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High-density lipoproteins in myocardial infarction survivors
High-Density
Lipoproteins in Myocardial Infarction Survivors
John J. Albers, Marion C. Cheung, and William R. Hazzard Subjects with existing coronary heart disease and those with many of the conditions associated with increased risk of coronary disease have reduced levels of high-density lipoprotein (HDL) cholesterol. Since HDL cholesterol is only one index of HDL composition, a reduction of HDL cholesterol could reflect a change in HDL composition and/or a decrease in all HDL constituents. Therefore the present studies assessed the major apolipoproteins of HDL, A-l and A-II, in addition to HDL cholesterol in 90 male myocardial infarction (Ml) survivors and their lipid-matched male controls. The MI survivors had significantly lower ( p < 0.01) A-l (112 f 2 mg/dl, mean + SEM), A-II (29 + 1 mg/dl), and HDL cholesterol (39 f 1 mg/dl) than the lipid-matched control group (A-l, 121 f 2; A-II, 33 i 1; HDL cholesterol, 43 f 1) and
than a population-based male control group (n = 172; A-l, 121 f 2; A-II, 33 zt 1; HDL cholesterol, 45 f 1). The HDL cholesterol/A-l ratio in the MI survivors was slightly lower than the ratio in the lipidmatched control group but significantly Iower(p < 0.02) than that in the population-based control group. The HDL cholesterol of both control groups was significantly negatively related to log triglyceride (r = -0.43). Similarly the HDL cholesterol of the Ml survivors was inversely correlated with log triglyceride (I = -0.51), but the slope of this relationship was significantly steeper in the MI survivors. These results are consistent with a relative decrease of HDL in MI survivors over and above that attributable to their increased triglyceride levels.
E
XISTING EVIDENCE suggests that high-density lipoprotein (HDL) may modulate the uptake of cholesterol by peripheral tissues’.’ and facilitate its transport to the liver for catabolism and excreti0n.j Many of the conditions associated with an increased risk of coronary heart disease, e.g., hypertriglyceridemia, obesity, diabetes mellitus. uremia, and hypercholesterolemia are associated with a reduced concentration of HDL cholestero1.4.5 Furthermore, subjects with existing coronary heart disease have lower levels of HDL cholesterol than healthy subjects in the same community.6*7 Thus it has been postulated that a reduction of plasma HDL concentration may accelerate the development of atherosclerosis and thereby increase the risk of coronary heart disease? HDL cholesterol is only one index of HDL concentration. A reduction in HDL cholesterol could reflect a change in composition of HDL, i.e., a decrease
From the Northwest Lipid Research Clinic, Department of Medicine. School of Medicine. University of Washington, Seattle, Washington. Received for publication June 20. 1977. Supported by NIH Contract NIHV 121574 (Lipid Metabolism Branch). Dr. Hazard is an investigator of the Howard Hughes Medical Institute. A preliminary report of part of this work has been published (Albers JJ, Cheung MC: Quanritation of apolipoprotein A-II of human plasma high density lipoproiein. Circulation 54 [Suppl 21: 367, 1976) and was presented at the 49th Annual Scientific Meeting of the American Heart Association, Miami, Florida. November 1976. Reprint requests should be addressed to Dr. John J. Albers. Northwest Lipid Research Clinic, 325 Ninth Ave.. Seattle, Wash. 98104. o 1978 by Grune & Stratton, Inc. 0026-0495/78/2704-09$01.00/0 Metabolism, Vol. 27, No. 4 (April), 1978
479
ALBERS,
480
CHEUNG,
AND HAZZARD
in the proportion of cholesterol relative to the other constituents including phospholipids and the principal apoproteins A-I and A-II. Alternatively, a reduction in HDL cholesterol could reflect proportional decreases in all constituents with no change in HDL composition. Moreover, a reduction of HDL cholesterol could be secondary to an increase in triglyceride,8 common in myocardial infarction survivors. In addition it is possible that lipoprotein A-I and/or A-II levels may correlate better with coronary disease risk than HDL cholesterol. Therefore the present studies were designed to compare A-I, A-II, and HDL cholesterol levels in male myocardial infarction survivors with those of a sex- and lipid-matched control group drawn from a population study. MATERIALS
AND METHODS
