti S, Opportuno A, Sorgato
F, Dorella M, Maiorino M, Ursini F. Effect of
monounsaturated and polyunsaturated fatty acids on
the susceptibility of plasma low density lipoproteins to oxidative
modification. Arterioscler Thromb Vasc Biol. 1992;12:529–533.
21. Jialal I, Norkus EP, Cristol L, Grundy SM. Carotene inhibits the oxidative modification of low-density lipoprotein. Biochim Biophys Acta. 1991;1086:134–138.[Medline] [Order article via Infotrieve]
22.
Stocker R, Bowry VW, Frei B. Ubiquinol-10 protects low
density lipoprotein more efficiently against lipid peroxidation than
does -tocopherol. Proc Natl Acad Sci
U S A.. 1991;88:1646–1650.
23. Kontush A, Hübner C, Finckh B, Kohlschütter A, Beisiegel U. Low density lipoprotein oxidizability by copper correlates to its initial ubiquinol-10 and polyunsaturated fatty acid content. FEBS Lett. 1994;341:69–73.[Medline] [Order article via Infotrieve]
24. Engelmann B, Bräutigam C, Thiery J. Plasmalogen phospholipids as potential protectors against lipid peroxidation of low density lipoproteins. Biochem Biophys Res Commun. 1994;204:1235–1242.[Medline] [Order article via Infotrieve]
25. Jürgens G, Fell A, Chen Q, Paltauf F. Delay of copper-catalyzed oxidation of low density lipoprotein by in vitro enrichment with choline or ethanolamine plasmalogen. Chem Phys Lipids. 1995;77:25–31.[Medline] [Order article via Infotrieve]
26. Thiery J, Seidel D. LDL apheresis: clinical experience and indications in the treatment of severe hypercholesterolemia. Transfus Sci. 1993;14:249–255.[Medline] [Order article via Infotrieve]
27. Wallin B, Rosengren B, Shertzer HG, Camejo G. Lipoprotein oxidation and measurement of thiobarbituric acid reacting substances formation in a single microtiter plate: its use for evaluation of antioxidants. Anal Biochem. 1993;208:10–15.[Medline] [Order article via Infotrieve]
28. Thiery J, Teupser D, Walli AK, Ivandic B, Nebendahl K, Setin O, Stein Y, Seidel D. Study of causes underlying the low atherosclerotic response to dietary hypercholesterolemia in a selected strain of rabbits. Atherosclerosis. 1996;121:63–73.[Medline] [Order article via Infotrieve]
29. Hess D, Keller HE, Oberlin B, Bonfanti R, Schüepp W. Simultaneous determination of retinol, tocopherols, carotenes, and lycopene in plasma by means of high-performance liquid chromatography on reversed phase. Int J Vit Nutr Res.. 1991;61:232–238.
30. Bligh EG, Dyer WJ. A rapid method of total lipid extraction and purification. Can J Biochem Physiol. 1959;37:912–917.
31. Broekhuyse RM. Quantitative two dimensional thin-layer chromatography of blood phospholipids. Clin Chim Acta. 1969;23:457–461.[Medline] [Order article via Infotrieve]
32. Engelmann B, Streich S, Schönthier UM, Richter WO, Duhm J. Changes of membrane phospholipid composition of human erythrocytes in hyperlipidemias, I: increased phosphatidylcholine and reduced sphingomyelin in patients with elevated levels of triacylglycerol-rich lipoproteins. Biochim Biophys Acta. 1992;1165:32–37.[Medline] [Order article via Infotrieve]
33. Morrison WR, Smith IM. Preparation of fatty acid methylesters and dimethylacetals from lipids with boron-trifluoride methanol. J Lipid Res. 1964;5:600–608.[Abstract]
34. Reiss D, Beyer K, Engelmann B. Delayed oxidative degradation of polyunsaturated diacyl phospholipids in the presence of plasmalogen phospholipids in vitro. Biochem J. 1997;323:807–814.
35. Havel RJ, Eder HA, Bragdon JH. The distribution and chemical composition of ultracentrifugally separated lipoproteins in human serum. J Clin Invest. 1955;34:1345–1353.
36. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976;72:248–254.[Medline] [Order article via Infotrieve]
37. Richter WO, Jacob BG, Ritter MM, Sühler K, Vierneisel K, Schwandt P. Three-year treatment of familial heterozygous hypercholesterolemia by extracorporal low-density-lipoprotein immunoadsorption with polyclonal apolipoprotein B antibodies. Metabolism. 1993;42:888–894.[Medline] [Order article via Infotrieve]
38.
