Lipoproteins and apolipoproteins in young male survivors of myocardial infarction

131

Atherosclerosis, 77 (1989) 131-138 Elsevier Scientific Publishers Ireland, Ltd.

ATH 04313

Lipoproteins and apolipoproteins in young male survivors of myocardial infarction N. Al-Muhtaseb ‘, N. Hayat ’ and M. Al-Khafaji * ’Department of Medicine, Fact&y of Medicine, Kuwait University, and 2 Al-Amiri Hospital, Kuwait (Kuwait) (Received 1 August, 1988) (Revised, received 25 November, 1988 and 16 January, 1989) (Accepted 20 January, 1989)

Plasma and lipoprotein cholesterol, triglycerides, apolipoproteins (apo) A-I, A-II, B and phospholipid concentrations were measured at 10 days and 4 months after myocardial infarction (MI) in 60 young Kuwaiti male MI survivors below the age of 40 years. Controls were matched for age, relative weights, smoking, dietary habits and physical activities. The young MI survivors had significantly higher levels of total and LDL-cholesterol, and ratios of LDL/HDL- and LDL/HDL,-cholesterol. Total VLDL and LDL triglycerides, and phospholipids were also elevated in MI survivors compared to controls. Similarly, plasma and LDL-apo B as well as the ratios of apo B/ape A-I were higher in the MI group. There was no significant change in the levels of VLDL and HDL,-cholesterol and of apo A-II in these patients compared to their controls. Concentrations of HDL- and HDL,-cholesterol and of plasma and HDL apo A-I were significantly lower in the young MI survivors compared to the control subjects. The better discriminating lipoproteins and apolipoproteins in MI patients in descending order were HDL,-cholesterol > apo B > apo A-I > VLDL-triglyceride > HDL-cholesterol > LDL/HDL,-cholesterol > triglycerides. The data indicate that measurement of HDL,-cholesterol, apo B and apo A-I may be useful indicators in assessing coronary artery disease risk than triglycerides (TG), total cholesterol (TC), LDL-cholesterol and HDL-cholesterol.

Key words: Lipoproteins;

Apolipoproteins

A-I, A-II, B; Myocardial infarction;

Introduction Numerous studies have described lipid and lipoprotein abnormalities associated with myocar-

Correspondence to: Dr. N. Al-Muhtaseb, Faculty of Meditine, Dept. of Medicine, P.O. Box 24923, Safat 13110, Kuwait. 0021-9150/89/$03.50

Young males

dial infarction (MI). Most of these findings have shown that decreased cholesterol concentration in the high density lipoprotein (HDL) fraction and an increased cholesterol in the low density lipoprotein (LDL) fraction are risk markers for the development of coronary artery diseases (CAD) [l-4]. The low HDL-cholesterol levels in these patients have been linked primarily to a reduction

0 1989 Elsevier Scientific Publishers Ireland, Ltd.

132 of the HDL,-cholesterol level and/or both HDL,and HDL,-cholesterol [1,5-81. Controversy surrounds elevated levels of total and VLDL triglycerides as independent risk factors for myocardial infarction [9-111. Recent studies indicate that the levels of plasma apo A-I (the major HDL protein) and apo B (the major LDL protein) are better discriminators for CAD than the levels of total or lipoprotein lipids [12-151. Low levels of apo A-I and high levels of apo B were reported to be positively correlated to CHD [14,16-181. Epidemiological studies in Kuwait revealed a relatively high frequency of CAD among young males aged below 40 years. The present study was undertaken to investigate the role of plasma lipids, lipoproteins and apo A-I, A-II and B in young male survivors of acute myocardial infarction (MI), which may provide additional information in assessing the risk of CAD in that community. Subjects and methods Sixty male patients, aged 28-40 years, who were admitted to the coronary care unit for a first myocardial infarction were selected for this study. Diagnosis was based on typical chest pain of 30 min duration or more, electrocardiographic (ECG) signs of infarction and elevated serum enzymes including AST, ALT, CPK, CK-MB and LDH. None of the patients was on medication prior to admission, and none had hypolipidemic therapy before or after the infarction. However, betablockers were prescribed 5-7 days after admission. Patients and controls had sedentary life styles and the majority (over 90%) smoked. No patient participated in regular physical exercise and only 5% had a family history of MI. The control subjects, selected from University personnel, were comparable for age, height, body weight and family history of MI. They were also free from other diseases and had life styles similar to those of the patients. Collection of blood samples and analytical methods Blood samples in EDTA tubes (1.5 mg/ml Monoject, Division of Sherwood Medical, St. Louis, MO, U.S.A.) were taken after an overnight fast at 10 days and 4 months after MI and sent