Subjects and Plasma Samples Blood was obtained from 90 adult male subjects 3 mo to 13 yi (mean f SD, 2 + 3 yr) after definite acute myocardial infarction (MI). The diagnosis of MI was established by chart review from clinical history. serial electrocardiograms, and blood chemistries according to established criteria.9 Blood was also obtained from two control groups without MI. The first group consisted of 172 males without history or symptoms of coronary heart disease who were selected at random from an industrial employee population; hence they were chosen without regard to their plasma lipid levels. The A-I and A-II levels had been previously measured.“” The second group consisted of 90 males, also without history or symptoms of coronary heart disease. who were matched with the male Ml survivors with regard to their age and total cholesterol and triglyceride levels. The control groups’ plasma samples were obtained from volunteers participating in a study of the prevalence of hyperlipoproteinemia in a population of industrial employees being conducted at the Northwest Lipid Research Clinic” and from men referred to the clinic for hyperlipidemia. Thirtyseven per cent of the lipid-matched subjects were chosen from among those in the first control group. No attempt was made to match either control group with the MI survivors with regard to other coronary risk factors or demographic characteristics. Venous blood was drawn from all subjects, after a 12-14 hr overnight fast, in Vacutainer tubes containing disodium EDTA. 1 mg/dl, according to standardized conditions.” The plasma was promptly separated by low-speed centrifugation at 4°C. Plasma samples were stored at -20°C with 0.05% (w/v) sodium azide in sealed Wheaton vials. (Freezing has previously been shown not 8.10 to affect apolipoprotein analysis. )
Immunoassay Procedures Plasma apolipo roteins A-l and A-II were determined by radial immunodiffusion assays as pre! viously described. .r” A-I and A-II were measured with a coefficient of variation of less than 5’;. Analyses of samples from the control and MI groups were performed at the same time, with the exception of the A-I results in the second control group, which had been measured during an earlier period.’
Lipid and Lipoprotein Analysis Cholesterol and glyceride were analyzed with the Technicon Auto Analyzer II.‘* For cholesterol analysis the coefficient of variation was less than 4O, and accuracy within 5”” of the target value; for glyceride analysis the coefficient of variation was less than 5’, and accuracy within lo”,,. To measure HDL cholesterol, 0.15 ml of 1 M MnC12 and 0.12 ml of sodium heparin (35 mg/ml, Riker) were added to a 3-ml aliquot of plasma, and cholesterol was measured in the supernatant fraction. For some samples precipitation was performed on I ml plasma.
Statistical Analyses The statistical significance of differences between mean values of various parameters was analyzed using Student’s t test. Pearson’s correlation coefficient, r. was used to show the degree
HDL IN MI SURVIVORS
481
of linear association among the different variables.13 Data were analyzed with (Statistical Package for Social Sciences) program’* on the CDC 6400 computer of Washington academic computer center.
a standard SPSS at the University
RESULTS
Age and Lipid Levels of MI Survivors and Matched Controls The MI subjects ranged in age from 31 to 61 yr, with a mean age of 52 yr. The means (and ranges) of cholesterol and triglyceride levels for the MI subjects were 231 mg/dl (156-330) and 180 mg/dl (56-1296) respectively. These values were significantly higher (p < 0.001) than the cholesterol and triglyceride levels of the adult males without MI (first control group, n = 172, mean age 44 yr): cholesterol 193 mg/dl (112-284) triglyceride 109 mg/dl (25-525). The lipid-matched control group had a mean age of 49 yr. The age of each MI subject differed from its matched control by a mean of 12%. The lipid-matched control group had a mean cholesterol level of 230 mg/dl and a mean triglyceride level of 175 mg/dl. The cholesterol and triglyceride levels of each MI subject differed from their matched control by means of 59, and 6’:,, respectively. Plasma Apolipoprotein A-I and A-II and HDL Cholesterol in MI Survivors The means and percentiles of HDL cholesterol and plasma A-I and A-II levels of the two control groups and the MI survivors are given in Table 1. Histograms depicting the distributions of plasma A-I and A-II and HDL cholesterol levels for the 90 male MI survivors and their lipid-matched controls are shown in Fig. 1. The population control group had an approximately normal A-I distribution (skewness, 0.57; kurtosis, 0.11) and a mean level of 120 mg/dl. The lipid-matched control group had a slightly skewed A-I distribution (skewness, 1.30; kurtosis, 0.82) but an almost identical mean level of 121 mg/dl (Table 1). For the MI survivors the frequency distribution of plasma A-I was unimodal and approximately normal (skewness, 0.39; kurtosis, 0.1 1) but shifted to lower values, with a mean level of 112 mg/dl, significantly lower than either control group (p < 0.001). Eighteen percent (16 of 90) of the MI surTable 1. A-l
and A-II
and HDL Cholesterol Levels
Apoprotein
Percentile
SubiectGrow
Mean f SEM
5th
10th
50th
Values
90th
95th
Apoiipoprotein A-l levels Population controls
120.5 f
90
96
121
148
158
Lipid-matched controls
121.0 h2.0
94
98
121
147
156
MI survivors
11 1.6 + 2.0
a3
87
110
138
140
Population controls
33.4 f 0.4
26
28
33
40
43
Lipid-matched controls
33.2 k 0.6
25
27
33
38
44
MI survivors
29.1 + 0.5
22
23
29
37
40
Population controls
45.0 + 0.9
29
32
44
59
67
Lipid-matched controls
43.4 l 1.2
27
32
43
56
63
MI survivors
38.9 f
22
27
39
52
57
1.5
Apolipoprotein A-II levels
HDL Cholesterol kvels
1 .o
482
ALBERS,
CHEUNG,
AND
HAZZARD
25
Apopofein
tiCL Cholesleml (mq/dll
Fig. 1. Distribution matched controls.
