© 1998 American Heart Association, Inc.
Original Contributions |
Sialic Acid Content of LDL in Coronary Artery Disease: No Evidence of Desialylation in Subjects With Coronary Stenosis and Increased Levels in Subjects With Extensive Atherosclerosis and Acute Myocardial Infarction
Relation Between Desialylation and In Vitro Peroxidation
From Laboratoire de Biochimie Appliquée, Faculté des Sciences Pharmaceutiques et Biologiques, Châtenay-Malabry, France (B.C., I.M.); and Laboratoire de Biochimie (B.C., E.F., I.M.), INSERM U-430 (I.M.), Département de Radiologie Vasculaire (B.B., J.C.G.), and Département de Cardiologie (F.L., J.L.G.), Hôpital Broussais, Paris, France.
Correspondence to Dr B. Chappey, Laboratoire de Biochimie Appliquée, Tour D4, 2ème étage, Faculté de Pharmacie, Paris Sud, 5 rue Jean-Baptiste Clément, 92296 Châtenay-Malabry, France.
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Abstract |
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Abstract—We recently showed that sialic acid content of LDL was not a marker of early cardiovascular disease (Arterioscler Thromb Vasc Biol. 1995;15:334–339). Here, we investigated this parameter in patients with advanced coronary artery disease (CAD). We first examined 100 patients having undergone coronary angiography. The distribution of LDL sialic acid values was very similar in subjects with no coronary stenosis (31.3±3.7 nmol/mg LDL protein, mean±SD) and those with
75% stenosis in at least one main coronary
artery or 50% stenosis in at least two main coronary
arteries (32.1±5.5 nmol/mg LDL protein). In contrast, LDL sialic acid
content was significantly increased in patients with both
coronary stenosis and peripheral
arterial atherosclerotic lesions compared with those with
either no lesion or only one or the other type of lesion. We then
examined LDL sialic acid content in 20 patients with acute myocardial
infarction. LDL sialic acid content was significantly higher (35.9±3.2
nmol/mg LDL protein) than that in the CAD(-) control group. These data
suggest that LDL sialic acid content increases with the extension of
atherosclerosis and its progression to acute
complications. To explain the discordance with Orekhov and coworkers
(Atherosclerosis. 1991;86:153–161), who
showed that LDL sialic acid content in patients with advanced CAD was
lower than that in healthy subjects, we studied the time courses of
sialic acid, TBARS, and vitamin E levels in LDL dialyzed in different
experimental conditions. A continuous decrease in both sialic acid and
vitamin E levels and an increase in TBARS levels were observed in LDL
samples containing less than 1 mmol/L EDTA, the intensity and
rapidity of which varied with the EDTA concentration in the buffer. Our
data support the idea that desialylation may result from in vitro
peroxidation of LDL.
Key Words: low-density lipoprotein • sialic acid • coronary artery disease • angiography • peroxidation
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Introduction |
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There is a strong relation between LDL cholesterol levels and the incidence of CAD.1 2 However, LDL comprises a continuum of particles differing in their chemicophysical3 4 5 6 and biological6 7 8 properties, and some subfractions have been more strongly incriminated than others in the development of atherosclerosis.9 10 Sialic acid is a component of both the protein and lipid moieties of LDL.11 12 13 14 In vitro, the sialic acid content of LDL can modulate its receptor-mediated cell uptake,15 16 17 its avidity for arterial proteoglycans,18 and its susceptibility to oxidation.19 20 Several investigators have reported large interindividual differences in LDL carbohydrate content.13 14 Thereby, the important question is whether these differences in LDL sialic acid content have implications for the development of atherosclerosis. Tertov et al14 21 and Orekhov et al16 17 22 reported that LDL isolated from plasma of patients with angiographically assessed advanced CAD had a total sialic acid content 40% to 75% lower than that from healthy subjects. This finding was explained by a greater proportion of desialylated LDL in patients with CAD. Based on these data, the authors propose LDL sialic acid content as a marker of the cardiovascular risk.23 To test whether LDL sialic acid content could be used early for effective prevention of atherosclerosis, we recently examined hypercholesterolemic subjects with and without subclinical atherosclerosis.24 Surprisingly, we found no relation between LDL sialic acid content and the prevalence of peripheral plaques and/or coronary calcifications detected respectively by high-resolution ultrasonography and ultrafast CT. As our population consisted of asymptomatic subjects, we postulated that desialylation of LDL might occur much later in the atherosclerotic process. Only two other groups studied LDL sialic acid content in patients with and without documented CAD. Ruelland et al25 found a far less marked decrease (23%) than that reported by Orekhov and coworkers. Melajärvi et al26 found no difference between patients with and without CAD, either in diabetic or nondiabetic individuals. In view of these contradictory findings, we decided to investigate again LDL sialic acid content in subjects with advanced CAD. Here, we examined angiographed patients with and without coronary stenosis on the one hand and patients with acute MI on the other hand.
