Int J Clin Lab Res (1997) 27:247-252 9 Springer-Verlag 1997
A. A d i o u n i 9 M. El Messal 9 N. G h a l i m 9 R. Sa'ile
Apolipoproteins and lipoprotein particles in Moroccan patients with previous myocardial infarction
Received: 14 July 1997 / Accepted: 16 October 1997
A b s t r a c t We investigated for the first time in the Moroc- can population the relationship between lipoprotein parti- cles and the progression of coronary atherosclerosis.
Plasma lipid variables, including total cholesterol, trigly- cerides, high-density lipoprotein-cholesterol, low-density lipoprotein-cholesterol, apolipoproteins AI and B, Lp AI, Lp A I : A I I , and Lp(a) were measured in 40 Moroccan adults who suffered a verified myocardial infarction be- fore the age of 50 years. The results were compared with a healthy control group. Plasma total cholesterol, triglyce- ride, and Lp AI : A I I levels of patients did not differ signif- icantly from control subjects. Patients had lower plasma high-density lipoprotein-cholesterol (P<0.05), apo AI (P < 0.05), and Lp AI (P < 0.001 ) than control subjects, sug- gesting that the cholesterol reverse transport system is al- tered in patients with previous myocardial infarction. How- ever, patients had higher plasma low-density lipoprotein- cholesterol (P<0.001), apo B (P<0.001), and Lp(a) (P<0.001). In all patients the best predictor of cardiovas- cular risk was the independent risk factor Lp(a) plasma level, and the Lp AI plasma level. In this study, the in- creased coronary atherosclerosis risk with elevated plasma levels of apo B and Lp(a), and with reduced Lp A1, was substantially modified by smoking habits, but not by fam- ily history of myocardial infarction.
K e y w o r d s Myocardial infarction 9 Apolipoprotein - Lipoprotein particles - Cholesterol -
Cardiovascular risk factor
A. Adlouni ([]) - R. Sa;ile
Laboratoire de Recherches sur les Lipoprotdines,
D~partement de Biologie. Facult6 des Sciences Ben M'sik, B.R 7955 Sidi Othman. Casablanca, Morocco
M. El Messal
Laboratoire de Biochimie, Biologie Cellulaire et Mol6culaire, Facult6 des Sciences A]n chock, Casablanca, Morocco N. Ghalim
Laboratoire de Biochimie, Institut Pasteur du Maroc.
Casablanca, Morocco
Introduction
Coronary artery disease (CAD) is the leading cause of death in both women and men after 40 years of age. Sev- eral prospective epidemiological studies have shown an in- dependent inverse relationship between the high-density lipoprotein-cholesterol (HDL-chol) concentration and the risk of future C A D [1]. It has been hypothesized that ei- ther HDL exerts a direct protective effect by facilitating cholesterol transport from peripheral tissus, including the arterial wall [2, 3], or that a low plasma H D L level reflects metabolic processes that are associated with the develop- ment of atherosclerosis [4]. Based on the presence of ap- olipoprotein at their surface, plasma H D L lipoproteins con- tain two main types of particles, one containing apolipo- protein (apo) AI and apo AII (Lp AI :AII) and one contain- ing apo AI but not apo AII (Lp AI) [5]. These two parti- cles are differently distributed in H D L subfractions, since H D L 2 comprise mainly Lp AI and H D L 3 mainly Lp AI :AII. The two particles may have different metabolic functions [6]. Lp AI appears to be the H D L fraction in- volved in reverse cholesterol transport [7, 8]. Moreover, Lp AI seems to be a more powerful marker for atheroma risk than Apo AI and HDL-chol [9]. However, this remains controversial [10, 11].
Recent studies have shown that increased levels of Lp(a) are associated with an increased incidence of C A D [12], but data are still controversial. Lp(a) displays remarkable genetic heterogeneity, with isoform frequency varying between different ethnic groups [13, 14]. A high serum level of Lp(a) is a strong independent risk factor for the development of ischemic heart disease. Thus, Lp(a) could be a link between atherosclerosis and thrombosis. How- ever, this hypothesis has not yet been validated, and Lp(a) may contribute to coronary heart disease (CHD) by dis- charging lipids in the arterial intima [15, 16]. Myocardial infarction is almost invariably accompanied by a throm- boembolic process in which Lp(a) could play a role.
In order to investigate the relationship between plasma
levels o f L p AI, Lp AI :AII, and Lp(a) and C A D with myo-
248 A. Adlouni et al.: Lipoprotein particles in Moroccan patients with myocardial infarction c a r d i a l i n f a r c t i o n as t h e p r i m a r y m a n i f e s t a t i o n , we s t u d i e d
M o r o c c a n p a t i e n t s w i t h p r e v i o u s m y o c a r d i a l i n f a r c t i o n . W e c o m p a r e d p l a s m a l i p i d s , a p o l i p o p r o t e i n s , a n d l i p o p r o ~ rein p a r t i c l e s , a d j u s t e d f o r e n v i r o n m e n t a l f a c t o r s , b e t w e e n a d u l t s w h o h a d s u f f e r e d a v e r i f i e d m y o c a r d i a l i n f a r c t i o n a n d c o n t r o l s u b j e c t s .