immediately to the laboratory. The samples were centrifuged at 4O C and 3000 rpm to separate the plasma which was used for the immediate analysis of total cholesterol, triglyceride, phospholipid, and of apo A-I, A-II and B. Ultracentrifugal fractionation of VLDL, LDL, total HDL, HDL, and HDL, fractions was performed sequentially. Lipoprotein separation Separation of lipoproteins was performed by a modified sequential ultracentrifugation method [19] in a preparative ultracentrifuge (Sorvall OTD 75.B, Biomedical Products Div., Washington, DC, U.S.A.) using a fixed-angle rotor (type TFT 48.6) with appropriate adaptors. Plasma samples were centrifuged at d = 1.006 g/ml (40000 rpm, for 18 h) and VLDL (d < 1.006 g/ml) was recovered as the top fraction. The bottom fraction was adjusted to d = 1.063 g/ml by addition of a NaCl/NaBr mixture (40000 rpm at 15” C for 18 h). After ultracentrifugation the top 2 ml were separated as LDL (d = 1.006-1.063 g/ml). The bottom fraction was taken to represent HDL (d = 1.063-1.21 g/ml). HDL, and HDL, subfractions were separated at a density of 1.125 g/ml (40000 rpm at 15” C for 40 h). After ultracentrifugation HDL, (d < 1.125 g/ml) was recovered as the top fraction. The remaining bottom fraction was adjusted to d = 1.21 g/ml and HDL, (d c 1.21 g/ml) was recovered as the top fraction (40000 rpm, 15°C 48 h). Density adjustments were made by addition of solutions of NaCl and NaBr and densities were monitored by measurement of refractive index (Reichert ABBE Mark II, Buffalo, NY, U.S.A.). Dilutions were taken into consideration during the calculations of lipoprotein concentration. The purity of the fractions was checked by electrophoresis using the Helena System (Helena Laboratories Saint leu La Foret, France) and immunodiffusion techniques. In the Helena cellulose acetate electrophoresis system, VLDL appeared as a band in the prebeta-globulin region. LDL showed as a band in the betaglobulin fraction. Total HDL banded in the alphaglobulin area, while HDL, and HDL, fractions showed 2 separate bands within the HDL region. Radial immunodiffusion plates with apo B and apo A-I antisera were used separately. Total HDL, HDL, and HDL, fractions showed precipitation rings only with apo A-I antiserum. LDL

133 showed a antiserum showed a LDL with

strong precipitation ring with apo B but was negative for apo A-I. VLDL much weaker precipitation ring than the apo B antiserum.

Results

Characteristics

of MI survivors

and controls (Table

1) Lipid and apolipoprotein estimations Plasma triglyceride [20], cholesterol [21], phospholipid [22] and HDL-,, HDL,and HDL,cholesterol were analysed in triplicate by enzymatic kits (Wako Pure Chemical Industries Ltd., Osaka, Japan) using an Abbott Super VP Analyzer (Abbot Diagnostic Products GmbH, Wiesbaden, F.R.G.). Control plasma pools were supplied by Ortho Diagnostics Co. and Center for Disease Control (CDC, Atlanta, GA, U.S.A.). Control values were within the recommended range and within and between batch coefficients of variation were less than 1.8% and less than 4.5% for all the above estimations across the entire concentration range measured. Apolipoproteins A-I, A-II and B [23] values were determined by radial immunodiffusion assay. Plates were purchased from Immuno AG and samples were analysed in duplicate including the standards and controls (Immuno AG, Vienna, Austria). Controls from CDC were also used and were found to be within the expected ranges. For apo A-I, inter- and intra-assay coefficient of variation was less than 3.5% at a level of 150 mg/dl and less than 4.5% at a level of 100 mg/dl. For apo A-II the coefficient of variation was less than 4.5% at a level of 70 mg/dl and less than 5.0% at a level of 40 mg/dl. For apo B it was less than 5.0% at a level of 100 mg/dl and less than 5.5% at a level of 70 mg/dl.