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Methods |
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Angiographed Subjects
The study was conducted in accordance with the Helsinki Declaration of 1975. Coronary angiography was always carried out for diagnostic purposes. Subjects had either a cardiac history or symptoms sufficient to warrant angiography. The different indications were cardiomyopathy, mitral or aortic valve disease, cardiac arrhythmias, history of MI, clinical evidence of angina pectoris, and suspect chest pain. Cardiac catheterization was also undertaken as a presurgical evaluation of heart disease in patients with peripheral arterial atherosclerosis. After excluding patients with acute or recent MI, subjects were selected for this study according to angiography data as follows: Control patients, designated CAD(-), are defined as those with strictly normal angiograms; subjects with significant CAD, designated CAD(+), are defined as those with
75% diameter reduction in at least one main coronary artery
or 50% diameter reduction in at least two main coronary
arteries; subjects with minor irregularities of the coronary
vasculature or a moderate diameter reduction (<50%) were excluded
from the study. Information regarding age, smoking habits, medications, and medical history were obtained during interviews with a physician. Blood pressure, serum lipid parameters, and BMI (weight/height2) were determined. Hypertension was defined by a systolic blood pressure of 160 mm Hg or more and/or a diastolic pressure of 95 mm Hg or more.27 Patients with diabetes or renal failure, and those receiving heparin or lipid-lowering drugs, were excluded from the study.
The final population included 85 men and 15 women selected from 136 consecutive patients undergoing angiography.
Patients With Acute MI
Patients were admitted to the intensive care unit in the
hospital within 24 hours after the onset of symptoms. The diagnosis of
first MI included enzyme assays and electrocardiographic examination.
Of the 20 patients selected for the study, 3 were treated with heparin
only and 2 by prehospital thrombolysis; the other 15
underwent direct angioplasty. Information on smoking, medication,
medical history, and clinical and biological parameters
were obtained as described above. Blood sampling was performed as part
of the medical follow-up.
Coronary Investigations
Coronary angiography was performed through the femoral
approach with Judkins 6F catheters. The left main, left anterior
descending, left circumflex, and right coronary arteries were
carefully examined, and images were stored on 35-mm cinefilm.
Coronary angiograms were reviewed independently by two
cardiologists. The number, location, and severity of lesions on each
arterial segment were recorded. All patients received
intravenous heparin (50 IU/kg body weight) after puncturing
the artery and inserting the catheter.
Blood Sampling
In angiographed patients, blood was drawn after an overnight
fast at the time of catheterization, prior to the
injection of heparin or fluids. In patients with acute MI, blood was
collected once during the acute phase (within 24 hours after the onset
of symptoms) and once again on the sixth day after the ischemic
injury.
Blood for lipid analyses was drawn into a tube with no anticoagulant. Blood aimed to LDL preparation was drawn into tubes (Vacutainer) containing disodium EDTA. Plasma and serum were separated by low-speed centrifugation (20 minutes at +15°C).
Analytical Methods
Serum was routinely analyzed for total
cholesterol, triglycerides, HDL
cholesterol, LDL cholesterol, and Lp(a) as
previously described.24 28 LDL
(d=1.019 to 1.063 g/mL) was isolated by sequential
ultracentrifugation in a Beckman L90
ultracentrifuge (Beckman Instruments Inc) according to a
procedure previously described in detail.24
Collected LDL was dialyzed for 24 hours at +4°C against four changes
of 100 vol 10 mmol/L Tris-HCl buffer, pH 7.4, containing 1
mmol/L EDTA, and was stored at +4°C in the dark. For in vitro
studies, LDL was isolated by preparative
ultracentrifugation from normolipidemic plasma pools
according to a procedure previously described in
detail6 29 and then dialyzed as indicated above.
