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Serum lipoproteins and apolipoproteins in young male survivors of myocardial infarction
Atherosclerosrs.
59 (1986)
Elsevier Scientific
223
223-235
Publishers
Ireland.
Ltd.
ATH 03729
Serum Lipoproteins
A. Hamsten Depurtments
and Apolipoproteins in Young Male Survivors of Myocardial Infarction
‘, G. Walldius of’ Medicine
2, G. Dahlkn 3, B. Johansson
and 4 CIm~cal Cheml.rtty,
’ Depurtment .’Department
of Medane.
of Clinrc~ul
Dundetyd
Karolinsku
Chemistty,
Hospitul; Hospital,
Central
4 and U. De Faire ’
King GustuJ V Reseurch Instrtute Stockholm;
Hospital,
und
cmd
Boden (Sweden)
(Received 21 May, 1985) (Revised, received 9 September, 1985) (Accepted 11 September. 1985)
Summary Concentrations of serum lipoprotein lipids and apolipoproteins A-I, A-II and B were determined 3-6 months after myocardial infarction in 116 males below the age of 45 and in 116 age-matched controls. Among single variables the sum of cholesterol concentration in VLDL and LDL divided by the HDL cholesterol level was the best discriminator between patients and controls. The concentrations of serum triglycerides, apolipoprotein B, VLDL triglycerides and cholesterol. serum cholesterol, HDL cholesterol and LDL triglycerides, in that order, were better discriminators than was LDL cholesterol level. Among variables reflecting HDL concentration and composition HDL cholesterol was the best discriminator followed by HDL, cholesterol, apolipoprotein A-I and the HDL cholesterol/apolipoprotein A-I ratio. Multivariate analysis indicated independent significance of elevated VLDL lipid and LDL cholesterol concentrations, and a decreased HDL cholesterol concentration, in relation to MI. The present data suggest that a disturbed triglyceride metabolism, in addition to elevated LDL and decreased HDL cholesterol levels, has an independent and pathogenetic significance for MI at a young age.
Key words:
Apolipoproteins
- Lipoproteins
- Mj~ocurdial infarction
- Young adults
Introduction This study was supported by grants from the Swedish National Association against Heart and Chest Diseases. the Swedish Medical Research Council (19x-204). the Tore Nilsson Foundation for Medical Research, and the Association of Swedish Oleomargarine Manufacturers for Research on Nutritional Physiology. Author’s address: Anders Hamsten, Department of Meditine, Danderyd Hospital. S-182 88 Danderyd. Sweden. 0165-0327/86/$03.50
fi> 1986 Elsevier Science Publishers
The relationship between serum lipoprotein concentrations and coronary heart disease (CHD) has been most evident for low density lipoprotein (LDL) cholesterol. [ 1,2]. High density lipoprotein (HDL) cholesterol concentrations have been even more strongly predictive in some studies [3,4]. Multivariate analyses of most prospective studies
B.V. (Biomedical
Division)
224 have implied that correlations between serum triglyceride level and CHD derive from secondary associations, whereas in one study triglycerides have emerged as a significant and independent risk factor [6,7]. In recent studies the serum concentrations of the apohpoproteins, have been considered even better discriminators between patients with CHD and healthy controls [8-141 than the levels of total or lipoprotein lipids. In the present study, which is part of a comprehensive clinical and metabolic research programme in young post-infarction patients, the independent relation to myocardial infarction (MI) and discriminative power of serum lipoprotein levels and composition with respect to lipids and apolipoproteins was investigated in representative young male survivors of MI from a geographically and demographically well characterized area. On the basis of previous results obtained in young post-infarction patients [15], and the finding of hypertriglyceridemia as the dominating lipoprotein disturbance in this material, special emphasis was placed on the multivariate evaluation of serum triglycerides and VLDL lipids. Materials and Methods Putients Between May 1980 and September 1982 137 male patients below the age of 45 were admitted to the 11 hospitals in the county of Stockholm with a diagnosis of definite myocardial infarction [16]. Thirty-five patients were immigrants (residence in Sweden, mean t_ SD, 12.5 5 8.0; range 1.0-36.5 years), of whom 10 were born in Finland. Ten patients died during the stay in hospital. One hundred twenty-seven consecutive male survivors were subsequently referred to the Department of Medicine, Danderyd Hospital, for metabolic and cardiological investigations. Of these patients 2 died in the early post-infarction period, 7 declined the metabolic investigation and for other reasons a complete evaluation was not performed in 2 patients. Thus 116 male patients (92% of male survivors) constitute the material of the present study. Six patients had a history of a previous MI 3-60 months (28 + 22 months) prior to the present MI. All patients were admitted as out-patients and
investigated 3-6 months after the MI. Clinical data were abstracted from the medical records acquired during the admission to the coronary care units. In addition a medical history was obtained by a structured interview and questionnaires at the time of the metabolic investigation. Controls For each patient a male control subject from the same residence area was randomly selected from a register of all residents in the county after matching with regard to age. Previous medical history and risk factors were established in the same way among controls as in the patient group. None of the controls had a history suggestive of angina pectoris or electrocardiographic signs indicative of CHD during a maximal exercise stress test (Minnesota codes 4.1 and 4.2). All controls were included in the study, irresspective of the presence of metabolic disturbances or additional risk indicators. General treatment and medication After discharge from the referring hospitals all patients were seen by 1 cardiologist within 2 weeks and then at regular intervals during follow-up. Fifty-five of 101 patients who were regular smokers at the time of the MI stopped smoking between the onset of infarction and the present study. No dietary information was given prior to the metabolic evaluation. General treatment with betablockers was not instituted in the coronary care units in this young post-infarction group. Instead, beta-blockers had been prescribed individually according to the local policy in the referring units. In most patients the indication for beta-blockade was cardioprotection after myocardial infarction. Previously instituted medication was not changed for this study, except for the restriction of ongoing beta-blockade to metoprolol at the first visit to the outpatients clinic more than 2 months before the lipoprotein determinations. If major side-effects attributable to beta-blocker treatment occurred with metoprolol and the indication for beta-blockade was strong, metoprolol was replaced by atenolol. At the time of the study 35 patients were entirely without medication and 60 were treated with metoprolol and 2 with atenolol only. Three and 5 patients, respectively, were treated with iso-
225 sorbide-dinitrate and niphedipine in addition to metoprolol. Four had been prescribed a combination of drugs from all 3 groups. Furosemide together with potassium supplementation was used in 20 patients, 7 of whom had no other medication. The majority of the patients had no (n = 58) or only mild angina (New York Heart Association classes I-II; n = 44) during the early post-infarction period. Severe peripheral atherosclerosis with incapacitating intermittent claudication was present in two patients and manifest diabetes in 6. Coronary artery by-pass surgery had not been performed in any patient in the period between MI and this investigation. One patient had been operated upon 6 years prior to the study. All patients were without clinical signs of cardiac decompensation at the time of the metabolic evaluation. No patient had used lipid-lowering drugs during 6 months before the lipoprotein determinations. There was no clinical or laboratory evidence of thyroid dysfunction in any of the patients or controls. Serum values for transaminases and creatinine were normal in all subjects. Blood sampling Blood samples were drawn 3-6 months after the MI (3.5 + 1.2, range 2.5-12 months), when the patients were considered to be in a stable clinical and metabolic state. Matched controls were investigated simultaneously with the patients to avoid the influence of seasonal variations on lipoprotein levels. In both patients and controls blood sampling was evenly distributed over the year. Antecubital vein puncture was performed between 8.00 h and 9.30 h after a lo-min rest in the supine position. All subjects had fasted for 12 h prior to blood sampling, during which time smokers were asked to refrain from smoking. Venous blood for analyses of apolipoproteins A-I, A-II and B was allowed to clot for 2 h and serum was then prepared by centrifugation at 1900 x g for 10 min and stored at -70°C until analyzed. Quantitation of total serum and lipoprotein lipids Venous blood was allowed to clot at room temperature for 2 h. Serum was recovered after centrifugation at 5000 X g and 5% Na-EDTA was
added to a final concentration of 0.05%. Lipoprotein analysis was performed by preparative ultracentrifugation and quantitative lipid analysis [17]. Cholesterol [18] and triglycerides [19] were determined on an Ultrolab” (LKB) after chloroform/methanol extraction [20] of whole serum, the VLDL fraction, the infranatant after ultracentrifugation (containing LDL and HDL) and of the supernatant (HDL) after precipitation. LDL values were calculated by difference. Only samples with a total recovery of cholesterol and triglycerides in the 3 lipoprotein classes of 100 + 10% have been used. HDL, was obtained as a bottom fraction in which cholesterol was determined after one preparative ultracentrifugal spin in a fixed angle rotor (Beckman model L 5-75) at density 1.125 kg/l [21]. Total HDL was obtained as described above. HDL, cholesterol was then calculated as the difference between total HDL and HDL, cholesterol. The intraassay variation was 1.8 standard deviation % (SD%) for low cholesterol values and 2.1 SD% for high cholesterol values. The corresponding values for low and high triglyceride values were 2.9 SD% and 2.2 SD%, respectively. The interassay variations were minimized by adjustment of observed values using correction coefficients obtained from simultaneous analyses of reference sera. Quantitation of apolipoproteins A -I and A -II Serum apolipoproteins A-I and A-II were determined by electroimmunoassay [22] using rabbit antisera rendered monospecific by immunoabsorption [23]. Polyethylenglycol 6000 at a final concentration of 40 g/l was included in the agarose gels used for determinations of apolipoprotein A-II in order to render the immunoprecipitates more distinct. The contents of apolipoproteins A-I and A-II in a standard serum obtained from healthy blood donors was assessed by comparison with a human HDL preparation with known amounts of apolipoproteins A-I and A-II as determined by SDS-polyacrylamide gel electrophoresis. The apparent concentrations of apolipoproteins A-I and A-II in the standard serum were 103 mg/lOO ml and 30 mg/lOO ml, respectively. The intraassay and interassay coefficients of variance for apolipoprotein A-I were 4.9% and 6.5%. respectively.