Materials and methods Patients and control subjects
A totai of 40 patients who had survived a first myocardial infarction before the age of 50 years were enrolled in this study. All patients were hospitalized in the "Service de Cardiologie de la Polyclinique CNSS de Casablanca", for CAD. Patients with polygenic or mono- genic hypercholesterolemia were excluded. Obese subjects, those with manifest diabetes mellitus, and those who were taking lipid- lowering medication or who were abusing alcohol were also exclud- ed. None of the patients studied have suffered reinfarction within the ensuing 6 months.
A coronary disease-free control group comprising 52 normolip- idemic subjects of similar age (mean age _+SD, 45__.3.2 years, range, 4 1 - 5 2 years) was recruited. The selection criteria employed for the control group were comparable to those for patients. A complete clin- ical summary was obtained for both case and control subjects, with emphasis on personal and family history of myocardial infarction, peripheral arterial disease, and vascular risk factors. All subjects with clinical evidence of cancer or acute or chronic inflammatory disease were excluded. There was no clinical or laboratory evidence of thy- roid dysfunction or other conditions that might lead to secondary hy- perlipoproteinemia in any of the patients or control subjects studied.
Table l shows the clinical characteristics of CAD patients and controls. None of the control subjects were using anticoagulants. No evidence of sustained anticoagulant use before the ischemic event was found in CAD patients. Patients were considered hypertensive if blood pressure levels were above 140/90 mmHg on more than
t w ooccasions. Smoking history was obtained in all cases, and subjects were classified as present or former smokers, or as nonsmokers. Sub- jects were considered smokers if they smoked more than ten cigar- ettes daily. A 10-ml blood sample was taken from 12-h fasting sub- jects by venipuncture into sodium' EDTA tubes. Plasma was separ- ated from whole blood by centrifugation at 3500xg for 15 min at 4~ and frozen at - 2 0 ~ prior to measurement of lipids, apolipop- roteins, and lipoprotein particles.
Lipid and lipoprotein analysis
Plasma total cholesterol (T-chol) and triglycerides were analyzed by enzymatic methods (Boehringer Mannheim, Germany) using a Ko- dak-Ektachem 500 Autoanalyzer. Intra-assay coefficients of varia- tion (CVs) for cholesterol and triglycerides were 4.3% and 3.8%, re- spectively. Interassay CVs were 5.1% and 6.4%, respectively. Cali- bration was carried out according to the manufacturer's recommen- dations. HDL-chol was determined enzymatically by separating Table 1 Clinical characteristics of coronary artery disease (CAD) patients and controls
CAD Control
(n=40) (n=52)
Age, years (range) 46 ( 4 2 - 5 0 ) 45 ( 4 1 - 5 2 )
Sex (M/F) 30/10 34/18
Present smokers (%) 18 11
Former smokers (%) 33 27
Hypertension (%) 59 48
HDL from the plasma by precipitation of the low-density lipopro- rein (LDL) plus very low-density lipoprotein fraction by a phospho- tungstic acid/magnesium chloride solution (Boehringer Mannheim).
LDL-chol was calculated according to the Friedewald formula [17].
Apolipoprotein and lipoprotein particle analysis
Plasma apo AI and apo B were measured by immunoenzymometric assays as described respectively by Koren et al. [18] and Fruchart eta[. [19]. Intra-assay CVs were 4.2% and 6.8%, respectively. Inter- assay CVs were 3.4% anf4.5%, respectively. For calibration, apo AI and B isolated from plasma from a normotipidemic fasting subject were used as primary standards. Using these primary standards, we measured the concentrations of apo AI and apo B in a selected nor- molipidemic plasma. This plasma was then used as a secondary stan- dard on each ELISA plate.
Lp AI was quantified by a differential electroimmunoassay as de- scribed by Parra et al. [201 using the Hydragel Lp A1 particle kit sup- plied by Sebia (France). Lp AI :AII was measured by a two-site im- munoenzymometric assay [21 ]. Briefly, the plates were coated with antibodies to apo AII. After incubation with samples, only lipopro- tein particles containing apo A1 associated with apo AII (Lp AI : AID were retained while Lp A] particles were removed during the wash- ing process. The amount of Lp AI : AII was evaluated using peroxi- dase-labelled antibodies to apo AI. The intra-assay CVs ranged between 6.8% and 9.4% for Lp AI: AII and between 2.4% and 3.8%
for Lp AI. These were estimated by 20 replicate analyses in three plasma samples containing high, medium, and low levels of both Lp AI:AII and LpAI. Interassay CVs were 7.2% and 5,2% for Lp A I : A I I and Lp AI, respectively. These were assessed by meas- uring Lp A I : A I I and Lp AI concentrations in the same sample for 6 consecutive working days.