Statistical methods Statistical significance for differences in lipoprotein and apolipoprotein compositions between groups was tested using one way analysis of variance and the two-tailed t-test. The relationships between variables were calculated using Pearson correlation coefficients. Stepwise discriminant analysis was performed to determine the sets of independent lipoprotein and apolipoprotein variables. All statistical tests were performed at the computer department of Kuwait Medical School using SPSS and BMDP6D programs.

The mean age and Body Mass Index (BMI) for MI survivors was comparable with their corresponding controls. No differences between the groups were observed with respect to blood pressure, level of regular exercises, smoking and family history of MI.

Plasma lipoprotein lipid concentrations in MI survivors and controls (Table 2) Comparison of lipoproteins between MI survivors at 10 days and 4 months showed no significant differences for all parameters. The comparison between control and MI survivors at 10 days and 4 months showed significantly elevated levels of total and LDL cholesterol while triglycerides were elevated in plasma VLDL and LDL fractions. Marked reduction in HDL- and HDL,cholesterol but not in HDL,-cholesterol was observed in the patients compared to controls. No significant difference was observed in VLDLcholesterol between MI and controls. Plasma phospholipids were significantly elevated in these patients compared to controls.

TABLE

1

CHARACTERISTICS TROL MEN

OF

MI

SURVIVORS

Variables

Controls (n = 60)

Age (yrs) Weight (kg) Height (cm) BMI Systolic BP (mm Hg) Diastolic BP

36.0+_ 68.25 170.4* 23.3+

(mm Hg) Smokers (Sg) Regular exer-

85.0+ 93

cisers (%) Family history

3.6

5.0

of MI (‘%)

6.0

5.6

AND

Patients (n = 60) 5.0 7.1 4.2 2.3

130.0 + 12.0 8.0

36.0* 67.6+ 172.0* 22.2+

4.5 6.0 6.1 1.8

135.0+ 13.0 81.0& 10.0 90

CON-

134 TABLE

2

PLASMA

LIPOPROTEIN

LIPID

CONCENTRATIONS

IN MI SURVIVORS

AND

CONTROL

MEN

Values are mean f SD. Lipoprotein densities: VLDL, d < 1.006 g/ml; LDL, d = 1.006-1.063 g/ml; HDL,, d = 1.125-1.21 g/ml. Conversion factor from mg/dl to SI units for cholesterol = 0.026; triglycerides HDL,, mg/dl

d = 1.063-1.125 = 0.011.

Controls (n = 60)

MI survivors 10 days (n = 60)

P value a

MI survivors 4 months (n = 60)

P value b

205.0 k 29.0 30.0 + 12.0 121.0+21.8 51.2& 7.1 22.1* 4.1 28.4+ 3.6

234.0 + 37.5 f 158.0* 38.8+ 12.1* 27.6+

48.7 10.5 31.5 3.1 2,l 3.1

< 0.0001 NS < 0.001 < 0.0001 c 0.0001 NS

235.0 + 37.7 + 158.0 f 38.9+ 11.4* 27.6*

< 0.0001 NS < 0.001 c 0.0001 < 0.0001 NS

118.Ok46.0 78.0 f 30.0 27.0* 9.0 16.1 f 9.0

202.0 + 140.0 f 39.0 f 21.0+

58.0 41.0 1.1.0 8.0

< i < <

192.0 + 39.0 144.0 + 30.0 38.8 f 12.6 19.5+ 5.9

< < < <

215.0+

245.Ok46.0

240.0+

< 0.0001

g/ml;