226 The corresponding figures for apohpoprotein were 5.8% and 8.3%.
A-II
@iuntitution of apo/ipoprotein B Antiserum against apolipoprotein B was prepared from rabbits. LDL in the density range 1.02-1.05 g/l which had been isolated from plasma of lipoprotein (a)-negative subjects by ultracentrifugation and gel filtration on a Bio-Gel A 15 M column was used as antigen. Antiserum from the same bleeding was used throughout the study. The antiserum gave a single precipitation line with LDL and different human serum samples. This monospecific antiserum was used to quantitate total apolipoprotein B in the serum samples by electroimmunoassay [22]. The purified LDL was used as apolipoprotein B-standard (kept at -70°C) and had a mean protein content of 20.7% as estimated by [24] with bovine albumin as standard. This purified LDL was used to standardize a serum sample using electroimmunoassay. The serum standard used throughout the study was kept at - 70°C in small aliquots and was regularly checked against the purified LDL standard. The intraassay and interassay coefficients of variance were 2.8% and 3.1%.
Lipoprotein electrophoresis Agarose gel lipoprotein electrophoresis [25] was performed on whole serum and on the top (VLDL) and the bottom (LDL + HDL) fractions obtained after ultracentrifugation at d = 1.006 kg/l. One percent agarose (Miles-Seravac) was used and staining was done with Sudan black (1%). The stained electrophoretic strips were projected on a screen in an overhead projector and inspected, without knowledge of diagnosis, for the presence of late pre-beta (LPbeta) bands [26] and the sinking pre-beta band (SPbeta). According to the mobility relative to the beta band, the LPbeta band was further subdivided into LPbeta I with mobility slower than pre-beta but clearly faster than,beta, LPbeta II in which the slower part of the band was in the beta region, and LPbeta III with beta mobility. The SPbeta band has a faster mobility than beta but usually slower than the pre-beta found in A < 1.006 kg/l serum and is often seen in whole serum.
Additional risk indicators Subjects who smoked at least 1 cigarette or an equivalent amount of tobacco each day were classified as present smokers. Subjects who had never smoked or had smoked continuously for less than 1 month were defined as non-smokers. Others were regarded as former smokers. In calculation of the average tobacco consumption 1 cigarette was considered equivalent to 1 g, 1 cigarillo to 2 g and 1 cigar to 5 g of tobacco. Tobacco consumption of pipe smokers was calculated by dividing by 7 the weekly consumption in grams. As a cumulative estimate of tobacco consumption prior to MI, cigarette-years was used [27]. Figures of alcohol consumption immediately prior to MI were based on a structured interview and questionnaires and expressed as g of absolute alcohol consumed during one month. Blood ‘pressure was measured in the supine position after a 5-min rest. Hypertension was defined as present either if antihypertensive treatment had been instituted prior to MI or in the immediate post-infarction period or if blood pressure was above 160 mm Hg systolic and/or 95 mm Hg diastolic. Manifest diabetes mellitus was diagnosed as fasting hyperglycemia (whole blood) of > 7.0 mmol/l. Oral glucose tolerance was assessed in all patients following ingestion of 1.75 g glucose/kg body weight [28]. Decreased oral glucose tolerance required the measurement of blood glucose values > 8.9 mmol/l at 60 min and > 6.7 mmol/l at 120 min following glucose intake. Weight was measured with subjects dressed in indoor clothing without jacket and shoes. The weight/height index was calculated (weight (kg)/(height (cm) - 100)) and overweight defined by values above 1.10. Statistical methods Proportions were compared using the chi-square test with Yates’ correction. Statistical significance for differences in lipoprotein composition between groups was tested using one way analysis of variance and two-tailed t-test. Coefficients of skewness and kurtosis were used to test deviations from a normal distribution. Values of total triglycerides, VLDL triglycerides and VLDL cholesterol were accordingly logarithmically transformed to achieve a distribution not significantly different from nor-
227 Ethical considerations Prior to the study informed consent was obtained from all subjects. The study protocol had been approved by the regional ethical committee.
ma1 prior to significance testing. Differences between groups were further studied by analysis of covariance enabling control for concomitant group differences in other variables such as tobacco consumption, alcohol consumption and body weight. Correlation analyses were performed according to standard methods. Since strong intercorrelations exist between several lipoproteins and apolipoproteins stepwise multiple discriminant analyses were performed to determine the sets of independent lipoprotein and apolipoprotein variables giving the best discrimination between patients and controls. The variable with the highest F-value was entered at each step until no variable remained with a value (F to enter) of 4 or more. Variables reflecting smoking habits, alcohol consumption and body stature were entered as forced variables in some of the discriminant analyses. All statistical analyses were performed at the Stockholm Computer Centre for University Education and Research using standard BMDP programmes [29].
TABLE
Results Clinical churacteristics and addition& risk indicutors As shown in Table 1 smoking habits among patients and controls were identical at the time of the metabolic evaluation, whereas alcohol consumption was higher in the control group. However, there was a marked overrepresentation of former smokers in the patient group. the majority of whom had stopped smoking at the time of the MI. Accordingly cumulative life-time tobacco consumption was much higher in patients. More patients had manifest diabetes or reduced oral glucose tolerance. Young male post-infarction patients taken as a group were shorter than matched controls, whereas body weight was similar in the two groups. The weight/height index was thus higher in the patient group.
1
BASIC CHARACTERISTICS
AT INVESTIGATION
Values are number
in group or means&standard
of subjects
IN PATIENTS
Patients (n=116) Age (~0 Smoking habits
39.x
present smoker (n)
Tobacco
(n)
+
3.9
39.9
*
P-value
3.9
45 30 I 41
n!,
< 0.001 h
consumption
present (g/day) cumulative. (cigarette-years) Hypertension (n) Diabetes
6.5 + 10.1 411 f 226 29
typeI type II(n) Decreased
Controls (n=116)
46 63 .’ I
former smoker (n) non-smoker
AND CONTROLS
deviation.
oral glucose tolerance
(n)
8.0 * 12.5 198 +218 12
2 4 35
0 01 10
< 0.001 h
+ 388 623 180.7 k 6.9 79.9 & 11.8 0.99* 0.13
Alcohol consumption (g/month) Height (cm) Weight (kg) Weight/height index (kg/cm - 100)
41s &381 175.9 k 6.2 80.5 i 11.1 1.06* 0.13
“ 55 patients stopped smoking at MI. h Group differences for distributions of categorical
data were tested by chl-square
analysis
ns < 0.001 < 0.001
< 0.001 < 0.001 ns < 0.001
with Yates’ correction.
228 Lipoprotein lipid and apolipoprotein concentrations There were marked elevations of the cholesterol and triglyceride concentrations in VLDL in patients compared to controls (Table 2). The LDL cholesterol and triglyceride levels were also elevated in the patient group, the elevation of LDL triglyceride level being the most pronounced. Both HDL, and HDL, cholesterol concentrations were reduced, the reduction of HDL, cholesterol level
TABLE
accounting for the major part of the group difference in total HDL cholesterol concentration. Serum levels of apolipoproteins A-I and A-II were decreased in the patient group, whereas serum apolipoprotein B level was increased. The prevalence of the visually registered electrophoretic Lpbeta band (Lpbeta I-II) was increased among the patient (24% vs 13%, P < 0.05), whereas the preva!ence of the visually documented
2
LIPOPROTEIN
LIPID
AND APOLIPOPROTEIN
Values are means * standard
Cholesterol (mmoI/I) Total VLDL LDL HDL HDL, HDL, Triglycerides (mmol/I) Total VLDL LDL HDL
Apolipoproteins (mg/lOO A-I A-II B
deviation
or number
CONCENTRATIONS of subjects
Patients (n =116)
Controls (n =116)
1.33 1.40 1.19 0.99 4.84 1.23 1.12 0.24 0.37 0.20 0.75 0.15
6.07 1.16 0.54 0.54 3.97 0.92 1.45 0.38 0.58 0.37 0.85 0.14
2.95 2.35 2.12 1.90 0.49 0.17 0.16 0.06
1.45 1.23 0.91 1 .oo 0.35 0.12 0.15 0.04
ml) 111.0 19.3 36.9 1.3 128.4 20.8
125.6 19.2 40.4 1.3 105.2 20.1
IN PATIENTS
AND
CONTROLS
in group. % difference
A
P-value
+21
-c 0.001
+120
< 0.001
+22
i 0.001
-23
< 0.001
-36
i 0.001
-12
< 0.001
+ 103
i 0.001
+132
< 0.001
+40
i 0.001
+7
ns
-12
< 0.001
-9
< 0.001
i-22
i 0.001
Electrophoreticpattem Lpbeta Lpbeta Spbeta * Patients
I II
compared
18 10 41 to controls.
10
5 28
< 0.05 ns
229
SPbeta trait was not significantly increased in the patient group (35% vs 25%, P < 0.10). Means and standard deviations for lipoprotein lipid and apolipoprotein levels were practically identical in patients with and without beta-blocker medication. No correlations were observed between the time interval between MI and blood sampling and lipoprotein lipid or apolipoprotein levels (r values 0.0220.11 in correlation analyses). When differences between the groups in present tobacco and alcohol consumption, as well as weight/height index, were controlled by covariante analysis, some changes in group means of lipoprotein and apolipoprotein values appeared. In particular the differences in groups means of VLDL lipid concentrations were reduced (adjusted means i SEM in patients compared to controls for VLDL cholesterol level 0.85 k 0.05 vs 0.46 k 0.04 mmol/l and for VLDL triglyceride level 1.58 _t 0.05 vs 0.76 f 0.04 mmol/l). With the exception of serum apolipoprotein A-II all differences between patients and controls observed in simple univariate analysis remained statistically highly significant (P < 0.001). The discriminatory power of HDL in relation to MI was further evaluated by separate comparisons of normolipidemic and hypertriglyceridemic patients with the normolipidemic and hypertriglyceridemic subgroups of the controls after adjustment for group differences in whole serum cholesterol and/or triglyceride levels. Adjusted apolipoprotein A-I levels tended to be lower in normolipidemic patients compared to normolipidemic controls (116.4 f 3.9 vs 126.2 f 2.3 mg/lOO ml. P < 0.05) whereas adjusted HDL cholesterol values were not significantly reduced (1.34 k 0.06 vs 1.49 + 0.04 mmol/l, P < 0.10). In hypertriglyceridemic patients both apolipoprotein A-I and HDL cholesterol levels were decreased compared to hypertriglyceridemic controls after adjustment for differing triglyceride values (107.4 + 3.0 vs 122.0 i 3.9 mg/lOO ml and 1.02 & 0.04 vs 1.22 ~fr 0.05 mmol/l, respectively, P < 0.01). In addition, a significant elevation of VLDL cholesterol concentration persisted in the hypertriglyceridemic patients (1.55 of:0.06 vs 1.48 f 0.08 mmol/l, P < 0.05). The adjusted HDLz cholesterol concentration, however, did not differ between hypertriglyceridemic patients and controls.