The absence of contaminating lipoproteins (VLDL and HDL) in LDL
preparations was checked by agarose gel electrophoresis.
Total LDL sialic acid content was determined as previously
described.24 Briefly, bound sialic acid was
released from sialoglycoconjugates by mild hydrolysis (15 minutes at
80°C in 0.05 mol/L
H2SO4). Warren's
periodate-TBA assay30 was used. A standard curve
was constructed using N-acetylneuraminic acid (No. A2388,
Sigma Chemical Company) treated in the same conditions. Values are
means of duplicate assays, and the within-assay coefficient of
variation was below 5%. Aldehydes, which also react with TBA to form a
chromophore with an absorption maximum at 532 nm, interfere with the
conventional absorbance measurement of sialic acid at 549 nm (spectra
overlap). To avoid this, second-derivative spectrophotometry, which
separates peaks, was also used after colorimetric
reaction with TBA. The second-derivative spectrum of LDL samples from
study subjects was very close to that of the sialic acid standard
(single peak at 549 nm). When aldehydes were present (in vitro
studies), calibration was carried out by constructing a standard curve
for the second derivative at 549 nm. LDL sialic acid content was also
determined by means of a chromatographic method with
fluorometric detection,31 and the results
obtained by the two methods correlated well (r=.85). TBARS
were measured by using a kit from Sobioda in a modified version of
Yagi's assay with fluorimetric detection.32
Vitamin E was determined by using high-performance liquid
chromatography as previously
described33 with
D-
-tocopherol acetate (Sigma) as internal
standard and DL--tocopherol (Sigma) as
reference standard. Total LDL protein was measured by using Peterson's
method34 with bovine serum albumin as
standard.
Statistical Analysis
The statistical analysis was carried out on a computer
(Apple Macintosh) with StatView II (Abacus Concepts, Inc) software.
Comparisons between groups were performed with one-way ANOVA and, when
appropriate, with Student's unpaired two-tailed t test.
Comparisons of categorical data (proportions of subjects) were made by
using analysis of frequencies. Student's paired two-tailed
t test was used to compare day 0 and day 5 samples in
patients with acute MI and samples collected before and after injection
of heparin in angiographed subjects. Correlations were analyzed
by calculating Pearson coefficients (normal variables) and
Spearman's rank order coefficients (nonnormal variables).
Logarithmic transformations were used when appropriate. Probability
values of <0.05 were considered significant.
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Results |
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The first study population included 100 angiographed patients who were classified into two groups according to the degree of coronary stenosis: 58 had significant coronary stenosis and formed the CAD(+) group, and the other 42 had strictly normal angiograms and constituted the CAD(-) control group. This population did not include patients with acute or recent MI. The characteristics of the groups are detailed in Table 1
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They were homogeneous regarding age and sex and comprised
statistically equivalent proportions of hypertensive subjects, smokers,
and patients on the different categories of medication. No difference
in the lipid parameters was observed between the groups.
Only the BMI in the CAD(+) group was significantly higher than that in
the CAD(-) group. The distribution of patients in each group was
studied according to their medical history: the control CAD(-) group
included a majority of subjects with nonischemic heart disease,
such as cardiomyopathy and mitral or aortic valve
disease; 28% of subjects in this group were referred to our
cardiology center with suspected angina pectoris, which
turned out to be unrelated to coronary stenosis. In
contrast, the CAD(+) group included a large proportion of subjects with
known or documented angina pectoris and only 5% of subjects with
cardiomyopathy or valve failure. Finally, both
groups contained about 30% of subjects with peripheral
arterial atherosclerotic lesions.
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Figure 1 compares LDL sialic acid content
in the two groups. The frequency distribution of LDL sialic acid
content ranged from 23.8 to 42.2 nmol/mg LDL protein in the CAD(-)
group and from 19.4 to 44.8 nmol/mg LDL protein in the CAD(+) group,
with mean values of 31.3±3.7 and 32.1±5.5 nmol/mg LDL protein,
respectively. No significant difference was found between the groups
(P=0.43).
