Atherosclerosis, 89 (1991) 109-l 16 9 1991 Elsevier Scientific Publishers AlIONIS
109 Ireland,
Ltd. 0021.9150/91/$03.50
002191509100140E
ATHERQ
04660
Effect of moderate hypertriglyceridemia on the relation of plasma total and LDL apo B levels Allan Sniderman, Hai Vu and Katherine Cianflone .McGill Unit for the Prel,ention of Cardiocascular Disease, McGill Unilzrsity, Montreal, Quebec (Cunadal (Received 26 December, 1990) (Revised, received 27 February, 1991) (Accepted 25 March, 1991)
Summary The risk of premature coronary artery disease is related to an important degree to the number of particles of low density lipoproteins (LDL) in plasma, an estimate given by measurement of LDL apo B. In clinical practise, though, it is total, not LDL apo B, which is measured. The purpose of the present study therefore was to compare plasma total and LDL apo B in the presence and absence of moderate hypertriglyceridemia. The results demonstrate that within the range of plasma triglyceride levels examined, i.e., values of triglyceride up to 500 mg/dl, there is close correspondence between total and LDL apo B, with the latter more than 90% of the former. VLDL composition was also examined and two patterns found in hypertriglyceridemic patients: those with normal apo B had markedly lipid enriched VLDL while those with elevated apo B had VLDL which was normal in composition except for a moderate increase in triglyceride content. Thus total apo B within the circumstances studied reflects principally LDL apo B. Moreover measurement of apo B allows distinction between two different forms of hypertriglyceridemia, only one of which - that with an increased LDL particle number - has previous work shown to be associated with increased coronary risk. Total apo B, therefore, provides additional information not available from conventional plasma and lipoprotein lipids which allows more precise physiologic classification and may lead to more rational choice of pharmacologic therapy in normolipidemic and hypertriglyceridemic patients.
Key words:
Hypertriglyceridemia,
apo B; Hyperapobetalipoproteinemia
Correspondence to: Allan D. Sniderman, M.D., Cardiology Division. Royal Victoria Hospital, 687 Pine Avenue West, Montreal, Quebec, H3A 1Al Canada. Tel.: (514) 842-1231 ext. 4631; Fax: (514) 9X2-0686.
Introduction The present clinical practise to determine the atherogenic risk posed by lipoproteins is based on
110
measurement of serum and lipoprotein lipids. Thus if total, and more particularly, low density lipoprotein (LDL) cholesterol, is markedly elevated, risk is markedly elevated, and pharmacologic therapy is justified if dietary therapy fails. If plasma triglycerides are elevated, but LDL cholesterol is not, there is still no clear consensus as to how urgent or aggressive therapy should be. Marked elevation of LDL cholesterol to a level greater than the 90th percentile of the general population occurs in only a minority of patients with premature coronary artery disease [l-4]. Numerous prospective cross-sectional studies have shown apo B to be superior to total or LDL cholesterol in separating those with from those without premature coronary artery disease [4-l 11. That is, there is a substantially larger number of coronary patients with an apo B above the 90th percentile compared to those with an LDL cholesterol above this level [12,13]. However, apo B-100 is an integral constituent of 3 lipoprotein classes: very low density lipoproteins (VLDL), intermediate density lipoproteins (IDL), and low density lipoproteins (LDL). IDL and LDL are much smaller lipoprotein particles than are VLDL and this difference in size likely explains why IDL and LDL gain access to the arterial wall from the vessel lumen with greater ease than VLDL. Under most circumstances, by far the greatest proportion of the total apo B is found in LDL [14] and the same relations between apo B and risk of disease have been shown for LDL apo B as for total apo B [5,6]. While there is still little data on these relationships in hypertriglyceridemic patients, it is known that only a proportion of hypertriglyceridemic subjects have an elevated apo B, and that those who do appear to be at higher risk of coronary disease than those who do not [15-191. The primary objective of this study was to determine the relations between total and LDL apo B in a group of patients, some of whom were moderately hypertriglyceridemic. In addition, the composition of VLDL (that is, the composition of the d < 1.006 g/ml supernate lipoproteins) has been compared in those hypertriglyceridemic patients with, and those without, elevated apo B.