Ratios of cholesterol to trigbcerides in nwjor lipoprotein clusses The average ratios of cholesterol to triglycerides in all 3 major lipoprotein classes were lower in the patients than in the controls (Table 3). This difference was particularly pronounced in HDL. The VLDL cholesterol/triglyceride ratio was significantly higher in Lpbeta-positive compared to Lpbeta-negative patients (P < 0.001). but it was still within the range of Lpbeta-negative controls. In the LDL fraction the ratio of cholesterol to triglycerides was lower in Lpbeta-positive than in Lpbeta-negative patients (P < 0.05). However. as a group Lpbeta-negative patients also differed significantly from Lpbeta-negative controls (P < 0.05) with respect to LDL cholesterol/triglyceride ratio. All differences related to the Lpbeta trait were more prominent if only Lpbeta II-positive patients were considered, in whom, in addition. serum apolipoprotein B levels were elevated (138.7 + 22.8 vs 127.7 f 16.6 mg/lOO ml, P < 0.001) in comparison to Lpbeta-negative patients. A further evaluation of the relation between the concentrations of cholesterol and triglyceride in the major lipoprotein classes was obtained by plotting the individual lipid values of the patients against each other and relating them to the regression line of the healthy controls (Figs. 1 A-C). In VLDL (Fig 1A) there was a strong positive correlation between the lipid values at triglyceride levels below 1.5 mmol/l. At higher VLDL triglyceride levels (1.5-2.5 mmol/l). but still within the range of the control group, however, the VLDL cholesterol values were more variable in relation to the triglyceride values and some, mostly Lpbetapositive, patients were located markedly above the regression line of the controls. In LDL the cholesterol values varied considerably in relation to the triglyceride values (Fig. 1 B). Since the apparent relation between cholesterol and triglycerides in LDL for the controls did not contain the origin, the average lowering of the ratio of cholesterol to triglycerides in the patient group did not necessarily imply a changed relation between the two lipids in LDL. Moreover, within the LDL triglyceride range of the control group (0.2-0.6 mmol/l) an elevated LDL cholesterol/ triglyceride ratio was indicated, whereas in a num-
230 TABLE RATIOS
3 OF CHOLESTEROL
TO TRIGLYCERIDES
IN THE MAJOR
Patients Lpbeta + (n = 28)
LIPOPROTEIN
CLASSES P-value ’
Controls Lpbeta (n = 88)
All (n = 116)
Lpbeta + (n=15)
Lpbeta (n = 101)
All (n = 116)
VLDL Chol VLDL Tg
0.66 0.15
0.55 0.09
0.58 0.19
0.72 0.15
0.61 0.10
0.62 0.11
< 0.01
LDL Chol LDL Tg
9.45 2.53
11.21 3.65
10.74 3.44
9.71 2.55
12.61 3.38
12.23 3.42
< 0.01
HDL Chol HDL Tg
8.09 3.67
8.27 4.27
8.20 4.08
9.61 4.23
11.23 5.20
11.02 5.10
i 0.01
a All patients compared to all controls. Lpbeta+ = Lpbeta I-III-positive; Lpbeta-
= Lpbeta
I-III-negative;
ber of mostly Lpbeta-positive, markedly hypertriglyceridemic, patients a very low LDL cholesterol/triglyceride ratio was noted. A change in the relation between cholesterol and triglycerides in HDL was apparent in the patient group (Fig. 1C) since the vast majority of patients were located below the regression line of the control group and well within the distribution of HDL triglyceride values for this group. Ratios between concentrations of total HDL cholesterol, cholesterol in HDL subclasses, and serum apolipoproteins A-I and A-II As expected from the data in Table 2 the HDL,/HDL, cholesterol ratio was reduced among the patients compared to the control subjects, whereas no difference between the study groups was observed in the apolipoprotein A-I/A-II ratio (Table 4). In addition the HDL cholesterol/apo-
Chol = cholesterol;
Tg = triglycerides.
LDL CHOL
-/L 80
I 5.
:
.*.... -*.
4.
: -
4a. .iL&f+&Y ;;;-.. .. 8.0
-.
2.0.
08
0.2
0.4
0.8
LOL
I.0
TG.h&t/L
HDLCHOL -IL 2.0 1
. 1.0~ .
.
. .
.*._-. . .
.
: . .
oc 0
am
0.20
0.30
HDL TG. -/L
Fig. 1. The relations between concentrations of triglycerides (TG) and cholesterol (CHOL) in (A) VLDL, (B) LDL and (C) HDL in the patient group. The shadowed area denotes the 95% confidence limites of the regression line of the control group. ??= Lpbeta-positive patients; 0 = Lpbeta-negative patients.
231 TABLE
4
RATIOS BETWEEN CONCENTRATIONS OF TOTAL HDL CHOLESTEROL, CHOLESTEROL IN HDL SUBCLASSES, AND SERUM APOLIPOPROTEINS A-I AND A-II Values are means f standard
deviation
Patients (n=I16)
Controls (n =116)
P-value
0.51 0.27
0.71 0.43
i 0.001
apo A-I ~apo A-II
3.06 0.38
3.16 0.41
“s
HDL Chol
0.40 0.06
0.46 0.09
< 0.001
HDL,
Chol
HDL,
Chol
apo A-I
Stepwise discriminant una&sis The relations of single lipoprotein variables and different apolipoproteins to MI were first compared by calculation of the F-values to enter the discriminant function when the measurements of tobacco and alcohol consumption. and the weight/height index. had been entered as forced
Chol = cholesterol:
Tg = triglycerides:
glyceride value (P < 0.001). The same significance level for difference in position of the regression lines of the respective groups was also recorded if only patients with VLDL triglyceride values corresponding to the range of control subjects were analysed. Thus for any given VLDL triglyceride value the concentration of HDL cholesterol was lower in patients compared to controls.
apo = apolipoprotein.
TABLE
lipoprotein group.
A-I ratio
was reduced
in the patient
5
LIPOPROTEIN AND APOLIPOPROTEIN SIGNIFICANTLY DISCRIMINATING TIENTS AND CONTROLS
Relation between VLDL triglyceride and HDL cholesterol concentrations The strongest correlation (r = -0.53) between concentrations of VLDL triglycerides and total HDL cholesterol, HDLz and HDL, cholesterol, and apolipoproteins A-I and A-II was demonstrated between VLDL triglyceride and total HDL cholesterol concentrations in the patient group (Fig. 2). The regression line of the patients was parallel to that of the controls, but located significantly lower irrespective of VLDL tri-
VARIABLES BETWFEK PA-
Lipoprotem/variable
F-value
Lipoprotein/ variable
~-Lalw
VLDL Chol+
112.73
LDL Tg
46.67
I 11.36
LDL Chol
37.x3
1 10.03
HIX,
36.29
LDL Chol
HDL Chol LDL Chol HDL (‘ho1 apo B
(‘hvl
apo A-I Total Tg
65.33
apo B
64.33
30.09
apo A-l HDL Chol
28.75
ap” A-l HDL CHOC
MMcqL 2.0
VLDL Tg
62.40
1
HDL,
(‘ho1
IX.45
HDL,ChoI VLDL Chol
56.04
apo A-II
x.13
Total Chol
55.38
ape A-I apo A-II
7.63
HDL Chol
52.57
LDL (‘ho1
6.26
LDL Tg apo B apo A-II
0 01
10
50.94
VLDL Chol VLDL Tg
6.06
a.0
VLDL TG. MYOL/L
Fig. 2. The concentration of HDL cholesterol in relation to VLDL triglyceride concentration (logarithmic scale) in the patient group. The shadowed area denotes the 95% confidence limits of the regression line of the control group.
F-values are F-values to enter the diacrimlnant function when measurements of tobacco and alcohol consumption. ah well as the weight/height index. had been entered as forced variables. A F-value of 4 or more is considered aignlflcant. Chol = cholesterol: Tg = triglycerides: apo = apolipoprotri~~.
232 TABLE
6
LIPOPROTEIN CRIMINATING IN STEPWISE
VARIABLES SIGNIFICANTLY DISBETWEEN PATIENTS AND CONTROLS MULTIPLE DISCRIMINANT ANALYSIS
A
F
Correct
classification
LDL Chol HDL Chol Total Tg
38.19 21.85 12.02
infarct control total
83.3 76.8 80.1
F
Correct
classification
LDL Chol
60.92
HDL Chol Total Tg
12.16
infarct control total
83.3 81.1 82.2
VLDL Chol
4.73
B
apo A-I
(S)
7.15
apo A-II VLDL Chol
5.05
C
F
Correct
classification
Total Chol HDL Chol
58.60 39.79
infarct control total
85.4 77.9 81.7
F
Correct
classification
62.10
infarct control total
85.4 83.2 84.3
Smoking Alcohol Weight/height
(%)
index
D
LDL Chol HDL Chol
(X)
8.93 0.57 0.28 (%)
12.92 5.53
Total Tg apo A-I apo A-II VLDL Chol
5.04
Smoking Alcohol Weight/height
9.88 0.06 0.18
index
A: Only serum lipids, lipoprotein lipids and apolipoproteins included in the analysis. B: Variables as in A, but for the inclusion of ratios between serum lipids, lipoprotein lipids and apolipoproteins. C: Variables as in A, tobacco and alcohol consumption and weight/height index at the time of the investigation were forced variables. D: Variables as in B, tobacco and alcohol consumption and weight/height index at the time of the investigation were forced variables. F-values indicated in the tables are the F-values when all selected variables had been entered in the equation (F to remove). Chol = cholesterol; Tg = triglycerides; apo = apolipoprotein.
variables (Table 5). The sum of cholesterol in VLDL and LDL divided by HDL cholesterol level had the strongest power to discriminate between patients and controls. The concentrations of serum triglycerides, apolipoprotein B, VLDL triglycerides and cholesterol, serum cholesterol, HDL cholesterol and LDL triglycerides, in that order, discriminated better between patients and controls than did the LDL cholesterol concentration. Among variables reflecting HDL concentration and composition HDL cholesterol was the best discriminator, followed by HDL, cholesterol, apolipoprotein A-I, and the HDL cholesterol/ apolipoprotein A-I ratio. A number of lipoprotein components were found to discriminate independently between patients and controls in the stepwise multiple discriminant analysis (Tables 6A-D). When only lipoprotein lipid and serum apolipoprotein concentrations were entered in the discriminant function (Table 6A), the levels of LDL cholesterol, HDL cholesterol, total serum triglycerides and VLDL cholesterol, in that order, were found to be the best set of independent discriminators. Adding ratios (Table 6B) resulted in the replacement of LDL and HDL cholesterol concentration by the LDL/HDL cholesterol and apolipoprotein AI/A-II ratios. If, instead, variables reflecting smoking habits, alcohol consumption and body stature were forced and ratios not included (Table 6C), total serum cholesterol and HDL cholesterol concentrations emerged as independent discriminators. When, ratios were added (Table 6D), however, the previous pattern of independent lipoprotein variables returned. A correct total classification in percent of patients and controls around SO-85% was obtained, with slightly higher values for patients. The inclusion of ratios and serum apolipoprotein concentrations added somewhat to the correct classification of controls obtained by whole serum and lipoprotein lipid concentrations alone.