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We also compared LDL sialic acid content in four groups of subjects
defined as follows by the combined results of clinical investigations:
coronary stenosis (absence or presence) with or without
extracoronary atherosclerotic lesions (Table 2).
LDL sialic acid content in patients with both coronary
stenosis and peripheral arterial
atherosclerotic lesions was significantly higher than that in the other
subgroups (P
0.01).
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We then examined LDL sialic acid content in 20 patients with acute MI.
Lipid determination and LDL isolation were performed once within the
first 24 hours (day 0 sample) and again 6 days after the
ischemic injury (day 5 sample). The characteristics of the MI
group are detailed in Table 3. A significant decrease in
total and LDL cholesterol and a significant increase in
triglycerides were observed in sera collected on day 5,
relative to those collected on day 0. Figure 2 shows LDL sialic acid content in MI
patients. LDL sialic acid content ranged from 31.6 to 43.6 nmol/mg LDL
protein in samples collected on day 0 and from 31.4 to 46.9 nmol/mg LDL
protein in samples collected on day 5, with mean values of 35.9±3.2
and 37.2±3.4, respectively. No significant difference was found
between day 0 and day 5 samples (P=0.08). In contrast, LDL
sialic acid content in MI patients was significantly higher than that
in both the CAD(-) control group and the CAD(+) group of angiographed
subjects (P<0.01).
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In the whole angiographed study population, as in MI patients, the
relation between LDL sialic acid content and clinical and biological
parameters was examined. LDL sialic acid content was not
related to sex, age, BMI, blood pressure, smoking status, therapeutic
status, or the different lipid parameters. We paid
particular attention to Lp(a) because it is highly sialylated and has a
density range that overlaps that of LDL. In our previous
study,24 we observed no correlation between LDL
sialic acid content and serum Lp(a) when the latter was below 2 g/L.
The present study confirms the absence of interference of Lp(a)
with the determination of LDL sialic acid content in these conditions,
as shown by the linear regression analysis after logarithmic
transformation of serum Lp(a) (Figure 3)
and the Spearman's rank test (
=0.01, P=0.92, n=115).
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Because heparin is systematically administrated during angiography procedure, we examined its effect on LDL sialic acid content. Ten patients undergoing angiography were sampled once at the time of catheterization, prior to the intravenous injection of a heparin bolus (50 IU/kg body weight), and again 60 minutes after administration of heparin, at the end of the angiography procedure. Mean sialic acid values in LDL isolated before and after injection of heparin were 32.9±2.6 and 32.2±2.2 nmol/mg LDL protein, respectively. Paired comparison of LDL sialic acid values showed no significant difference (P=0.37).
Given the discordance with data obtained by Tertov et al14 21 and Orekhov et al,16 17 22 we examined LDL sialic acid content during LDL dialysis and storage in different experimental conditions chosen according to those described by these authors for the isolation of desialylated LDL. LDL prepared from plasma pools was dialyzed against either PBS or Tris-HCl buffer (10 mmol/L) containing, in each case, various EDTA concentrations (0, 10, and 100 µmol/L and 1 mmol/L). LDL dialyzed against Tris buffer containing 1 mmol/L EDTA was representative of our own experimental conditions. At various times up to 7 days, each LDL solution was assayed for sialic acid, TBARS, and vitamin E contents.
Figure 4 shows the concomitant time
courses of sialic acid, vitamin E, and TBARS levels in LDL in the
different experimental conditions. No change with time was observed in
these three parameters, in either PBS or Tris buffer
containing 1 mmol/L EDTA. As regards LDL samples dialyzed against
PBS, a large and continuous decrease in both sialic acid and vitamin E
levels was observed in LDL samples containing 0 and 10 µmol/L
EDTA. In these dialysis conditions, sialic acid and vitamin E contents
of LDL were respectively reduced by 20% to 32% and 33% to 43%
within the first 24 hours and by 62% to 71% and 90% to 95% after 7
days of dialysis. TBARS levels increased after the third day,
corresponding to a loss of 70% vitamin E in LDL, and were fourfold to
fivefold higher on the 7th day compared with LDL samples containing
1 mmol/L EDTA. Intermediate kinetics were observed in LDL sample
containing 100 µmol/L EDTA, showing a 37% reduction in sialic
acid content, a 68% reduction in vitamin E, and twofold higher TBARS
levels after 7 days of dialysis. As regards LDL samples dialyzed
against Tris buffer, similar trends were observed but were less
pronounced. After 7 days of dialysis, we found a 50% to 55% reduction
in sialic acid levels, a 67% to 73% reduction in vitamin E content,
and threefold higher levels of TBARS in LDL samples containing 0 and
10 µmol/L EDTA compared with LDL sample containing 1 mmol/L
EDTA. Our data show that the intensity of LDL peroxidation is inversely
proportional to the EDTA concentration in buffers and suggest greater
protective power of Tris buffer than PBS against oxidation.