of HDL cholesterol
A-i bngldiJ
and apoproteins
AwwMin
A-llhq/dl)
A-l and A-II in MI survivors
and lipid-
vivors had A-I levels below 94 mg/dl, the fifth percentile cutoff point for the matched controls. The population control group had an approximately normal but somewhat skewed A-II distribution (skewness, 0.59; kurtosis, 0.31), with a mean level of 33 mg/dl. Similarly, the A-II distribution for the lipid-matched controls was slightly skewed (skewness, 1.07; kurtosis, 0.84), with an identical mean level (33 mg/dl). The A-II levels for the MI survivors were unimodal but shifted to lower values, with a mean of 29 mg/dl, significantly lower than either control group (p < 0.001). Twenty-eight percent (25 of 90) of the MI survivors had A-II
25
,31
1 20
40 HDL
60 cholesterol
100 (mg/:)
Fig.2. Relationship between loglo total plasma triglyceride and HDL cholesterol for MI survivors and lipid-matched controls.
HDL
IN
MI
SURVIVORS
483
Table 2.
High-Density
Lipoprotein
Composition Molar Ratio
Subject
Group
Population
controls
lipid-matched
controls
MI survivors Mean
& SEM.
Molecular
weights
HDLCH*/A-I
HDL CH/A-II
A-I/A-II
27.2
zt 0.47
60.4
zt 1 .O
2.23
f
0.021:
26.2
f
0.5
58.8
zt 1.2
2.25
f
0.021
25.7
f
0.5
60.3
+
2.36
ziz 0.03
of A-l, A-II, and cholesterol
1.3
are assumed
to be 28,300,
17,400
and
386,
respectively.
lHDl tp
CH refers
to cholesterol
< 0.02
versus
Ml survivors.
$p < 0.01
versus
Ml survivors.
in the heparin-manganese
supernatant.”
levels below the fifth percentile cutoff of 26 mg/dl among the population controls. The mean HDL cholesterol level of the MI survivors was significantly lower than that of the population-based control group (p < 0.01) and the lipidmatched controls (p < 0.01). The mean HDL cholesterol level of the population control group was significantly inversely related to log total plasma triglyceride (slope, -0.008; r = -0.413; p < 0.01). Similarly, the HDL cholesterol levels of both the matched controls and the MI survivors were inversely correlated with log triglyceride (p < 0.01; Fig. 2). However, the slope of the regression line for the MI survivors was significantly steeper (p < 0.05) than for either control group. Of note, the A-I and A-II levels of the MI survivors and their control groups did not correlate significantly with log triglyceride. The HDL cholesterol/A-I ratio of the population control group was significantly higher (p < 0.02) than those of the MI survivors and the lipidmatched control group (Table 2). The HDL cholesterol/A-II ratio of the MI survivors was similar to that of both control groups. The A-I/A-II ratio of the MI survivors was statistically significantly higher (p < 0.01) than that found in either control group. DISCUSSION
Recent epidemiologic data have indicated an inverse relationship between HDL cholesterol levels and the prevalence6 and incidence’ of coronary heart disease. Furthermore, an inverse relationship between HDL cholesterol levels and cholesterol pool size was found among hypercholesterolemic subjects.4 An above-average life expectancy was demonstrated by subjects with high levels of HDL cholesterol.‘5 These data suggest that low HDL levels may lead to an increased risk of coronary heart disease. Conversely, high levels may exert a protective effect. Consistent with this concept is our preliminary observation that MI survivors have lower levels of HDL cholesterol and apolipoprotein A-I levels’ than population-based controls. In the present study we have extended these observations and showed that MI survivors have lower levels of apolipoproteins A-I and A-II and HDL cholesterol than healthy adult males. Since raised triglyceride levels were common in our subjects with coronary heart disease (237: had levels greater than 210 mg/dl) and since we found a reciprocal relationship between HDL cho-
ALBERS,
484
CHEUNG,
AND HAZZARD