Discussion Predisposing metabolic risk factors should be more apparent in patients with premature coronary heart disease than in middle-aged or elderly popu-
233 Iations. In a younger patient group, the confounding influence of age per se and coexisting age-dependent degenerative and inflammatory diseases can be considered insignificant. Since all medical departments within the region participated in this study and the number of dropouts was marginal, the present material is representative of young male infarction survivors in the Stockholm area. In healthy subjects the levels of several lipoprotein lipids are highly intercorrelated. In particular, close correlations exist among serum triglycerides and VLDL lipids [30]. In the young post-infarction patients, however, these latter interrelations were found to be altered as indicated by results from the multivariate analyses, where both serum triglyceride and VLDL cholesterol levels appeared as independent variables. Furthermore the intralipoprotein relations of cholesterol and triglyceride seemed to be altered in the patient group. Our results thus emphasize independent significances of the concentrations of VLDL, LDL and HDL in relation to myocardial infarction. As a single variable serum total apolipoprotein B concentration was a better discriminator than LDL cholesterol level. The same applied for the concentrations of total triglycerides, VLDL lipids, HDL cholesterol and total cholesterol. However, the stepwise multiple discriminant analysis indicated that the independent relations of LDL and HDL cholesterol levels to MI were closer than those of serum triglycerides or VLDL cholesterol. These results extend the findings of a similar study using multivariate analysis from Gothenburg [15] in which, however, the lipoprotein evaluation was restricted to measurement of total serum lipid levels and concentrations of apolipoproteins A-I, A-II, B and D. Multivariate analysis indicated that the levels of apolipoprotein A-II and total serum triglycerides correlated independently with myocardial infarction. When smoking had been included in the regression equation altogether only 28% of the variation of the dependent variable was accounted for. The relationship between VLDL and CHD is not well defined. In previous studies of post-infarction patients, the magnitude of the VLDL cholesterol elevation was the most pronounced change in lipoprotein levels [31-331. Multivariate analyses of prospecti;e data, as applied in the
Framingham and Gothenburg studies [3,5], have implied that the correlation of whole serum triglyceride concentration with risk derives from secondary associations, whereas in the Stockholm Prospective Study triglycerides emerged as a significant and independent risk factor for MI (6,7]. In the present study concentrations of both whole serum triglycerides, or VLDL triglycerides, and VLDL cholesterol were found to be independently related to MI. The indicated independent contribution to risk of both VLDL cholesterol and triglyceride levels in young post-infarction patients is at first sight astonishing since, in normolipoproteinemia and in all primary hyperlipoproteinemias except type III, a close relation has been demonstrated between the concentrations of cholesterol and triglycerides in the VLDL fraction. Among the young patients, however, the Lp-beta band which may represent remnant particles and is associated with a cholesterol-rich VLDL [26], was more frequently observed than in the control subjects. This may contribute to the independent risk indicated in VLDL cholesterol. In comparison, an increased frequency of Lpbeta-positive patients was not found in a group of older post-infarction patients [31]. Possibly the independent relation of VLDL cholesterol concentration to MI is specific for young patients and due to an increased occurrence of Lpbeta-positive patients with a cholesterol-rich VLDL. In the present study as well as in other studies of post-infarction patients [31,33] increased LDL lipid concentrations were noted. It has been assumed that the measurement of LDL by determining its cholesterol content provides an accurate assessment of total LDL particle concentration, particularly the LDL, concentration, LDL, being reflected by LDL triglycerides. However, a syndrome of hyperapobetalipoproteinemia was recently delineated in normolipidemic patients with coronary artery disease and an abnormal LDL composition [13]. The relative importance in our material of increased number of LDL particles versus altered LDL composition cannot be thoroughly evaluated since only whole serum apolipoprotein B was determined. In univariate analysis apolipoprotein B concentration was a better discriminator between patients and controls than LDL cholesterol level,
234 whereas in multivariate analysis LDL cholesterol was the better discriminator. This result from the multivariate analysis contrasts with several recent reports [10,11,14]. However, in previous studies apolipoprotein B concentration seemed to be more predictive at lower serum cholesterol levels [34]. The young patients in this study usually exhibited moderately elevated serum cholesterol levels, which might account for this discrepancy. HDL data emerging from this study showed that, as a single variable, HDL cholesterol provided a better means of discrimination between patients and controls than did HDL, cholesterol, apolipoprotein A-I and ratios between HDL subfractions or HDL apolipoproteins. In multivariate analysis including ratios of lipoproteins and apolipoproteins, however, the serum apolipoprotein ‘A-I/A-II ratio apparently gave a better representation of HDL changes relevant to MI. Whether this reflects the importance of the structural proteins of HDL or an inability of the HDL, and HDL, cholesterol determinations to accurately reflect the true relations between the HDL subclasses cannot be assessed from this study, since the HDL apolipoproteins were not determined in the HDL subfractions. The relation between HDL metabolism and triglyceride metabolism is not fully understood. Apparently the differences in HDL constituents between patients and controls in this study were not a mere reflection of disturbances in triglyceride metabolism, since the same differences persisted for the whole material using multivariate analyses and after controlling for triglyceride levels in the normolipidemic and hypertriglyceridemic subjects, respectively. The findings in this study should be interpreted with due regard to its cross-sectional design. Significant alterations in the associations between lipoproteins and MI may occur during the time between the onset of disease and the metabolic evaluation. However, such alterations are less probable in these young patients, who were as a rule without cardiac symptoms prior to MI and generally not severely incapacitated post MI. Sufficient time was shown to have elapsed between MI and blood sampling to allow for the temporary effects of acute phase reactions on lipoproteins to disappear [35,36]. Since no differences in lipopro-
tein levels or composition were observed between patients with or without betablockers or furosemide, medication decidedly cannot account for the differences demonstrated between the patient and control groups. The present study thus demonstrates the importance of elevated serum triglyceride and VLDL cholesterol levels, in addition to increased LDL and decreased HDL cholesterol concentrations, in relation to MI at young age. References
9
10
11
12
Keys, A., Seven Countries, Harvard University Press, Cambridge, 1980. Gordon, T.. Kannel, W.B., Castelli. W.P. and Dawber, T.R., Lipoproteins, cardiovascular disease and death The Framingham Study, Arch. Intern. Med., 141 (1981) 1128. Gordon, T., Castelli, W.P., Hjortland, M.C., Kannel. W.B. and Dawber, T.R., High density lipoprotein as a protective factor against coronary heart disease - The Framingham Study, Amer. J. Med., 62 (1977) 707. Miller, N.E., Forde, O.H., Thelle, D.S. and Mjos, O.B., The Tromso Heart Study: High-density lipoprotein and coronary heart disease - A prospective case-control study, Lancet. i (1977) 965. Wilhelmsen, L., Wedel, H. and Tibblin, G., Multivariate analysis of risk factors for coronary heart disease, Circulation, 48 (1973) 950. Carlson L.A. and B&tiger, L.E., lschemic heart disease in relation to fasting values of plasma triglycerides and cholesterol, Lancet, i (1972) 865. Carlson, L.A., Bottinger, L.E. and Ahfeldt, P.E., Risk factors for myocardial infarction in the Stockholm prospective study A 1Cyear follow-up focussing on the role of plasma triglycerides and cholesterol. Acta Med. Stand.. 206 (1979) 351. Ishikawa, T., Fidge, N., and Thelle, D.S., The Thorns@ Heart Study - Serum apolipoprotein A-l concentration in relation to future coronary heart disease, Europ. J. Clin. Invest., 8 (1978) 1979. Avogaro, P., Cazzolato, G., Bittolo Bon, G. and Quinci, G.B., Are apohpoproteins better discriminators than lipids for atherosclerosis?, Lancet, i (1979) 901. Avogaro, P., Bittolo Bon, G., Cauolato, G. and Rorai, E., Relationship between apolipoprotein and chemical components of lipoproteins in survivors of myocardial infarction, Atherosclerosis, 37 (1980) 69. Onitiri, A.C. and Jover, E., Comparative serum apolipoprotein studies in ischemic heart disease and control subjects, Clin. Chim. Acta, 108 (1980) 25. Riesen. W.F., Mordasini, R.. Salzman, C.. Theler. A. and Gurtner, H.P.. Apoproteins and lipids as discriminators of severity of coronary heart disease, Atherosclerosis. 37 (1980) 157.