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), 10 µmol/L (
), 100
µmol/L (
), and 1 mmol/L (
). On day 0, the LDL preparation,
isolated from normolipidemic plasma pool, was divided into eight
samples. Each was dialyzed for 24 hours at +4°C against four changes
of 100 vol buffer corresponding to one of the different experimental
conditions. On the first day, and then every day up to 7 days, an
aliquot of each LDL solution was removed from the dialysis bag and
assayed for the three parameters. Each experimental point
represents the mean of duplicate determinations. Two
independent experiments, carried out on separate LDL preparations, were
performed, with similar results. |
Discussion |
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Our data clearly show that patients in whom significant coronary stenosis has been assessed by angiography have an LDL sialic acid content within the range of values in subjects with normal coronary arteries. These results are in keeping with our recent study showing no link between LDL sialic acid content and the prevalence of coronary calcifications.24 They are also in accordance with findings more recently reported by Melajärvi et al,26 who found no difference in LDL sialic acid content between patients with and without CAD, either in diabetic or nondiabetic individuals. In contrast, our data showed that LDL sialic acid content was increased in patients with both coronary stenosis and peripheral atherosclerotic lesions, relative to those with either no lesion or only one or the other type of lesion, and in patients with acute MI, relative to both the CAD(-) control group and the CAD(+) group of angiographed subjects. This finding suggests that LDL sialic acid content increases with the extension of atherosclerosis and its progression to acute complications. The reasons for this increase remain to be discovered. It appears unrelated to serum lipid levels, as LDL sialic acid content did not vary between days 0 and 5 in patients with MI, in spite of the lipid changes that occurred during this period. It is also unrelated to the presence of aldehydes interfering in sialic acid determination. Indeed, we measured TBARS in LDL of MI patients because malondialdehyde-conjugated LDL had been described in these patients.35 TBARS levels were always below 1 nmol/mg LDL protein, ie, within the range of values in control subjects (data not shown). Interestingly, Melajärvi et al26 also found an increase in LDL sialic acid content in diabetics relative to nondiabetics, irrespective of CAD. Taken together, these observations and our data suggest a possible relation between LDL sialic acid content and the inflammatory process, as already described for sialic acid in serum.36
The present study confirms the heterogeneous distribution of LDL sialic acid values among individuals, as observed by other groups13 14 and ourselves.24 Our data showed no relation between LDL sialic acid content and the different clinical and biological parameters in the study populations, in keeping with the recent report by Melajärvi et al.26 According to Senn et al,12 37 gangliosides (sialic acid–containing glycosphingolipids) are excreted by the liver into the circulation along with apolipoprotein B–containing lipoproteins, and these sialylated lipids are then distributed among the different lipoprotein classes, especially LDL. La Belle and Krauss13 have also reported that the heterogeneity in LDL sialic acid content is directly related to the total lipid content of LDL. Only a minor portion of LDL-associated gangliosides are truly anchored in the LDL outer monolayer, whereas the bulk is absorbed to the lipoprotein surface.38 Gangliosides are also released by cells into the circulation, and the equilibrium between bound and unbound gangliosides can be shifted in some pathological conditions.36 38 Accordingly, it may be that differences between individuals related to the synthesis and intravascular processing of LDL precursors on the one hand and to pathological circumstances on the other hand contribute to the formation of various LDL subspecies that differ in their sialic acid content.