lesterol and triglyceride levels in all groups, the lower HDL cholesterol levels could, in part, reflect the higher triglyceride levels in MI subjects, as suggested level by Carlson and Ericsson. I6 However, whereas the mean HDL cholesterol of the lipid-matched control group was 4% lower than that of the non-lipidmatched controls, the mean HDL cholesterol of the MI survivors was an additional 10% lower. In addition, the normotriglyceridemic MI survivors (below the 95th percentile of the population-based male control group) had signiticantly lower HDL cholesterol (mean 39.9 mg/dl, lower by 8%) than the corresponding normotriglyceridemic controls (mean 43.2 mg/dl). Thus only a minority of the decrease in HDL cholesterol levels found in the MI survivors could be attributed to their higher triglyceride levels. Of note, A-I and A-II levels were significantly depressed in the MI survivors (by 8% and 9%, respectively) relative to the corresponding levels in lipidmatched controls, which were in turn nearly identical to those of the population-based male control group. This is consistent with the observation that raised triglyceride levels have a greater effect upon HDL cholesterol than HDL apoprotein levels.’ Thus whereas a portion of the reduced HDL cholesterol in MI survivors may reflect the selective loss of cholesterol from HDL molecules of relatively normal A-I and A-II content (related to their degree of hypertriglyceridemia), the remainder would appear likely to reflect reduction in both the principal apolipoproteins and their bound cholesterol. If atherogenesis relates more closely to the latter phenomenon [as might be inferred from the yetnormal health of the lipid- (and approximately age-) matched controls], then A-I and A-II levels may prove more potent indices of coronary risk than total HDL cholesterol levels. However, especially since the present observations were confined to MI survivors (and an infarction may conceivably cause a decrease in HDL and/or its constituents), the roles of apolipoproteins A-I and A-II as predictors of coronary disease independent of HDL cholesterol levels must be appraised in prospective studies. We previously reported that MI subjects had a low molar ratio of HDL cholesterol to A-I.* In the present study the ratio of HDL cholesterol to A-I was only slightly lower in the MI survivors than the matched controls. Both were significantly lower (p < 0.02) than the population-based male control group, suggesting that hyperlipidemia per se alters the HDL cholesterol/A-I ratio. The amount of HDL cholesterol associated with A-I or A-II was similar for MI survivors and their matched controls, although the MI survivors had a slightly higher A-I/A-II ratio. We have previously shown that the density 1.063-1.10 HDL fraction has a significantly higher A-I/A-II ratio than the density 1.10P1.21 fraction. lo Thus this small but statistically significant difference in total apoprotein compositon could reflect a redistribution of HDL subclasses and/or compositional alteration of the HDL molecules. Additional studies are needed to assess the factors that alter HDL polypeptide composition. ACKNOWLEDGMENT We thank G. Russell Warnick and the Northwest Lipid Research Clinic Core for assistance in lipid analysis. We are indebted to Dr. Arno Motulsky and Medical Genetics for the plasma samples from the MI survivors.
Laboratory staff the Division of
HDL IN MI SURVIVORS
485
REFERENCES 1. Stein 0, Stein Y: High density lipoproteins reduce the uptake of low density lipoproteins by human endothelial cells in culture. Biochim Biophys Acta 431:363-368. 1976 2. Carew TE, Koschinsky T. Hayes SB, et al: A mechanism by which high-density lipoproteins may slow the atherogenic process. Lancet 2:1315-1376, 1976 3. Glomset JA: The plasma lecithin:cholesterol acyltransferase reaction. J Lipid Res 9:155-167, 1968 4. Miller GJ, Miller NE: Plasma-high-density-lipoprotein concentration and development of ischaemic heart-disease. Lancet 1: 16-19, 1975 5. Bagdade JD, Albers JJ: Plasma-highdensity lipoprotein concentrations in chronic hemodialysis and renal transplant patients. N Engl J Med 296:1436-1439, 1977 6. Rhoads GG, Gulbrandsen CL, Kagan A: Serum lipoproteins and coronary heart disease in a population study of Hawaii Japanese men. N Engl J Med 294293-298, 1976 7. Gordon T, Castelli WP, Hjortland MC, et al: High density lipoprotein as a protective factor against coronary heart disease. The Framingham study. Am J Med 62:7077714, 1977 8. Albers JJ, Wahl PW, Cabana VG, et al: Quantitation of apolipoprotein A-I of human plasma high density lipoprotein. Metabolism 25:6333644, 1975 9. Goldstein JL. Hazzard WR, Schrott HG, et al: Hyperlipidemia in coronary heart disease.