235 13 Sniderman. A., Shapiro, S., Marpole, D., Skinner. B., Teng, B. and Kwiterovich. Jr, P.O.. Association of coronary atherosclerosis with hyperapobetalipoproteinemia (Increased protein but normal cholesterol levels in human plasma low density (p) lipoprotein), Proc. Nat. Acad. Sci. (USA), 77 (1980) 604. 14 De Backer, G., Rosseneu, M. and Deslypere. J.P.. Discriminative value of lipids and apoproteins in coronary heart disease, Atherosclerosis, 42 (1982) 197. 15 Fager, G., Wiklund, O., Olofsson, S-O.. Wiihelmsen, L. and Bondjers, G.. Multivariate analyses of serum apolipoproteins and risk factors in relation to acute myocardial infarction. Arteriosclerosis. 1 (1981) 273. 16 Nomenclature and criteria for diagnosis of ischemic heart disease, Circulation. 59 (1979) 607. 17 Carlson. K., Lipoprotein fractionation, J. Clin. Path.. 26 (1973) 32. 18 Zlatkis, A.. Zak. B. and Boyle. A.. A new method for direct determination of serum cholesterol, J. Lab. Clin. Med.. 41 (1953) 486. 19 Fletcher, M.J., A calorimetric method for estimating serum triglycerides. Clin. Chim. Acta, 22 (1968) 393. 20 Carlson, L.A., Determination of serum triglycerides. J. Atheroscler. Res.. 3 (1963) 334. 21 Kirstein, P. and Carlson, K., Determination of the cholesterol content of high density lipoprotein subfractions without contamination of Lp(a) in HDL, and HDL,. human plasma, Clin. Chim. Acta. 113 (1981) 123. 22 Laurell, C.B.. Quantitative estimation of proteins by electrophoresis in agarose gel containing antibodies, Anal. Biothem., 15 (1966) 45. 23 Ekman. R.. Johansson. B.G. and Pettersson, B.G., Immunochemical studies of lipoproteins in cholestasis, Stand. J. Immunol.. 4 (1975) 13. 24 Lowry. O.H., Rosebrough, N.J.. Farr, A.L. and Randall. R.J.. Protein measurement with rhe Folin phenol reagent. J. Biol. Chem.. 193 (1951) 265. 25 Noble. R.P.. Electrophoretic separation of plasma lipoproteins in agarose gel, J. Lipid Res.. 9 (1968) 693. 26 Carlson. K. and Carlson, L.A., Comparison of behaviour of very low density lipoproterns of type III hyperlipopro-
27
28
29 30
31
32
33
34
35
36
teinemia on electrophoresis on paper and on agarose gel with a note on a late (slow) pre-beta VLDL lipoprotein. Stand. J. Clin. Lab. Invest., 35 (1975) 655. Lundman. T.. Smoking in relation to coronary heart disease and lung functions in twins - A co-twin control study, Acta Med. Stand., Suppl. 455. 1966. Efkndic, S.. WaJngot, A., Cerasi. E. and Luft. R., Insulin reiease, Insulin sensitivity and glucose tolerance, Proc. Nat. Acad. Sci. (USA), 77 (1980) 7425. BMDP Statistical Software. 1983. University of California Press. Berkeley. 1983. Olsson. A.G., Walldius, G., RGssner. S.. Callmer. E. and Kaijser, L.. Studies on serum lipoproteins and lipid metabolism - Analysis of a random sample of 40-year-old men, Acta Med. Stand., 637 (1980) 1. Carlson, L.A. and Ericsson, M.. Quantitative and qualitative serum lipoprotem analysis. Part 2 (Studies in male survivors of myocardial infarction., Atherosclerosis. 21 (1975) 435. Lewis, B.. Chait. A., Oakley, C.M.O.. Wootton, I.D.P., Krikler, D.M., Onitiri, A., Sigurdsson. G. and February, A.. Serum lipoprotein abnormalities in patients with ischemic heart disease - Comparison with a control population, Brit. Med. J., 9 (1974) 489. Ballantyne, F.C.. Clark, R.S.. Simpson, H.S. and Ballantyne. D., High density and low density lipoprotein subfractions in survivors of myocardial infarction and m control subjects, Metabolism, 31 (1982) 433. Whayne, T.F., Alaupovic. P., Curry, M.D.. Lee, ET.. Andersson, P.S. and Schechter, E., Plasma apolipoprotein B and VLDL- and HDL-cholesterol as risk factors in the development of coronary artery disease in male patients examined by angiography. Atherosclerosis, 39 (1981) 1411. McCall M., Elmfeldt, D.. Vedin. A.. Wilhemsson. C.. Wedel. H. and Wilhelmsen L.. Influence of a myocardial infarction in blood pressure and serum cholesterol, Acta Med. Stand.. 206 (1979)477. Stubbe, I.. Gustafsson. A. and Nilsson-Ehle. D., Alterations in plasma proteins and lipoproteins in acute myocardial infarction: effects on activation of lipoprotein lipase. Stand. J. Clin. Lab. Invest., 42 (1982) 437.
59 (1986)
Elsevier Scientific
223
223-235
Publishers
Ireland.
Ltd.
ATH 03729
Serum Lipoproteins
A. Hamsten Depurtments
and Apolipoproteins in Young Male Survivors of Myocardial Infarction
‘, G. Walldius of’ Medicine
2, G. Dahlkn 3, B. Johansson
and 4 CIm~cal Cheml.rtty,
’ Depurtment .’Department
of Medane.
of Clinrc~ul
Dundetyd
Karolinsku
Chemistty,
Hospitul; Hospital,
Central
4 and U. De Faire ’
King GustuJ V Reseurch Instrtute Stockholm;
Hospital,
und
cmd
Boden (Sweden)
(Received 21 May, 1985) (Revised, received 9 September, 1985) (Accepted 11 September. 1985)
Summary Concentrations of serum lipoprotein lipids and apolipoproteins A-I, A-II and B were determined 3-6 months after myocardial infarction in 116 males below the age of 45 and in 116 age-matched controls. Among single variables the sum of cholesterol concentration in VLDL and LDL divided by the HDL cholesterol level was the best discriminator between patients and controls. The concentrations of serum triglycerides, apolipoprotein B, VLDL triglycerides and cholesterol. serum cholesterol, HDL cholesterol and LDL triglycerides, in that order, were better discriminators than was LDL cholesterol level. Among variables reflecting HDL concentration and composition HDL cholesterol was the best discriminator followed by HDL, cholesterol, apolipoprotein A-I and the HDL cholesterol/apolipoprotein A-I ratio. Multivariate analysis indicated independent significance of elevated VLDL lipid and LDL cholesterol concentrations, and a decreased HDL cholesterol concentration, in relation to MI. The present data suggest that a disturbed triglyceride metabolism, in addition to elevated LDL and decreased HDL cholesterol levels, has an independent and pathogenetic significance for MI at a young age.
Key words:
Apolipoproteins
- Lipoproteins
- Mj~ocurdial infarction
- Young adults
Introduction This study was supported by grants from the Swedish National Association against Heart and Chest Diseases. the Swedish Medical Research Council (19x-204). the Tore Nilsson Foundation for Medical Research, and the Association of Swedish Oleomargarine Manufacturers for Research on Nutritional Physiology. Author’s address: Anders Hamsten, Department of Meditine, Danderyd Hospital. S-182 88 Danderyd. Sweden. 0165-0327/86/$03.50
fi> 1986 Elsevier Science Publishers
The relationship between serum lipoprotein concentrations and coronary heart disease (CHD) has been most evident for low density lipoprotein (LDL) cholesterol. [ 1,2]. High density lipoprotein (HDL) cholesterol concentrations have been even more strongly predictive in some studies [3,4]. Multivariate analyses of most prospective studies
B.V. (Biomedical
Division)
224 have implied that correlations between serum triglyceride level and CHD derive from secondary associations, whereas in one study triglycerides have emerged as a significant and independent risk factor [6,7]. In recent studies the serum concentrations of the apohpoproteins, have been considered even better discriminators between patients with CHD and healthy controls [8-141 than the levels of total or lipoprotein lipids. In the present study, which is part of a comprehensive clinical and metabolic research programme in young post-infarction patients, the independent relation to myocardial infarction (MI) and discriminative power of serum lipoprotein levels and composition with respect to lipids and apolipoproteins was investigated in representative young male survivors of MI from a geographically and demographically well characterized area. On the basis of previous results obtained in young post-infarction patients [15], and the finding of hypertriglyceridemia as the dominating lipoprotein disturbance in this material, special emphasis was placed on the multivariate evaluation of serum triglycerides and VLDL lipids. Materials and Methods Putients Between May 1980 and September 1982 137 male patients below the age of 45 were admitted to the 11 hospitals in the county of Stockholm with a diagnosis of definite myocardial infarction [16]. Thirty-five patients were immigrants (residence in Sweden, mean t_ SD, 12.5 5 8.0; range 1.0-36.5 years), of whom 10 were born in Finland. Ten patients died during the stay in hospital. One hundred twenty-seven consecutive male survivors were subsequently referred to the Department of Medicine, Danderyd Hospital, for metabolic and cardiological investigations. Of these patients 2 died in the early post-infarction period, 7 declined the metabolic investigation and for other reasons a complete evaluation was not performed in 2 patients. Thus 116 male patients (92% of male survivors) constitute the material of the present study. Six patients had a history of a previous MI 3-60 months (28 + 22 months) prior to the present MI. All patients were admitted as out-patients and
investigated 3-6 months after the MI. Clinical data were abstracted from the medical records acquired during the admission to the coronary care units. In addition a medical history was obtained by a structured interview and questionnaires at the time of the metabolic investigation. Controls For each patient a male control subject from the same residence area was randomly selected from a register of all residents in the county after matching with regard to age. Previous medical history and risk factors were established in the same way among controls as in the patient group. None of the controls had a history suggestive of angina pectoris or electrocardiographic signs indicative of CHD during a maximal exercise stress test (Minnesota codes 4.1 and 4.2). All controls were included in the study, irresspective of the presence of metabolic disturbances or additional risk indicators. General treatment and medication After discharge from the referring hospitals all patients were seen by 1 cardiologist within 2 weeks and then at regular intervals during follow-up. Fifty-five of 101 patients who were regular smokers at the time of the MI stopped smoking between the onset of infarction and the present study. No dietary information was given prior to the metabolic evaluation. General treatment with betablockers was not instituted in the coronary care units in this young post-infarction group. Instead, beta-blockers had been prescribed individually according to the local policy in the referring units. In most patients the indication for beta-blockade was cardioprotection after myocardial infarction. Previously instituted medication was not changed for this study, except for the restriction of ongoing beta-blockade to metoprolol at the first visit to the outpatients clinic more than 2 months before the lipoprotein determinations. If major side-effects attributable to beta-blocker treatment occurred with metoprolol and the indication for beta-blockade was strong, metoprolol was replaced by atenolol. At the time of the study 35 patients were entirely without medication and 60 were treated with metoprolol and 2 with atenolol only. Three and 5 patients, respectively, were treated with iso-