Cholesterol Total VLDL LDL HDL HDL, HDL,

32.0 14.3 22.2 3.3 4.1 4.5

Triglycerides Total VLDL LDL HDL Phosphohpids

35.0

a 10 days MI survivors compared to controls. b 4 months MI survivors compared to controls. MI survivors at 10 days compared to M.I. survivors

NS

at 4 months

Plasma apolipoprotein concentrations in MI survivors and controls (Table 3) Unlike lipids and lipoproteins, levels of apo A-I for MI survivors at 10 days compared to MI survivors at 4 months were significantly decreased in plasma and HDL fraction, while levels of apo B were significantly elevated in plasma and LDL fraction. This was also true when controls were compared with either the 10 days or 4 months MI TABLE

3

PLASMA

APOLIPOPROTEIN

CONCENTRATIONS

0.0001 0.0001 0.05 0.05

were not significant

38.0

0.0001 O.O@Ol 0.0001 0.02

for all the above parameters.

survivors. Series of experiments were done for MI survivors after 4 months which did not show any more changes in the apolipoproteins (results not shown). Accordingly, the statistical analysis for comparison was carried out between the control subjects and 4 months MI survivors. Ratios of lipids and apolipoproteins in MI survivors at 4 months and controls (Table 4) Table 4 shows the ratios of different parameters in MI survivors and control subjects. LDL-

IN MI SURVIVORS

AND

CONTROL

MEN

Values are means f SD. Controls (n = 60)

mg/dl Plasma: Apo Apo Apo LDL HDL HDL

A-I A-II B Apo B Apo A-I Apo A-II

a MI survivors b MI survivors ’ MI survivors

150.0 f 49.0+ 102.0 + 88.4 + 125.0 f 42.4+

16.0 5.5 15.6 13.5 10.4 6.5

MI survivors 10 days (n = 60)

P value a

MI survivors 4 months (n = 60)

P value b

P value ’

126.0+13.5 48.Ok 5.7 125.0* 17.1 108.0+ 13.4 106.0 f 10.3 41.9+ 5.3

< 0.001 NS < 0.001 < 0.0001 < 0.001 NS

110.0* 48.Ok 136.0 i118.0* 88.0* 43.0*

< 0.0001 NS < 0.0001 < 0.0001 < 0.0001 NS

< 0.0001 NS i 0.0001 < 0.0001 < 0.0001 NS

10 days compared to controls. 4 months compared to controls. 10 days compared to MI survivors

4 months.

7.4 3.3 11.2 11.2 8.4 4.7

135 TABLE

4

RATIOS OF LIPIDS AND APOLIPOPROTEINS SURVIVORS AND CONTROL MEN Values are mean + S.D. C = cholesterol; Controls (n = 60)

LDL-C/HDLC HDL,-C/HDL,-C LDL-C/HDL,-C Apo B/APO A-I Apo A-I/APO A-II

2.41 f 0.56 0.78 kO.15 5.69kl.44 0.68kO.12 4.00 f 0.65

IN

HDL,-cholesterol and apo A-I/ape significantly decreased in MI survivors pared to the controls.

MI

apo = apolipoprotein.

MI survivors 4 months (n = 60)

P value

4.08 k 0.65 0.41 f 0.09 15.06k4.70 1.24kO.21 2.97 kO.35

i < < < <

Pearson correlation coefficients between lipids and apolipoproteins in MI survivors at 4 months (Table 5) Apolipoprotein A-I correlated positively with HDL-cholesterol and negatively with total and LDL triglyceride, and with apo B. Apolipoprotein A-II correlated negatively with LDL- and VLDLcholesterol and positively with triglyceride, while apo B correlated positively with LDLand VLDL-cholesterol, total and LDL triglycerides. Negative correlations were observed for both HDL-cholesterol and HDL,-cholesterol compared to LDL- and VLDL-cholesterol and triglycerides. A strong positive correlation between HDL-

0.0001 0.0001 0.0001 0.0001 0.0001

cholesterol/HDL-cholesterol, LDL-cholesterol/HDL,-cholesterol and apo B/ape A-I were significantly elevated while HDL,-cholesterol/ TABLE