I. Lipid levels in 500 survivors of myocardial infarction. J Clin Invest 52: 1533- 1577, 1973 IO. Cheung MC, Albers JJ: The measurement of apolipoprotein A-I and A-II levels in men and women by immunoassay. J Clin Invest 60:43-50, 1977 1 I. Hoover JJ, Tyroler HA: Epidemiologic concepts of the LRC program-Report prepared by the LRC Prevalence Committee. Paper presented at the American Public Health Association Meeting, Chicago, November 19. 1975 (unpublished) 12. Lipid Research Clinics Program Manual of Laboratory Operations, vol I. Washington. D.C., U.S. GPO, DHEW Publication No. (NIH) 75-628, 1974 13. Snedecor GW, Cochran WC: Statistical Methods (ed 6). Ames. Iowa, Iowa State Univ Pr, 1967 14. Nie NH. Hull CH, Jenkins JG, et al: SPSS (Statistical Package for the Social Sciences) for the CDC (Control Data Corporation) 6400 Series (ed 2). New York, McGrawHill, 1975 15. Glueck CJ, Fallat RW, Millett F, et al: Familial hyper-alpha-lipoproteinemia: Studies in eighteen kindreds. Metabolism 24: 124331265. 1975 16. Carlson LA, Ericsson M: Quantitative and qualitative serum lipoprotein analysis. Part 2. Studies in male survivors of myocardial infarction. Atherosclerosis 21:435-450. 1975
Lipoproteins in Myocardial Infarction Survivors
John J. Albers, Marion C. Cheung, and William R. Hazzard Subjects with existing coronary heart disease and those with many of the conditions associated with increased risk of coronary disease have reduced levels of high-density lipoprotein (HDL) cholesterol. Since HDL cholesterol is only one index of HDL composition, a reduction of HDL cholesterol could reflect a change in HDL composition and/or a decrease in all HDL constituents. Therefore the present studies assessed the major apolipoproteins of HDL, A-l and A-II, in addition to HDL cholesterol in 90 male myocardial infarction (Ml) survivors and their lipid-matched male controls. The MI survivors had significantly lower ( p < 0.01) A-l (112 f 2 mg/dl, mean + SEM), A-II (29 + 1 mg/dl), and HDL cholesterol (39 f 1 mg/dl) than the lipid-matched control group (A-l, 121 f 2; A-II, 33 i 1; HDL cholesterol, 43 f 1) and
than a population-based male control group (n = 172; A-l, 121 f 2; A-II, 33 zt 1; HDL cholesterol, 45 f 1). The HDL cholesterol/A-l ratio in the MI survivors was slightly lower than the ratio in the lipidmatched control group but significantly Iower(p < 0.02) than that in the population-based control group. The HDL cholesterol of both control groups was significantly negatively related to log triglyceride (r = -0.43). Similarly the HDL cholesterol of the Ml survivors was inversely correlated with log triglyceride (I = -0.51), but the slope of this relationship was significantly steeper in the MI survivors. These results are consistent with a relative decrease of HDL in MI survivors over and above that attributable to their increased triglyceride levels.
E
XISTING EVIDENCE suggests that high-density lipoprotein (HDL) may modulate the uptake of cholesterol by peripheral tissues’.’ and facilitate its transport to the liver for catabolism and excreti0n.j Many of the conditions associated with an increased risk of coronary heart disease, e.g., hypertriglyceridemia, obesity, diabetes mellitus. uremia, and hypercholesterolemia are associated with a reduced concentration of HDL cholestero1.4.5 Furthermore, subjects with existing coronary heart disease have lower levels of HDL cholesterol than healthy subjects in the same community.6*7 Thus it has been postulated that a reduction of plasma HDL concentration may accelerate the development of atherosclerosis and thereby increase the risk of coronary heart disease? HDL cholesterol is only one index of HDL concentration. A reduction in HDL cholesterol could reflect a change in composition of HDL, i.e., a decrease
From the Northwest Lipid Research Clinic, Department of Medicine. School of Medicine. University of Washington, Seattle, Washington. Received for publication June 20. 1977. Supported by NIH Contract NIHV 121574 (Lipid Metabolism Branch). Dr. Hazard is an investigator of the Howard Hughes Medical Institute. A preliminary report of part of this work has been published (Albers JJ, Cheung MC: Quanritation of apolipoprotein A-II of human plasma high density lipoproiein. Circulation 54 [Suppl 21: 367, 1976) and was presented at the 49th Annual Scientific Meeting of the American Heart Association, Miami, Florida. November 1976. Reprint requests should be addressed to Dr. John J. Albers. Northwest Lipid Research Clinic, 325 Ninth Ave.. Seattle, Wash. 98104. o 1978 by Grune & Stratton, Inc. 0026-0495/78/2704-09$01.00/0 Metabolism, Vol. 27, No. 4 (April), 1978