225 sorbide-dinitrate and niphedipine in addition to metoprolol. Four had been prescribed a combination of drugs from all 3 groups. Furosemide together with potassium supplementation was used in 20 patients, 7 of whom had no other medication. The majority of the patients had no (n = 58) or only mild angina (New York Heart Association classes I-II; n = 44) during the early post-infarction period. Severe peripheral atherosclerosis with incapacitating intermittent claudication was present in two patients and manifest diabetes in 6. Coronary artery by-pass surgery had not been performed in any patient in the period between MI and this investigation. One patient had been operated upon 6 years prior to the study. All patients were without clinical signs of cardiac decompensation at the time of the metabolic evaluation. No patient had used lipid-lowering drugs during 6 months before the lipoprotein determinations. There was no clinical or laboratory evidence of thyroid dysfunction in any of the patients or controls. Serum values for transaminases and creatinine were normal in all subjects. Blood sampling Blood samples were drawn 3-6 months after the MI (3.5 + 1.2, range 2.5-12 months), when the patients were considered to be in a stable clinical and metabolic state. Matched controls were investigated simultaneously with the patients to avoid the influence of seasonal variations on lipoprotein levels. In both patients and controls blood sampling was evenly distributed over the year. Antecubital vein puncture was performed between 8.00 h and 9.30 h after a lo-min rest in the supine position. All subjects had fasted for 12 h prior to blood sampling, during which time smokers were asked to refrain from smoking. Venous blood for analyses of apolipoproteins A-I, A-II and B was allowed to clot for 2 h and serum was then prepared by centrifugation at 1900 x g for 10 min and stored at -70°C until analyzed. Quantitation of total serum and lipoprotein lipids Venous blood was allowed to clot at room temperature for 2 h. Serum was recovered after centrifugation at 5000 X g and 5% Na-EDTA was
added to a final concentration of 0.05%. Lipoprotein analysis was performed by preparative ultracentrifugation and quantitative lipid analysis [17]. Cholesterol [18] and triglycerides [19] were determined on an Ultrolab” (LKB) after chloroform/methanol extraction [20] of whole serum, the VLDL fraction, the infranatant after ultracentrifugation (containing LDL and HDL) and of the supernatant (HDL) after precipitation. LDL values were calculated by difference. Only samples with a total recovery of cholesterol and triglycerides in the 3 lipoprotein classes of 100 + 10% have been used. HDL, was obtained as a bottom fraction in which cholesterol was determined after one preparative ultracentrifugal spin in a fixed angle rotor (Beckman model L 5-75) at density 1.125 kg/l [21]. Total HDL was obtained as described above. HDL, cholesterol was then calculated as the difference between total HDL and HDL, cholesterol. The intraassay variation was 1.8 standard deviation % (SD%) for low cholesterol values and 2.1 SD% for high cholesterol values. The corresponding values for low and high triglyceride values were 2.9 SD% and 2.2 SD%, respectively. The interassay variations were minimized by adjustment of observed values using correction coefficients obtained from simultaneous analyses of reference sera. Quantitation of apolipoproteins A -I and A -II Serum apolipoproteins A-I and A-II were determined by electroimmunoassay [22] using rabbit antisera rendered monospecific by immunoabsorption [23]. Polyethylenglycol 6000 at a final concentration of 40 g/l was included in the agarose gels used for determinations of apolipoprotein A-II in order to render the immunoprecipitates more distinct. The contents of apolipoproteins A-I and A-II in a standard serum obtained from healthy blood donors was assessed by comparison with a human HDL preparation with known amounts of apolipoproteins A-I and A-II as determined by SDS-polyacrylamide gel electrophoresis. The apparent concentrations of apolipoproteins A-I and A-II in the standard serum were 103 mg/lOO ml and 30 mg/lOO ml, respectively. The intraassay and interassay coefficients of variance for apolipoprotein A-I were 4.9% and 6.5%. respectively.
226 The corresponding figures for apohpoprotein were 5.8% and 8.3%.
A-II
@iuntitution of apo/ipoprotein B Antiserum against apolipoprotein B was prepared from rabbits. LDL in the density range 1.02-1.05 g/l which had been isolated from plasma of lipoprotein (a)-negative subjects by ultracentrifugation and gel filtration on a Bio-Gel A 15 M column was used as antigen. Antiserum from the same bleeding was used throughout the study. The antiserum gave a single precipitation line with LDL and different human serum samples. This monospecific antiserum was used to quantitate total apolipoprotein B in the serum samples by electroimmunoassay [22]. The purified LDL was used as apolipoprotein B-standard (kept at -70°C) and had a mean protein content of 20.7% as estimated by [24] with bovine albumin as standard. This purified LDL was used to standardize a serum sample using electroimmunoassay. The serum standard used throughout the study was kept at - 70°C in small aliquots and was regularly checked against the purified LDL standard. The intraassay and interassay coefficients of variance were 2.8% and 3.1%.
Lipoprotein electrophoresis Agarose gel lipoprotein electrophoresis [25] was performed on whole serum and on the top (VLDL) and the bottom (LDL + HDL) fractions obtained after ultracentrifugation at d = 1.006 kg/l. One percent agarose (Miles-Seravac) was used and staining was done with Sudan black (1%). The stained electrophoretic strips were projected on a screen in an overhead projector and inspected, without knowledge of diagnosis, for the presence of late pre-beta (LPbeta) bands [26] and the sinking pre-beta band (SPbeta). According to the mobility relative to the beta band, the LPbeta band was further subdivided into LPbeta I with mobility slower than pre-beta but clearly faster than,beta, LPbeta II in which the slower part of the band was in the beta region, and LPbeta III with beta mobility. The SPbeta band has a faster mobility than beta but usually slower than the pre-beta found in A < 1.006 kg/l serum and is often seen in whole serum.
Additional risk indicators Subjects who smoked at least 1 cigarette or an equivalent amount of tobacco each day were classified as present smokers. Subjects who had never smoked or had smoked continuously for less than 1 month were defined as non-smokers. Others were regarded as former smokers. In calculation of the average tobacco consumption 1 cigarette was considered equivalent to 1 g, 1 cigarillo to 2 g and 1 cigar to 5 g of tobacco. Tobacco consumption of pipe smokers was calculated by dividing by 7 the weekly consumption in grams. As a cumulative estimate of tobacco consumption prior to MI, cigarette-years was used [27]. Figures of alcohol consumption immediately prior to MI were based on a structured interview and questionnaires and expressed as g of absolute alcohol consumed during one month. Blood ‘pressure was measured in the supine position after a 5-min rest. Hypertension was defined as present either if antihypertensive treatment had been instituted prior to MI or in the immediate post-infarction period or if blood pressure was above 160 mm Hg systolic and/or 95 mm Hg diastolic. Manifest diabetes mellitus was diagnosed as fasting hyperglycemia (whole blood) of > 7.0 mmol/l. Oral glucose tolerance was assessed in all patients following ingestion of 1.75 g glucose/kg body weight [28]. Decreased oral glucose tolerance required the measurement of blood glucose values > 8.9 mmol/l at 60 min and > 6.7 mmol/l at 120 min following glucose intake. Weight was measured with subjects dressed in indoor clothing without jacket and shoes. The weight/height index was calculated (weight (kg)/(height (cm) - 100)) and overweight defined by values above 1.10. Statistical methods Proportions were compared using the chi-square test with Yates’ correction. Statistical significance for differences in lipoprotein composition between groups was tested using one way analysis of variance and two-tailed t-test. Coefficients of skewness and kurtosis were used to test deviations from a normal distribution. Values of total triglycerides, VLDL triglycerides and VLDL cholesterol were accordingly logarithmically transformed to achieve a distribution not significantly different from nor-
227 Ethical considerations Prior to the study informed consent was obtained from all subjects. The study protocol had been approved by the regional ethical committee.
ma1 prior to significance testing. Differences between groups were further studied by analysis of covariance enabling control for concomitant group differences in other variables such as tobacco consumption, alcohol consumption and body weight. Correlation analyses were performed according to standard methods. Since strong intercorrelations exist between several lipoproteins and apolipoproteins stepwise multiple discriminant analyses were performed to determine the sets of independent lipoprotein and apolipoprotein variables giving the best discrimination between patients and controls. The variable with the highest F-value was entered at each step until no variable remained with a value (F to enter) of 4 or more. Variables reflecting smoking habits, alcohol consumption and body stature were entered as forced variables in some of the discriminant analyses. All statistical analyses were performed at the Stockholm Computer Centre for University Education and Research using standard BMDP programmes [29].
TABLE
Results Clinical churacteristics and addition& risk indicutors As shown in Table 1 smoking habits among patients and controls were identical at the time of the metabolic evaluation, whereas alcohol consumption was higher in the control group. However, there was a marked overrepresentation of former smokers in the patient group. the majority of whom had stopped smoking at the time of the MI. Accordingly cumulative life-time tobacco consumption was much higher in patients. More patients had manifest diabetes or reduced oral glucose tolerance. Young male post-infarction patients taken as a group were shorter than matched controls, whereas body weight was similar in the two groups. The weight/height index was thus higher in the patient group.
1
BASIC CHARACTERISTICS
AT INVESTIGATION
Values are number
in group or means&standard
of subjects
IN PATIENTS
Patients (n=116) Age (~0 Smoking habits
39.x
present smoker (n)
Tobacco
(n)
+
3.9
39.9
*
P-value
3.9
45 30 I 41
n!,
< 0.001 h
consumption
present (g/day) cumulative. (cigarette-years) Hypertension (n) Diabetes
6.5 + 10.1 411 f 226 29
typeI type II(n) Decreased
Controls (n=116)
46 63 .’ I
former smoker (n) non-smoker
AND CONTROLS
deviation.
oral glucose tolerance
(n)
8.0 * 12.5 198 +218 12
2 4 35
0 01 10
< 0.001 h
+ 388 623 180.7 k 6.9 79.9 & 11.8 0.99* 0.13
Alcohol consumption (g/month) Height (cm) Weight (kg) Weight/height index (kg/cm - 100)
41s &381 175.9 k 6.2 80.5 i 11.1 1.06* 0.13
“ 55 patients stopped smoking at MI. h Group differences for distributions of categorical
data were tested by chl-square
analysis
ns < 0.001 < 0.001
< 0.001 < 0.001 ns < 0.001
with Yates’ correction.