The Lp AI assay was calibrated according to the manufacturer's recommendations. Sample and control values were read from the standard curve. For L p A I : A I I calibration, a pure fraction of Lp AI:AII particles isolated by immunoaffinity chromatography on a column specific for apo All was used as a primary standard. After concentration under reduced pressure, dilutions of the primary stan- dard were used with known concentrations of apo AI to prepare the primary standard curve for Lp AI : AII. This primary standard was used to measure the Lp AI:AII concentration in a lyophilized nor- molipidemic plasma which then acted as a secondary standard on the ELISA plates.
Lp(a) was quantified by an electroimmunoassay using the Hydragel Lp(a) kit supplied by Sebia. The intra-assay CVs ranged between 4% and 6%. These were estimated by 15 replicate analyse in four plasma samples containing Lp(a) levels between 8 and 56 mg/dl. Interassay CVs were assessed by measuring Lp(a) in two samples for 6 consecutive working days (Lp(a) concentrations of 14 and 58 mg/dl) and were 7.2% and 8.1%, respectively. The Lp(a) assay was calibrated according to the manufacturer's recommenda- tions (Sebia).
Statistical analysis
Student's t-test was employed to determine differences in plasma lev- els of lipids, apolipoproteins, and lipoprotein particles. Correlations were determined by Pearson's correlation coefficients. Logistic re- gression analysis was used to estimate the probability of developing CAD. Values were considered to be significantly different if P<0.05.
All data were expressed as mean plus or minus SD or as percentage.
Results
T a b l e 2 s h o w s t h e l i p i d a n d a p o l i p o p r o t e i n l e v e l s in C A D
a n d c o n t r o l g r o u p s . B o t h T - c h o l a n d t r i g l y c e r i d e l e v e l s
w e r e n o t s i g n i f i c a n t l y d i f f e r e n t in C A D s u b j e c t s a n d
c o n -A. Adlouni et al.: Lipoprotein particles in Moroccan patients with myocardial infarction T a b l e 2 P l a s m a lipids and apolipoproteins (Apos) in C A D and con-
trol groups (HDL h i g h - d e n s i t y lipoprotein, LDL low-density lipop- rotein a
C A D Control
( n = 4 0 ) ( n = 5 2 ) Total cholesterol (mg/dl) 189 -+ 30 178 -+ 25
Triglycerides (mg/dl) 142 -+ 36 128 -+ 30
HDL-cholesterol (mg/dl) 25 -+ 14 * 40 -+ 10 LDL-cholesterol (mg/dl) 132 -+ 36 * 109 -+ 20
Apo AI (g/l) 1.17 -+0.30 * 1.27 -+0.20
Apo B (g/l) 1.14__.0.33"* 0.67_.+0.30
Apo AI/Apo B 1.04-+0.37"* 1.89-+0.67
* P < 0 . 0 5 ; ** P < 0 . 0 0 1 c o m p a r e d with control a Results are m e a n -+SD
trols. Whereas LDL-chol was significantly (P<0.05) in- creased (132+36 mg/dl vs. 109___20 mg/dl control sub- jects) and HDL-chol levels were significantly (P<0.05) decreased in CAD patients. The plasma concentrations of apo AI were decreased (P<0.05), while those of apo B were significantly increased (P<0.001). However, the apo A I / a p o B ratio was lower in CAD subjects.
Table 3 shows the plasma lipoprotein particles levels in CAD and control groups. Plasma levels of Lp AI and Lp(a) were significantly different between the CAD and control groups. Plasma L p A I was lower in the CAD group than controls (0.29+0.05g/I vs.
0.46_+0.13g/1, P<0.001), whereas plasma L p A I I : A I
Fig, 1 Distribution of Lp(a) levels in coronary artery disease (CAD, n = 4 0 ) and control ( n = 5 2 ) groups
249 T a b l e 3 Plasma lipoprotein particles in C A D and control groups =
C A D Control
( n = 4 0 ) ( n = 5 2 ) Lp AI (g/l) 0 . 2 9 + 0 . 0 5 * 0.46-+0.13
4 - ")
Lp A I I : A I (g/l) 0.85 -+0.24 0.74 _ 0 . - 0
Lp (a) b (mg/dl) 36* 9
* P < 0 . 0 0 1 c o m p a r e d with control a Results are m e a n __.SD b Results are median
was not significantly different in the CAD group (0.85_0.24 vs. 0.74+0.20 g/l).