479
ALBERS,
480
CHEUNG,
AND HAZZARD
in the proportion of cholesterol relative to the other constituents including phospholipids and the principal apoproteins A-I and A-II. Alternatively, a reduction in HDL cholesterol could reflect proportional decreases in all constituents with no change in HDL composition. Moreover, a reduction of HDL cholesterol could be secondary to an increase in triglyceride,8 common in myocardial infarction survivors. In addition it is possible that lipoprotein A-I and/or A-II levels may correlate better with coronary disease risk than HDL cholesterol. Therefore the present studies were designed to compare A-I, A-II, and HDL cholesterol levels in male myocardial infarction survivors with those of a sex- and lipid-matched control group drawn from a population study. MATERIALS
AND METHODS
Subjects and Plasma Samples Blood was obtained from 90 adult male subjects 3 mo to 13 yi (mean f SD, 2 + 3 yr) after definite acute myocardial infarction (MI). The diagnosis of MI was established by chart review from clinical history. serial electrocardiograms, and blood chemistries according to established criteria.9 Blood was also obtained from two control groups without MI. The first group consisted of 172 males without history or symptoms of coronary heart disease who were selected at random from an industrial employee population; hence they were chosen without regard to their plasma lipid levels. The A-I and A-II levels had been previously measured.“” The second group consisted of 90 males, also without history or symptoms of coronary heart disease. who were matched with the male Ml survivors with regard to their age and total cholesterol and triglyceride levels. The control groups’ plasma samples were obtained from volunteers participating in a study of the prevalence of hyperlipoproteinemia in a population of industrial employees being conducted at the Northwest Lipid Research Clinic” and from men referred to the clinic for hyperlipidemia. Thirtyseven per cent of the lipid-matched subjects were chosen from among those in the first control group. No attempt was made to match either control group with the MI survivors with regard to other coronary risk factors or demographic characteristics. Venous blood was drawn from all subjects, after a 12-14 hr overnight fast, in Vacutainer tubes containing disodium EDTA. 1 mg/dl, according to standardized conditions.” The plasma was promptly separated by low-speed centrifugation at 4°C. Plasma samples were stored at -20°C with 0.05% (w/v) sodium azide in sealed Wheaton vials. (Freezing has previously been shown not 8.10 to affect apolipoprotein analysis. )
Immunoassay Procedures Plasma apolipo roteins A-l and A-II were determined by radial immunodiffusion assays as pre! viously described. .r” A-I and A-II were measured with a coefficient of variation of less than 5’;. Analyses of samples from the control and MI groups were performed at the same time, with the exception of the A-I results in the second control group, which had been measured during an earlier period.’
Lipid and Lipoprotein Analysis Cholesterol and glyceride were analyzed with the Technicon Auto Analyzer II.‘* For cholesterol analysis the coefficient of variation was less than 4O, and accuracy within 5”” of the target value; for glyceride analysis the coefficient of variation was less than 5’, and accuracy within lo”,,. To measure HDL cholesterol, 0.15 ml of 1 M MnC12 and 0.12 ml of sodium heparin (35 mg/ml, Riker) were added to a 3-ml aliquot of plasma, and cholesterol was measured in the supernatant fraction. For some samples precipitation was performed on I ml plasma.
Statistical Analyses The statistical significance of differences between mean values of various parameters was analyzed using Student’s t test. Pearson’s correlation coefficient, r. was used to show the degree
HDL IN MI SURVIVORS
481
of linear association among the different variables.13 Data were analyzed with (Statistical Package for Social Sciences) program’* on the CDC 6400 computer of Washington academic computer center.