228 Lipoprotein lipid and apolipoprotein concentrations There were marked elevations of the cholesterol and triglyceride concentrations in VLDL in patients compared to controls (Table 2). The LDL cholesterol and triglyceride levels were also elevated in the patient group, the elevation of LDL triglyceride level being the most pronounced. Both HDL, and HDL, cholesterol concentrations were reduced, the reduction of HDL, cholesterol level
TABLE
accounting for the major part of the group difference in total HDL cholesterol concentration. Serum levels of apolipoproteins A-I and A-II were decreased in the patient group, whereas serum apolipoprotein B level was increased. The prevalence of the visually registered electrophoretic Lpbeta band (Lpbeta I-II) was increased among the patient (24% vs 13%, P < 0.05), whereas the preva!ence of the visually documented
2
LIPOPROTEIN
LIPID
AND APOLIPOPROTEIN
Values are means * standard
Cholesterol (mmoI/I) Total VLDL LDL HDL HDL, HDL, Triglycerides (mmol/I) Total VLDL LDL HDL
Apolipoproteins (mg/lOO A-I A-II B
deviation
or number
CONCENTRATIONS of subjects
Patients (n =116)
Controls (n =116)
1.33 1.40 1.19 0.99 4.84 1.23 1.12 0.24 0.37 0.20 0.75 0.15
6.07 1.16 0.54 0.54 3.97 0.92 1.45 0.38 0.58 0.37 0.85 0.14
2.95 2.35 2.12 1.90 0.49 0.17 0.16 0.06
1.45 1.23 0.91 1 .oo 0.35 0.12 0.15 0.04
ml) 111.0 19.3 36.9 1.3 128.4 20.8
125.6 19.2 40.4 1.3 105.2 20.1
IN PATIENTS
AND
CONTROLS
in group. % difference
A
P-value
+21
-c 0.001
+120
< 0.001
+22
i 0.001
-23
< 0.001
-36
i 0.001
-12
< 0.001
+ 103
i 0.001
+132
< 0.001
+40
i 0.001
+7
ns
-12
< 0.001
-9
< 0.001
i-22
i 0.001
Electrophoreticpattem Lpbeta Lpbeta Spbeta * Patients
I II
compared
18 10 41 to controls.
10
5 28
< 0.05 ns
229
SPbeta trait was not significantly increased in the patient group (35% vs 25%, P < 0.10). Means and standard deviations for lipoprotein lipid and apolipoprotein levels were practically identical in patients with and without beta-blocker medication. No correlations were observed between the time interval between MI and blood sampling and lipoprotein lipid or apolipoprotein levels (r values 0.0220.11 in correlation analyses). When differences between the groups in present tobacco and alcohol consumption, as well as weight/height index, were controlled by covariante analysis, some changes in group means of lipoprotein and apolipoprotein values appeared. In particular the differences in groups means of VLDL lipid concentrations were reduced (adjusted means i SEM in patients compared to controls for VLDL cholesterol level 0.85 k 0.05 vs 0.46 k 0.04 mmol/l and for VLDL triglyceride level 1.58 _t 0.05 vs 0.76 f 0.04 mmol/l). With the exception of serum apolipoprotein A-II all differences between patients and controls observed in simple univariate analysis remained statistically highly significant (P < 0.001). The discriminatory power of HDL in relation to MI was further evaluated by separate comparisons of normolipidemic and hypertriglyceridemic patients with the normolipidemic and hypertriglyceridemic subgroups of the controls after adjustment for group differences in whole serum cholesterol and/or triglyceride levels. Adjusted apolipoprotein A-I levels tended to be lower in normolipidemic patients compared to normolipidemic controls (116.4 f 3.9 vs 126.2 f 2.3 mg/lOO ml. P < 0.05) whereas adjusted HDL cholesterol values were not significantly reduced (1.34 k 0.06 vs 1.49 + 0.04 mmol/l, P < 0.10). In hypertriglyceridemic patients both apolipoprotein A-I and HDL cholesterol levels were decreased compared to hypertriglyceridemic controls after adjustment for differing triglyceride values (107.4 + 3.0 vs 122.0 i 3.9 mg/lOO ml and 1.02 & 0.04 vs 1.22 ~fr 0.05 mmol/l, respectively, P < 0.01). In addition, a significant elevation of VLDL cholesterol concentration persisted in the hypertriglyceridemic patients (1.55 of:0.06 vs 1.48 f 0.08 mmol/l, P < 0.05). The adjusted HDLz cholesterol concentration, however, did not differ between hypertriglyceridemic patients and controls.
Ratios of cholesterol to trigbcerides in nwjor lipoprotein clusses The average ratios of cholesterol to triglycerides in all 3 major lipoprotein classes were lower in the patients than in the controls (Table 3). This difference was particularly pronounced in HDL. The VLDL cholesterol/triglyceride ratio was significantly higher in Lpbeta-positive compared to Lpbeta-negative patients (P < 0.001). but it was still within the range of Lpbeta-negative controls. In the LDL fraction the ratio of cholesterol to triglycerides was lower in Lpbeta-positive than in Lpbeta-negative patients (P < 0.05). However. as a group Lpbeta-negative patients also differed significantly from Lpbeta-negative controls (P < 0.05) with respect to LDL cholesterol/triglyceride ratio. All differences related to the Lpbeta trait were more prominent if only Lpbeta II-positive patients were considered, in whom, in addition. serum apolipoprotein B levels were elevated (138.7 + 22.8 vs 127.7 f 16.6 mg/lOO ml, P < 0.001) in comparison to Lpbeta-negative patients. A further evaluation of the relation between the concentrations of cholesterol and triglyceride in the major lipoprotein classes was obtained by plotting the individual lipid values of the patients against each other and relating them to the regression line of the healthy controls (Figs. 1 A-C). In VLDL (Fig 1A) there was a strong positive correlation between the lipid values at triglyceride levels below 1.5 mmol/l. At higher VLDL triglyceride levels (1.5-2.5 mmol/l). but still within the range of the control group, however, the VLDL cholesterol values were more variable in relation to the triglyceride values and some, mostly Lpbetapositive, patients were located markedly above the regression line of the controls. In LDL the cholesterol values varied considerably in relation to the triglyceride values (Fig. 1 B). Since the apparent relation between cholesterol and triglycerides in LDL for the controls did not contain the origin, the average lowering of the ratio of cholesterol to triglycerides in the patient group did not necessarily imply a changed relation between the two lipids in LDL. Moreover, within the LDL triglyceride range of the control group (0.2-0.6 mmol/l) an elevated LDL cholesterol/ triglyceride ratio was indicated, whereas in a num-
230 TABLE RATIOS
3 OF CHOLESTEROL
TO TRIGLYCERIDES
IN THE MAJOR
Patients Lpbeta + (n = 28)
LIPOPROTEIN
CLASSES P-value ’
Controls Lpbeta (n = 88)
All (n = 116)
Lpbeta + (n=15)
Lpbeta (n = 101)
All (n = 116)
VLDL Chol VLDL Tg
0.66 0.15
0.55 0.09
0.58 0.19
0.72 0.15
0.61 0.10
0.62 0.11
< 0.01
LDL Chol LDL Tg
9.45 2.53
11.21 3.65
10.74 3.44
9.71 2.55
12.61 3.38
12.23 3.42
< 0.01
HDL Chol HDL Tg
8.09 3.67
8.27 4.27
8.20 4.08
9.61 4.23
11.23 5.20
11.02 5.10
i 0.01
a All patients compared to all controls. Lpbeta+ = Lpbeta I-III-positive; Lpbeta-
= Lpbeta
I-III-negative;
ber of mostly Lpbeta-positive, markedly hypertriglyceridemic, patients a very low LDL cholesterol/triglyceride ratio was noted. A change in the relation between cholesterol and triglycerides in HDL was apparent in the patient group (Fig. 1C) since the vast majority of patients were located below the regression line of the control group and well within the distribution of HDL triglyceride values for this group. Ratios between concentrations of total HDL cholesterol, cholesterol in HDL subclasses, and serum apolipoproteins A-I and A-II As expected from the data in Table 2 the HDL,/HDL, cholesterol ratio was reduced among the patients compared to the control subjects, whereas no difference between the study groups was observed in the apolipoprotein A-I/A-II ratio (Table 4). In addition the HDL cholesterol/apo-
Chol = cholesterol;
Tg = triglycerides.
LDL CHOL
-/L 80
I 5.
:
.*.... -*.
4.
: -
4a. .iL&f+&Y ;;;-.. .. 8.0
-.
2.0.
08
0.2
0.4
0.8
LOL
I.0
TG.h&t/L
HDLCHOL -IL 2.0 1
. 1.0~ .
.
. .
.*._-. . .
.
: . .
oc 0
am
0.20
0.30
HDL TG. -/L
Fig. 1. The relations between concentrations of triglycerides (TG) and cholesterol (CHOL) in (A) VLDL, (B) LDL and (C) HDL in the patient group. The shadowed area denotes the 95% confidence limites of the regression line of the control group. ??= Lpbeta-positive patients; 0 = Lpbeta-negative patients.
231 TABLE
4
RATIOS BETWEEN CONCENTRATIONS OF TOTAL HDL CHOLESTEROL, CHOLESTEROL IN HDL SUBCLASSES, AND SERUM APOLIPOPROTEINS A-I AND A-II Values are means f standard
deviation
Patients (n=I16)
Controls (n =116)
P-value
0.51 0.27
0.71 0.43
i 0.001
apo A-I ~apo A-II
3.06 0.38
3.16 0.41
“s
HDL Chol
0.40 0.06
0.46 0.09
< 0.001
HDL,
Chol
HDL,
Chol
apo A-I
Stepwise discriminant una&sis The relations of single lipoprotein variables and different apolipoproteins to MI were first compared by calculation of the F-values to enter the discriminant function when the measurements of tobacco and alcohol consumption. and the weight/height index. had been entered as forced
Chol = cholesterol:
Tg = triglycerides:
glyceride value (P < 0.001). The same significance level for difference in position of the regression lines of the respective groups was also recorded if only patients with VLDL triglyceride values corresponding to the range of control subjects were analysed. Thus for any given VLDL triglyceride value the concentration of HDL cholesterol was lower in patients compared to controls.
apo = apolipoprotein.
TABLE
lipoprotein group.
A-I ratio
was reduced
in the patient
5
LIPOPROTEIN AND APOLIPOPROTEIN SIGNIFICANTLY DISCRIMINATING TIENTS AND CONTROLS
Relation between VLDL triglyceride and HDL cholesterol concentrations The strongest correlation (r = -0.53) between concentrations of VLDL triglycerides and total HDL cholesterol, HDLz and HDL, cholesterol, and apolipoproteins A-I and A-II was demonstrated between VLDL triglyceride and total HDL cholesterol concentrations in the patient group (Fig. 2). The regression line of the patients was parallel to that of the controls, but located significantly lower irrespective of VLDL tri-
VARIABLES BETWFEK PA-
Lipoprotem/variable
F-value
Lipoprotein/ variable
~-Lalw
VLDL Chol+
112.73
LDL Tg
46.67
I 11.36
LDL Chol
37.x3
1 10.03
HIX,
36.29
LDL Chol
HDL Chol LDL Chol HDL (‘ho1 apo B
(‘hvl
apo A-I Total Tg
65.33
apo B
64.33
30.09
apo A-l HDL Chol
28.75
ap” A-l HDL CHOC
MMcqL 2.0
VLDL Tg
62.40
1
HDL,
(‘ho1
IX.45
HDL,ChoI VLDL Chol
56.04
apo A-II
x.13
Total Chol
55.38
ape A-I apo A-II
7.63
HDL Chol
52.57
LDL (‘ho1
6.26
LDL Tg apo B apo A-II
0 01
10
50.94
VLDL Chol VLDL Tg
6.06
a.0
VLDL TG. MYOL/L
Fig. 2. The concentration of HDL cholesterol in relation to VLDL triglyceride concentration (logarithmic scale) in the patient group. The shadowed area denotes the 95% confidence limits of the regression line of the control group.