5

PEARSON MONTHS

CORRELATION

LDL C HDL C TG VLDL-C LDL TG Apo B For For For

A-II were when com-

COEFFICIENTS

BETWEEN

LIPIDS

AND

APOLIPOPROTEINS

IN MI SURVIVORS

A-I

A-II

B

HDL-C

HDL,-C

VLDL TG

- 0.05 0.36 - 0.45 0.01 - 0.29 - 0.25

- 0.26 0.03 0.26 - 0.29 0.13 0.10

0.43 -0.15 0.38 0.15 0.23 1.00

-0.35 1.00 - 0.26 - 0.29 - 0.01 -0.15

-0.39 0.53 -0.32 -0.31 - 0.29 0.07

0.26 - 0.28 0.85 0.09 0.79 0.23

AT 4

> 0.27 and i 0.35, P < 0.05. > 0.35 and < 0.60, P < 0.001. > 0.61, P < 0.0001.

TABLE

6

STEPWISE DISCRIMINANT ANALYSIS AND APOLIPOPROTEIN VARIABLES

BETWEEN

coefficients

AND

F

Significant variables

Classification

HDL,-C Apo B

1.82 0.47

0.94 0.61

149.84 74.05

Apo A-I VLDL-TG HDL-C T-TG LDL-C/HDL,-C

0.85 0.88 2.00 0.01 1.13

0.70 0.75 1.67 -0.05 1.65

59.01 50.20 43.80 38.62 38.37

Controls

function

CONTROLS

Patients

MI 4 MONTHS

Percent

correctly

SURVIVORS

classified

Case (sensitivity)

Controls (specificity)

90%

92%

FOR LIPOPROTEIN

Total

91%

Stepwise discriminant analysis is a form of multivariate analysis. The discriminant function selects from a set of variables those significantly (P < 0.001) classifying cases and controls as indicated by an F value above 8 and list these variables in descending order of significance. C = cholesterol; apo = apolipoprotein; TG = triglyceride; T = total serum.

136 cholesterol and HDL,-cholesterol was observed. Finally VLDL triglyceride was positively correlated to LDL-cholesterol, triglycerides and LDL triglyceride, while it correlated negatively with HDL-cholesterol.

Stepwise discriminant analysis between controls and MI survivors at 4 months for lipid lipoprotein and apolipoprotein variables (Table 6) Using stepwise discriminant analysis between controls and MI survivors at 4 months showed that the levels of plasma HDL,-cholesterol, apo B, apo A-I, VLDL triglyceride, HDL-cholesterol and plasma triglyceride in this order discriminated better between patients and controls. 92% and 90% were correctly classified by these functions in controls (specificity) and patients (sensitivity), respectively. The results in a total percentage had correctly classified 91% of subjects.

Discussion Most of the published reports on MI survivors have shown that lipids, lipoproteins and apolipoproteins do not change for 4 months after infarction. Our data are in agreement with these studies [24,25]. However, in the present study apo A-I, A-II and B showed a sharp change between 10 days and 4 months but remained stable after that. The reason for these changes is not clear but could be due to different genetic and/or environmental influences, diet, exercises and infarction size, which have been shown to affect the concentration of plasma lipids, lipoproteins and apolipoproteins [3,16,26-291. Beta-blockers used in this study had no clear effects on lipoprotein levels. The young MI survivors in the present study had higher levels of total VLDL and LDL triglycerides than the control subjects which agrees with most published reports [2,30,31]. The association of triglycerides and particularly in VLDL with CAD as risk factor is still controversial [2,9-111. The high level of triglyceride could either be caused by increased production of VLDL triglyceride (as VLDL is the main endogenous triglyceride carrier) or due to a decrease in its catabolic rate [2,32,33].