a standard SPSS at the University
RESULTS
Age and Lipid Levels of MI Survivors and Matched Controls The MI subjects ranged in age from 31 to 61 yr, with a mean age of 52 yr. The means (and ranges) of cholesterol and triglyceride levels for the MI subjects were 231 mg/dl (156-330) and 180 mg/dl (56-1296) respectively. These values were significantly higher (p < 0.001) than the cholesterol and triglyceride levels of the adult males without MI (first control group, n = 172, mean age 44 yr): cholesterol 193 mg/dl (112-284) triglyceride 109 mg/dl (25-525). The lipid-matched control group had a mean age of 49 yr. The age of each MI subject differed from its matched control by a mean of 12%. The lipid-matched control group had a mean cholesterol level of 230 mg/dl and a mean triglyceride level of 175 mg/dl. The cholesterol and triglyceride levels of each MI subject differed from their matched control by means of 59, and 6’:,, respectively. Plasma Apolipoprotein A-I and A-II and HDL Cholesterol in MI Survivors The means and percentiles of HDL cholesterol and plasma A-I and A-II levels of the two control groups and the MI survivors are given in Table 1. Histograms depicting the distributions of plasma A-I and A-II and HDL cholesterol levels for the 90 male MI survivors and their lipid-matched controls are shown in Fig. 1. The population control group had an approximately normal A-I distribution (skewness, 0.57; kurtosis, 0.11) and a mean level of 120 mg/dl. The lipid-matched control group had a slightly skewed A-I distribution (skewness, 1.30; kurtosis, 0.82) but an almost identical mean level of 121 mg/dl (Table 1). For the MI survivors the frequency distribution of plasma A-I was unimodal and approximately normal (skewness, 0.39; kurtosis, 0.1 1) but shifted to lower values, with a mean level of 112 mg/dl, significantly lower than either control group (p < 0.001). Eighteen percent (16 of 90) of the MI surTable 1. A-l
and A-II
and HDL Cholesterol Levels
Apoprotein
Percentile
SubiectGrow
Mean f SEM
5th
10th
50th
Values
90th
95th
Apoiipoprotein A-l levels Population controls
120.5 f
90
96
121
148
158
Lipid-matched controls
121.0 h2.0
94
98
121
147
156
MI survivors
11 1.6 + 2.0
a3
87
110
138
140
Population controls
33.4 f 0.4
26
28
33
40
43
Lipid-matched controls
33.2 k 0.6
25
27
33
38
44
MI survivors
29.1 + 0.5
22
23
29
37
40
Population controls
45.0 + 0.9
29
32
44
59
67
Lipid-matched controls
43.4 l 1.2
27
32
43
56
63
MI survivors
38.9 f
22
27
39
52
57
1.5
Apolipoprotein A-II levels
HDL Cholesterol kvels
1 .o
482
ALBERS,
CHEUNG,
AND
HAZZARD
25
Apopofein
tiCL Cholesleml (mq/dll
Fig. 1. Distribution matched controls.
of HDL cholesterol
A-i bngldiJ
and apoproteins
AwwMin
A-llhq/dl)
A-l and A-II in MI survivors
and lipid-
vivors had A-I levels below 94 mg/dl, the fifth percentile cutoff point for the matched controls. The population control group had an approximately normal but somewhat skewed A-II distribution (skewness, 0.59; kurtosis, 0.31), with a mean level of 33 mg/dl. Similarly, the A-II distribution for the lipid-matched controls was slightly skewed (skewness, 1.07; kurtosis, 0.84), with an identical mean level (33 mg/dl). The A-II levels for the MI survivors were unimodal but shifted to lower values, with a mean of 29 mg/dl, significantly lower than either control group (p < 0.001). Twenty-eight percent (25 of 90) of the MI survivors had A-II
25
,31
1 20
40 HDL
60 cholesterol
100 (mg/:)
Fig.2. Relationship between loglo total plasma triglyceride and HDL cholesterol for MI survivors and lipid-matched controls.
HDL
IN
MI
SURVIVORS
483
Table 2.
High-Density
Lipoprotein
Composition Molar Ratio
Subject
Group
Population
controls
lipid-matched
controls
MI survivors Mean
& SEM.
Molecular
weights
HDLCH*/A-I
HDL CH/A-II
A-I/A-II
27.2
zt 0.47
60.4
zt 1 .O
2.23
f
0.021:
26.2
f
0.5
58.8
zt 1.2
2.25
f
0.021
25.7
f
0.5
60.3
+
2.36
ziz 0.03
of A-l, A-II, and cholesterol
1.3
are assumed
to be 28,300,
17,400
and
386,
respectively.
lHDl tp
CH refers
to cholesterol
< 0.02
versus
Ml survivors.