F-values are F-values to enter the diacrimlnant function when measurements of tobacco and alcohol consumption. ah well as the weight/height index. had been entered as forced variables. A F-value of 4 or more is considered aignlflcant. Chol = cholesterol: Tg = triglycerides: apo = apolipoprotri~~.
232 TABLE
6
LIPOPROTEIN CRIMINATING IN STEPWISE
VARIABLES SIGNIFICANTLY DISBETWEEN PATIENTS AND CONTROLS MULTIPLE DISCRIMINANT ANALYSIS
A
F
Correct
classification
LDL Chol HDL Chol Total Tg
38.19 21.85 12.02
infarct control total
83.3 76.8 80.1
F
Correct
classification
LDL Chol
60.92
HDL Chol Total Tg
12.16
infarct control total
83.3 81.1 82.2
VLDL Chol
4.73
B
apo A-I
(S)
7.15
apo A-II VLDL Chol
5.05
C
F
Correct
classification
Total Chol HDL Chol
58.60 39.79
infarct control total
85.4 77.9 81.7
F
Correct
classification
62.10
infarct control total
85.4 83.2 84.3
Smoking Alcohol Weight/height
(%)
index
D
LDL Chol HDL Chol
(X)
8.93 0.57 0.28 (%)
12.92 5.53
Total Tg apo A-I apo A-II VLDL Chol
5.04
Smoking Alcohol Weight/height
9.88 0.06 0.18
index
A: Only serum lipids, lipoprotein lipids and apolipoproteins included in the analysis. B: Variables as in A, but for the inclusion of ratios between serum lipids, lipoprotein lipids and apolipoproteins. C: Variables as in A, tobacco and alcohol consumption and weight/height index at the time of the investigation were forced variables. D: Variables as in B, tobacco and alcohol consumption and weight/height index at the time of the investigation were forced variables. F-values indicated in the tables are the F-values when all selected variables had been entered in the equation (F to remove). Chol = cholesterol; Tg = triglycerides; apo = apolipoprotein.
variables (Table 5). The sum of cholesterol in VLDL and LDL divided by HDL cholesterol level had the strongest power to discriminate between patients and controls. The concentrations of serum triglycerides, apolipoprotein B, VLDL triglycerides and cholesterol, serum cholesterol, HDL cholesterol and LDL triglycerides, in that order, discriminated better between patients and controls than did the LDL cholesterol concentration. Among variables reflecting HDL concentration and composition HDL cholesterol was the best discriminator, followed by HDL, cholesterol, apolipoprotein A-I, and the HDL cholesterol/ apolipoprotein A-I ratio. A number of lipoprotein components were found to discriminate independently between patients and controls in the stepwise multiple discriminant analysis (Tables 6A-D). When only lipoprotein lipid and serum apolipoprotein concentrations were entered in the discriminant function (Table 6A), the levels of LDL cholesterol, HDL cholesterol, total serum triglycerides and VLDL cholesterol, in that order, were found to be the best set of independent discriminators. Adding ratios (Table 6B) resulted in the replacement of LDL and HDL cholesterol concentration by the LDL/HDL cholesterol and apolipoprotein AI/A-II ratios. If, instead, variables reflecting smoking habits, alcohol consumption and body stature were forced and ratios not included (Table 6C), total serum cholesterol and HDL cholesterol concentrations emerged as independent discriminators. When, ratios were added (Table 6D), however, the previous pattern of independent lipoprotein variables returned. A correct total classification in percent of patients and controls around SO-85% was obtained, with slightly higher values for patients. The inclusion of ratios and serum apolipoprotein concentrations added somewhat to the correct classification of controls obtained by whole serum and lipoprotein lipid concentrations alone.
Discussion Predisposing metabolic risk factors should be more apparent in patients with premature coronary heart disease than in middle-aged or elderly popu-
233 Iations. In a younger patient group, the confounding influence of age per se and coexisting age-dependent degenerative and inflammatory diseases can be considered insignificant. Since all medical departments within the region participated in this study and the number of dropouts was marginal, the present material is representative of young male infarction survivors in the Stockholm area. In healthy subjects the levels of several lipoprotein lipids are highly intercorrelated. In particular, close correlations exist among serum triglycerides and VLDL lipids [30]. In the young post-infarction patients, however, these latter interrelations were found to be altered as indicated by results from the multivariate analyses, where both serum triglyceride and VLDL cholesterol levels appeared as independent variables. Furthermore the intralipoprotein relations of cholesterol and triglyceride seemed to be altered in the patient group. Our results thus emphasize independent significances of the concentrations of VLDL, LDL and HDL in relation to myocardial infarction. As a single variable serum total apolipoprotein B concentration was a better discriminator than LDL cholesterol level. The same applied for the concentrations of total triglycerides, VLDL lipids, HDL cholesterol and total cholesterol. However, the stepwise multiple discriminant analysis indicated that the independent relations of LDL and HDL cholesterol levels to MI were closer than those of serum triglycerides or VLDL cholesterol. These results extend the findings of a similar study using multivariate analysis from Gothenburg [15] in which, however, the lipoprotein evaluation was restricted to measurement of total serum lipid levels and concentrations of apolipoproteins A-I, A-II, B and D. Multivariate analysis indicated that the levels of apolipoprotein A-II and total serum triglycerides correlated independently with myocardial infarction. When smoking had been included in the regression equation altogether only 28% of the variation of the dependent variable was accounted for. The relationship between VLDL and CHD is not well defined. In previous studies of post-infarction patients, the magnitude of the VLDL cholesterol elevation was the most pronounced change in lipoprotein levels [31-331. Multivariate analyses of prospecti;e data, as applied in the
Framingham and Gothenburg studies [3,5], have implied that the correlation of whole serum triglyceride concentration with risk derives from secondary associations, whereas in the Stockholm Prospective Study triglycerides emerged as a significant and independent risk factor for MI (6,7]. In the present study concentrations of both whole serum triglycerides, or VLDL triglycerides, and VLDL cholesterol were found to be independently related to MI. The indicated independent contribution to risk of both VLDL cholesterol and triglyceride levels in young post-infarction patients is at first sight astonishing since, in normolipoproteinemia and in all primary hyperlipoproteinemias except type III, a close relation has been demonstrated between the concentrations of cholesterol and triglycerides in the VLDL fraction. Among the young patients, however, the Lp-beta band which may represent remnant particles and is associated with a cholesterol-rich VLDL [26], was more frequently observed than in the control subjects. This may contribute to the independent risk indicated in VLDL cholesterol. In comparison, an increased frequency of Lpbeta-positive patients was not found in a group of older post-infarction patients [31]. Possibly the independent relation of VLDL cholesterol concentration to MI is specific for young patients and due to an increased occurrence of Lpbeta-positive patients with a cholesterol-rich VLDL. In the present study as well as in other studies of post-infarction patients [31,33] increased LDL lipid concentrations were noted. It has been assumed that the measurement of LDL by determining its cholesterol content provides an accurate assessment of total LDL particle concentration, particularly the LDL, concentration, LDL, being reflected by LDL triglycerides. However, a syndrome of hyperapobetalipoproteinemia was recently delineated in normolipidemic patients with coronary artery disease and an abnormal LDL composition [13]. The relative importance in our material of increased number of LDL particles versus altered LDL composition cannot be thoroughly evaluated since only whole serum apolipoprotein B was determined. In univariate analysis apolipoprotein B concentration was a better discriminator between patients and controls than LDL cholesterol level,
234 whereas in multivariate analysis LDL cholesterol was the better discriminator. This result from the multivariate analysis contrasts with several recent reports [10,11,14]. However, in previous studies apolipoprotein B concentration seemed to be more predictive at lower serum cholesterol levels [34]. The young patients in this study usually exhibited moderately elevated serum cholesterol levels, which might account for this discrepancy. HDL data emerging from this study showed that, as a single variable, HDL cholesterol provided a better means of discrimination between patients and controls than did HDL, cholesterol, apolipoprotein A-I and ratios between HDL subfractions or HDL apolipoproteins. In multivariate analysis including ratios of lipoproteins and apolipoproteins, however, the serum apolipoprotein ‘A-I/A-II ratio apparently gave a better representation of HDL changes relevant to MI. Whether this reflects the importance of the structural proteins of HDL or an inability of the HDL, and HDL, cholesterol determinations to accurately reflect the true relations between the HDL subclasses cannot be assessed from this study, since the HDL apolipoproteins were not determined in the HDL subfractions. The relation between HDL metabolism and triglyceride metabolism is not fully understood. Apparently the differences in HDL constituents between patients and controls in this study were not a mere reflection of disturbances in triglyceride metabolism, since the same differences persisted for the whole material using multivariate analyses and after controlling for triglyceride levels in the normolipidemic and hypertriglyceridemic subjects, respectively. The findings in this study should be interpreted with due regard to its cross-sectional design. Significant alterations in the associations between lipoproteins and MI may occur during the time between the onset of disease and the metabolic evaluation. However, such alterations are less probable in these young patients, who were as a rule without cardiac symptoms prior to MI and generally not severely incapacitated post MI. Sufficient time was shown to have elapsed between MI and blood sampling to allow for the temporary effects of acute phase reactions on lipoproteins to disappear [35,36]. Since no differences in lipopro-
tein levels or composition were observed between patients with or without betablockers or furosemide, medication decidedly cannot account for the differences demonstrated between the patient and control groups. The present study thus demonstrates the importance of elevated serum triglyceride and VLDL cholesterol levels, in addition to increased LDL and decreased HDL cholesterol concentrations, in relation to MI at young age. References
9
10
11
12
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