Quinn MT, Parthasarathy S, Steinberg D.
Lysophosphatidylcholine: a chemotactic factor for human monocytes and
its potential role in atherogenesis. Proc Natl Acad Sci
U S A.. 1988;85:2805–2809.
39. Meyer DF, Nealis AS, Macphee CH, Groot PH, Suckling KE, Bruckdorfer KR, Perkins SJ. Time-course studies by synchrotron X-ray solution scattering of the structure of human low-density lipoprotein during Cu(2+)-induced oxidation in relation to changes in lipoprotein composition. Biochem J. 1996;319:217–227.
40.
Neuzil J, Thomas SR, Stocker R. Requirement for,
promotion, or inhibition by -tocopherol of
radical-induced initiation of plasma lipoprotein lipid peroxidation.
Free Radic Biol Med. 1997;22:57–71.[Medline]
[Order article via Infotrieve]
41. Ylä-Herttuala S, Palinski W, Rosenfeld ME, Parthasarathy S, Carew TE, Butler S, Witztum JL, Steinberg D. Evidence for the presence of oxidatively modified low density lipoprotein in atherosclerotic lesions of rabbit and man. J Clin Invest. 1989;84:1086–1095.
42. Smith C, Mitchinson MJ, Aruoma OI, Halliwell B. Stimulation of lipid peroxidation and hydroxyl-radical generation by the contents of human atherosclerotic lesions. Biochem J. 1992;286:901–905.
43.
Mukhopadhyay CK, Mazumder B, Lindley PF, Fox, PL.
Identification of the prooxidant site of human ceruloplasmin: a model
for oxidative damage by copper bound to protein surfaces. Proc
Natl Acad Sci U S A.. 1997;94:11546–11551.
44. Heinecke JW. Oxidants and antioxidants in the pathogenesis of atherosclerosis: implications for the oxidized low density lipoprotein hypothesis. Atherosclerosis. 1998;141:1–15.[Medline] [Order article via Infotrieve]
45.
Zoeller RA, Morand OH, Raetz CRH. A possible role for
plasmalogens in protecting animal cells against photosensitized
killing. J Biol Chem. 1988;263:11590–11596.
46. Zommara M, Tachibana N, Mitsui K, Nakatani N, Sakono M, Ikeda I, Imaizumi K. Inhibitory effect of ethanolamine plasmalogen on iron- and copper-dependent lipid peroxidation. Free Radic Biol Med. 1995;18:599–602.[Medline] [Order article via Infotrieve]
47. Frei B, Gaziano JM. Content of antioxidants, preformed lipid hydroperoxides, and cholesterol as predictors of the susceptibility of human LDL to metal ion-dependent and -independent oxidation. J Lipid Res. 1993;34:2135–2145.[Abstract]
48. Engelmann B, Bräutigam C, Kulschar R, Duhm J, Prenner E, Hermetter A, Richter WO, Thiery J, Seidel D. Reversible reduction of phospholipid bound arachidonic acid after low density lipoprotein apheresis: evidence for rapid incorporation of plasmalogen phosphatidylethanolamine into the red blood cell membrane. Biochim Biophys Acta. 1994;1196:154–164.[Medline] [Order article via Infotrieve]
49. Bräutigam C, Engelmann B, Reiss D, Reinhardt U, Thiery J, Richter WO, Brosche T. Plasmalogen phospholipids in plasma lipoproteins of normolipidemic donors and patients with hypercholesterolemia treated by LDL apheresis. Atherosclerosis. 1996;119:77–88.[Medline] [Order article via Infotrieve]
50. Kugiyama K, Kerns SA, Morrisett JD, Roberts R, Henry P. Impairment of endothelial-dependent arterial relaxation by lysolecithin in modified low-density lipoproteins. Nature. 1990;344:160–162.[Medline] [Order article via Infotrieve]
51. Kume N, Gimbrone MA Jr. Lysophosphatidylcholine transcriptionally induces growth factor gene expression in cultured human endothelial cells. J Clin Invest. 1994;93:907–911.
52. Stadler RW, Ibrahim SF, Lees RS. Peripheral vasoactivity in familial hypercholesterolemic subjects treated with heparin-induced extracorporal LDL precipitation (HELP). Atherosclerosis. 1997;128:241–249.[Medline] [Order article via Infotrieve]
53. Mellwig KP, Baller D, Gleichmann U, Moll D, Betker S, Weise R, Notohamiprodjo G. Improvement of coronary vasodilation capacity through single LDL apheresis. Atherosclerosis. 1998;139:173–178.[Medline] [Order article via Infotrieve]
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