$p < 0.01
versus
Ml survivors.
in the heparin-manganese
supernatant.”
levels below the fifth percentile cutoff of 26 mg/dl among the population controls. The mean HDL cholesterol level of the MI survivors was significantly lower than that of the population-based control group (p < 0.01) and the lipidmatched controls (p < 0.01). The mean HDL cholesterol level of the population control group was significantly inversely related to log total plasma triglyceride (slope, -0.008; r = -0.413; p < 0.01). Similarly, the HDL cholesterol levels of both the matched controls and the MI survivors were inversely correlated with log triglyceride (p < 0.01; Fig. 2). However, the slope of the regression line for the MI survivors was significantly steeper (p < 0.05) than for either control group. Of note, the A-I and A-II levels of the MI survivors and their control groups did not correlate significantly with log triglyceride. The HDL cholesterol/A-I ratio of the population control group was significantly higher (p < 0.02) than those of the MI survivors and the lipidmatched control group (Table 2). The HDL cholesterol/A-II ratio of the MI survivors was similar to that of both control groups. The A-I/A-II ratio of the MI survivors was statistically significantly higher (p < 0.01) than that found in either control group. DISCUSSION
Recent epidemiologic data have indicated an inverse relationship between HDL cholesterol levels and the prevalence6 and incidence’ of coronary heart disease. Furthermore, an inverse relationship between HDL cholesterol levels and cholesterol pool size was found among hypercholesterolemic subjects.4 An above-average life expectancy was demonstrated by subjects with high levels of HDL cholesterol.‘5 These data suggest that low HDL levels may lead to an increased risk of coronary heart disease. Conversely, high levels may exert a protective effect. Consistent with this concept is our preliminary observation that MI survivors have lower levels of HDL cholesterol and apolipoprotein A-I levels’ than population-based controls. In the present study we have extended these observations and showed that MI survivors have lower levels of apolipoproteins A-I and A-II and HDL cholesterol than healthy adult males. Since raised triglyceride levels were common in our subjects with coronary heart disease (237: had levels greater than 210 mg/dl) and since we found a reciprocal relationship between HDL cho-
ALBERS,
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CHEUNG,
AND HAZZARD
lesterol and triglyceride levels in all groups, the lower HDL cholesterol levels could, in part, reflect the higher triglyceride levels in MI subjects, as suggested level by Carlson and Ericsson. I6 However, whereas the mean HDL cholesterol of the lipid-matched control group was 4% lower than that of the non-lipidmatched controls, the mean HDL cholesterol of the MI survivors was an additional 10% lower. In addition, the normotriglyceridemic MI survivors (below the 95th percentile of the population-based male control group) had signiticantly lower HDL cholesterol (mean 39.9 mg/dl, lower by 8%) than the corresponding normotriglyceridemic controls (mean 43.2 mg/dl). Thus only a minority of the decrease in HDL cholesterol levels found in the MI survivors could be attributed to their higher triglyceride levels. Of note, A-I and A-II levels were significantly depressed in the MI survivors (by 8% and 9%, respectively) relative to the corresponding levels in lipidmatched controls, which were in turn nearly identical to those of the population-based male control group. This is consistent with the observation that raised triglyceride levels have a greater effect upon HDL cholesterol than HDL apoprotein levels.’ Thus whereas a portion of the reduced HDL cholesterol in MI survivors may reflect the selective loss of cholesterol from HDL molecules of relatively normal A-I and A-II content (related to their degree of hypertriglyceridemia), the remainder would appear likely to reflect reduction in both the principal apolipoproteins and their bound cholesterol. If atherogenesis relates more closely to the latter phenomenon [as might be inferred from the yetnormal health of the lipid- (and approximately age-) matched controls], then A-I and A-II levels may prove more potent indices of coronary risk than total HDL cholesterol levels. However, especially since the present observations were confined to MI survivors (and an infarction may conceivably cause a decrease in HDL and/or its constituents), the roles of apolipoproteins A-I and A-II as predictors of coronary disease independent of HDL cholesterol levels must be appraised in prospective studies. We previously reported that MI subjects had a low molar ratio of HDL cholesterol to A-I.* In the present study the ratio of HDL cholesterol to A-I was only slightly lower in the MI survivors than the matched controls. Both were significantly lower (p < 0.02) than the population-based male control group, suggesting that hyperlipidemia per se alters the HDL cholesterol/A-I ratio. The amount of HDL cholesterol associated with A-I or A-II was similar for MI survivors and their matched controls, although the MI survivors had a slightly higher A-I/A-II ratio. We have previously shown that the density 1.063-1.10 HDL fraction has a significantly higher A-I/A-II ratio than the density 1.10P1.21 fraction. lo Thus this small but statistically significant difference in total apoprotein compositon could reflect a redistribution of HDL subclasses and/or compositional alteration of the HDL molecules. Additional studies are needed to assess the factors that alter HDL polypeptide composition. ACKNOWLEDGMENT We thank G. Russell Warnick and the Northwest Lipid Research Clinic Core for assistance in lipid analysis. We are indebted to Dr. Arno Motulsky and Medical Genetics for the plasma samples from the MI survivors.
Laboratory staff the Division of
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