Elevated total and LDL-cholesterol but not VLDL-cholesterol levels observed in this study have been previously reported [2,12,31,34]. These changes may be due to changes in the intercomposition of LDL particles as a consequence of increased flux of VLDL through the plasma, diet or exercises. Alternatively, a decreased rate of LDL catabolism arising from LDL receptor activity could also explain this observation [26,27,35]. Elevated apo B levels and their correlation with LDL-cholesterol and other lipids reported in the present study indicates a possible metabolic and functional association among them [12,31,34]. In contrast to apo B, the finding of low levels of apo A-I and normal apo A-II among our young MI survivors is in agreement with other studies [14,16]. Low levels of total HDL- and HDL,-cholesterol but not of HDL,-cholesterol were observed in our patients. Further, reduction in HDL-cholesterol, seen in this study, occurs predominantly in the HDL, subfraction which confirms previous reports [2,5,8,36]. Discriminant analysis indicates that HDL,-cholesterol had a stronger association with MI than did either HDL-cholesterol or HDL,-cholesterol. Further, the inverse correlation seen in the present study between HDL, HDL,cholesterol and apo A-I with VLDL triglyceride may indicate that HDL and VLDL are metabolically linked to each other and could be regulated by the same mechanism [2,31,32]. A review of the literature showed that there is no general agreement on lipid indices as better discriminators for CAD [1,2,37]. Some investigators consider that HDL-cholesterol is a better discriminator of CAD than HDL,-cholesterol and apo A-I, while others reported the opposite [2,34,36,38]. Previous studies on MI survivors indicated that apo A-I and B are as effective discriminators as were total cholesterol, triglyceride, HDL- and HDL,-cholesterol (37,39-41). In our patients, HDL,-cholesterol, apo B and A-I showed stronger association with CAD. Other indices including VLDL triglyceride, HDL-cholesterol, the ratio of LDL-cholesterol/HDL-cholesterol and triglycerides were also useful predictors. In apo A-II levels no difference is observed between these patients and the controls which is in line with the findings of others [42]. Increased lipoprotein ratios of LDL-cholester-

137 LDL-cholesterol/ HDL,ol/ HDL-cholesterol, cholesterol and apo B/ape A-I [3,24,43] and decreased ratios of HDL,-cholesterol/HDL,-cholesterol and apo A-I/ape A-II were shown to be risk factors in CAD patients [2,34,42]. In the current study, similar ratios were seen in CAD patients. In conclusion, measurement of lipids and lipoproteins in MI survivors at 10 days and 4 months showed no significant changes, while apo B and A-I changed significantly at 4 months compared to 10 days after infarction. Our results emphasize the importance of HDL,-cholesterol, apo B and apo A-I as more useful indicators in assessing CAD risk than TG, TC, LDL-C and HDL-C. These factors should be investigated in detail in young MI patients and should be assessed in prospective studies. Acknowledgements We are especially grateful to Mr. Abdul Hamid Parker for his excellent technical assistance. We also thank Miss Basma Al-Ghotti, Miss Nabila Abdulhadi, Mrs. Suad Sobho and Mrs. Anagha Kamik for their technical assistance, and Mrs. Grace D’Silva and Mrs. Augustilia G. Pinto for their secretarial assistance. References Miller, N.E., High density lipoprotein as a predictor of clinical coronary heart disease. In: Fidge, N.H. and Nestel, P.J. (Eds.), Atherosclerosis, VII, Elsevier, Amsterdam, 1986, p. 61. Hamsten, A., Walldius, G., Dahlen, G., Johansson, B. and De Faire& Serum lipoproteins and apolipoproteins in young male survivors of myocardial infarction, Atherosclerosis, 59 (1986) 223. Castelli, W.P., Doyle, J.T., Gordon, T., Hames, C.G., Hjortland, M.C., Hulley, S.B., Kagan, A. and Zukel, W.J., HDL cholesterol and other lipids in coronary heart disease. The cooperative lipoprotein phenotyping study, Circulation, 55 (1977) 767. Miller, N.E., Forde, O.H., Thelle, D.S., et al., The Tromse heart study. High density lipoprotein and coronary heart disease. A prospective case-control study, Lancet, 1 (1977) 965. 5 Kaukola, S., Manninen, V. and Halonen, P.I., Serum lipids with special reference to HDL cholesterol and triglycerides in young male survivors of acute myocardial infarction, Acta Med. Stand., 208 (1980) 41.

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