Our data differ largely from those obtained by Tertov et al14 21 and Orekhov et al.16 17 22 This disagreement is not related to ethnic origin, because in one of their reports,17 these authors compared LDL isolated from subjects in Moscow and subjects in Houston and concluded that the properties of the LDL were strictly similar. The fact that we chose the same degree of coronary stenosis as the inclusion criterion for the CAD(+) group and a comparable number of subjects, together with the fact that we used the same method (Warren's TBA assay) to determine LDL sialic acid content, rules out a methodological explanation for this discordance.
Only the disease-free control subjects differed between the two studies: in ours, all subjects were angiographed, and the control group consisted of subjects with no coronary artery stenosis and not of apparently healthy subjects with no evidence of CAD based on clinical examination and medical history.17 22 Accordingly, one possible explanation was the fact that the discordance was inherent in the angiographic conditions. A first concern was the potential effect of heparin on LDL sialic acid content, because some studies have suggested that the release of lipases after an intravenous bolus injection of heparin influences the metabolic pathways of triglyceride-rich lipoproteins.39 Our data clearly showed that LDL sialic acid content was not modified after heparin injection. Another concern was the clinical circumstances of the coronary angiography, because there is abundant evidence that lipid levels change substantially in patients hospitalized for acute MI.40 As mentioned earlier, we found not a decrease but rather an increase in LDL sialic acid content in these patients.
The lack of an apparent explanation for the discordance led us to consider possible artifactual desialylation of LDL related to the experimental conditions used for isolation, handling, and storage of LDL. This is why we focused on a possible link between desialylation and in vitro peroxidation of LDL and examined the concomitant time courses of sialic acid, TBARS, and vitamin E levels in LDL dialyzed against PBS or Tris buffer with various EDTA concentrations. Our data clearly showed that only LDL in buffers containing 1 mmol/L EDTA exhibited unchanged contents of sialic acid, vitamin E, and TBARS, even after 7 days' dialysis. In contrast, exposure of LDL to dialysis buffers containing lower EDTA concentrations for various times resulted in partial oxidation of LDL, as assessed by the decrease in vitamin E and the increase in TBARS levels, the intensity and rapidity of which varied with the EDTA concentration in the buffer. This autoxidation of LDL is slower than that previously reported by Esterbauer et al.41 This finding may be explained by the fact that the buffers were not continuously gassed with oxygen in our experimental conditions. As regards TBARS levels, only slight increases were observed. This observation may be explained by the fact that LDL still contained vitamin E and that free MDA (the main component of TBARS) can be released from the LDL particle during dialysis.41 42 The most important information given by these experiments is that the oxidative changes coincided with a marked decrease in LDL sialic acid content. The loss of sialic acid may be related to a subsequent alteration of lipoprotein integrity.
Some of the experimental conditions described above were used by Tertov et al 14 43 44 and Orekhov et al17 for handling and storage of LDL. By contrast with these authors, we always used 1 mmol/L EDTA in our buffers. This approach supports the idea that their observations may result from an artifactual modification of LDL related to in vitro peroxidation, an hypothesis that could explain some of the characteristics of the desialylated LDL described by this group, evocative of peroxidatively modified LDL,42 such as higher levels of lysophosphatidylcholine and oxysterols, lower contents of vitamin E and free lysine amino groups, and greater electrophoretic mobility than sialylated LDL. This latter property is at the basis of the recent report by Tertov et al44 showing a strong similarity between desialylated LDL and the oxidized LDL fraction, named LDL(-), isolated from human plasma by Cazzolato et al45 by using ion-exchange chromatography. LDL peroxidation could explain the paradoxical increase in negative charge of desialylated LDL. Indeed, sialic acid is a negatively charged component46 and several groups,11 15 18 19 including ourselves,20 have shown that desialylation of LDL by neuraminidase treatment reduces its electrophoretic migration in agarose. One outstanding question is why the LDL desialylation was observed only in subjects with CAD. A possible explanation is the greater susceptibility to oxidation of LDL in these subjects.47 48
In conclusion, the present work confirms the heterogeneous distribution of LDL sialic acid content among individuals and shows that this parameter increases with the extension of atherosclerosis and the occurrence of acute coronary complications.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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We thank N. Poulain, I. Souletie, and M. Le Stunff for their excellent technical assistance.
Received June 7, 1996; accepted December 2, 1997.
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