Plasma Lp(a) was more than twofold higher in the CAD group than controls (39---17 vs. 17_+ 10 mg/dl, P<0.001).
The median values were 9 mg/dl and 36 mg/dl for control and CAD groups, respectively. Figure 1 shows the distri- bution of Lp(a) levels in CAD and control groups; 55 ,C/c of CAD patients had a Lp(a) level of more than 30 mg/dl com- pared with 15% of the control group. There was a wider range of plasma Lp(a) concentrations in the CAD group (between 12 and 98 mg/dl).
Table 4 shows the risk factors for CAD in Moroccan subjects. The risk for CAD was increased with elevated levels of apo B and Lp (a) and reduced levels of Lp AI.
CAD correlated with smoking: there were more smokers in the CAD group than in control group. We found no cor- relation with hypertension, family history of CAD, or al- coholism. Multivariate analysis confirms that in our study only Lp AI, Lp(a), apo B, and smoking habits contributed to progression of coronary atherosclerosis. The calculated probabilities of developing CAD by logistic regression are
70
60
50
O 40
30
20
10
0-10 11-20
b L =
21-30 31-40 41-50 51-60 61-70 71-80 81-90
Lp(a) mg/dl
mCAD 9 Control
m
91-100
250
T a b l e 4
Risk factors for CAD in CAD and control groups
CAD Control
(n =40) (n = 52)
(%) (%)
Table 5 Multivariate analysis of risk factors for CAD in Moroccan patients ~ (logistic regression analysis) (CI confidence intervals)
Variable Odds ratio b 95% CI
Smoking 4.95
Lp (a) 3.75
Lp AI 3.54
Apo B 3.02
A. Adlouni et at.: Lipoprotein particles in Moroccan patients with myocardial infarction has been considered a more accurate predictor of CAD than T-chol or even LDL-chol. In the present study, apo B was shown to be an important risk factor for CAD develop- ment. Apo B and LDL-chol were higher in the CAD group than controls. We note that in the control group the levels of apo B were low relatively to other studies. This may be due to the diet of the Moroccan population since it is.
known that the Mediterranean diet improves lipid metab- olism and could protect against CAD, in comparison with other populations.
Apo AI and HDL-chol are lower in subjects who have suffered a myocardial infarction. The large difference in the apo AI levels between CAD subjects and controls is particularly striking and confirms the results of several studies which suggest that apo AI levels might be more strongly associated with CAD than are total and HDL-chol levels [22, 23]. A previous longitudinal study has con- firmed that apo B and apo AI levels are predictive of CAD risk on univariate analysis. However, levels of these apol- ipoproteins ceased to be predictive after adjustment for lev- els of lipids, HDL-chol, age, and smoking [24].
2.34, 12.25 The Lp AI and Lp A I : A I I levels in the control group 1.70, 10.27 are in agreement with those obtained in other studies 1.02, 8.40 [9, 25]. In the CAD group, the Lp AI but not the Lp AI :AII 1.27, 12.52 level was lower than in the control group, as in previous studies [26]. The interpretation of such changes is difficult because the decrease in Lp AI may be due to increased ca- tabolism or decreased synthesis. It has been shown that pa- tients with coronary atherosclerosis have a lower mean level of Lp AI than those without coronary lesions [27].
The low level of Lp AI observed in the CAD group could thus partly explain the higher risk of atherosclerosis in this group as confirmed by the multiple regression analysis.
However, Lp AI as the best discriminatory marker of CAD risk remains controversial. In a recent study, the concen- tration ofLp AI was lower in normolipidemic patients with angiographically proven CAD, whereas Lp A I : A I I was unchanged compared with controls [27]. However, in a similar study where the patient group had higher triglyce- ride levels than controls, both Lp AI and Lp A I : A I I were reduced to a similar degree in CAD patients, suggesting that the decrease in Lp AI :AII particles in hypertriglycer- idemic patients could be related to the decreased forma- tion of such particles during lipolysis [26]. However, in our study patients did not have higher triglyceride levels than controls and this may explain the differences between our data and the last hypothesis. Moreover, it has recently been observed that the level of Lp AI but not Lp A I : A I I in children whose parents suffer from premature CHD is lower than in a control group with no family history of CHD [28]. Since Lp AI may represent the particle that is involved in cholesterol efflux from peripheral cells, we suggest that the lower levels o f L p AI in CAD subjects may reflect an impairment of cholesterol transport that results in increased CAD risk. Thus, Lp AI may protect against atherosclerosis, as suggested by previous studies [7].
Studies in normolipidemic subjects indicate that high plasma levels of Lp(a) are associated with myocardial in- farction [29, 30]. The association between Lp(a) and risk
a