Methods
Fasting plasma samples were obtained from 56 patients. Their average age was 57.6 years; 32 were female, 24 male. Plasma was separated by centrifugation at 2500 x g at 4 ’ C. To obtain the d < 1.006 lipoproteins, 1 ml plasma was added to 4 ml of a salt solution of density 1.006 g/ml in an ultra clear centrifuge tube; VLDL was separated by ultracentrifugation at 40 000 rpm for 18 h in an SW50 rotor at 4” C [20] and the d < 1.006 g/ml supernate collected. The following parameters were measured: on plasma, total cholesterol, triglycerides, and apo B; on the d < 1.006 g/ml supernate, total and free cholesterol, triglycerides, phospholipids and apo B; on the d > 1.006 g/ml infranatant, apo B. Triglycerides, free and total cholesterol and phospholipids were measured by enzymatic kits (Boehringer, Mannheim) while apo B was measured by an ELISA immunoassay [21] as modified by Ortho Diagnostics, La Jolla, California. HDL cholesterol was measured by heparin manganese precipitation while LDL cholesterol was quantitated as the difference between total plasma cholesterol and the sum of VLDL and HDL cholesterol. VLDL apo B was defined as the apo B measured in the d < 1.006 g/ml supernate, while for the purposes of this study, LDL apo B was defined as the apo B measured in the d > 1.006 g/ml infranate. This would include intermediate density lipoproteins within the LDL fraction. LDL 3-20% gradient gel PAGE was performed by the method of Laemmli [22]. Statistical analysis was performed by r-test of 2 means or by ANOVA. Linear regression was performed by least-squares linear regression. Results
The average plasma lipid, lipoprotein lipid, and total apo B results for the total group are given in Table 1. The total group is also subdivided into those with a fasting plasma triglyceride < 200 mg/dl (the normotriglyceridemic patients) and those with a fasting plasma triglyceride > 200 mg/dl (the hypertriglyceridemic group). There
111 TABLE
I
Normo-triglyceridemia (n = 27)
Total (n = 57)
54.4 (18.8) 33F/24M 207.9 (101.9) 142.1 (96.3) 136.3 (66.8) 9.8
Age Sex Plasma TG VLDL TG Plasma
apo B
VLDL apo B
Chol
LDL Chol HDL Chol
53.3 (21.6) 16F/14M 289.6 (69.7) 214.1 (78.0) 155.1 (76.2) 12.1 (11.4) 143 (69.6) 271.3 (45.9) I65 (45.7) 35.6 (9.8)
55.5
(4.2) 108.1 (47.1) 264.7 (53.8) 185.9 (58.1) 45.1 (14.7)
0
r
a~08
lmg/d’l
100
50
150
PI-ASMA
200
250
300
, 353
apoB (mg/dl)
Fig. 2. Plasma apo B vs. LDL apo B in 27 normal triglyceridemic samples (plasma triglycerides < 200 mg/dl). Regression: LDL apo B = I.011 plasma apo B - 8.477; r’ = 0.992.
were 27 patients in the former group and 29 in the latter. The first issue examined was the relation between total apo B and LDL apo B in the entire group. These results are shown in Fig. 1. The
LDL
--
01
Normotriglyceridemia = plasma triglycerides < 200 mg/dl; Hypertriglyceridemia = plasma triglycerides > 200 mg/dl. TG = triglycerides (mg/dl); apo B = apoiipoprotein B (mg/dl); Chol = cholesterol (mg/dl).
35”
~DOB (mg/dli
Hyper-triglyceridemia (n = 30)
(15.6) 17F/lOM 117 (28.8) 62.1 (25.5) 115.3 (46.4) 7.2
(9.1) 126.5 (62.5) 268.2 (49.9) 174.9 (53.0) 40.1 (13.2)
LDL apo B Plasma
LDL ,-
350
i
linear regression obtained was y = 0.927x + 0.127 which indicates that, overall, LDL apo B accounted for just under 93% of the total apo B. The correlation coefficient was high with an r’ of 0.985 and inspection of the data indicates that this relationship held throughout the range of values. The results in the two subgroups are shown in Figs. 2 and 3. In the normotriglyceridemic group (Fig. 21, there is again a strong relation between plasma and LDL apo B with a slope of 1.011 and an r2 of 0.992. This, of course, is not unexpected. The results in Fig. 3 are more critical to the present study. Again however, a close relation
LDL
awB
(mg/dli
350,
300
i’>ll
I
250
:
zoo/ 150
100
, i_~L.._._ 0
50
/
I 100
150
PLASMA
200
250
300
350
apoB (mg/dl)
Fig. 1. Plasma apo B vs. LDL apo B in 57 normal and hypertriglyceridemic samples. Regression: LDL apo B = 0.927 plasma apo B + 0.128; r2 = 0.985.
_1
0
100
150
PLASMA
200
250
333
350
apoB (mg/dt)
Fig. 3. Plasma apo B vs. LDL apo B in 30 hypertriglyceridemic samples (plasma triglycerides > 200 mg/dl). Regression: LDL apo B = 0.9058 plasma apo B + 2.485; r’ = 0.983.
112 TABLE
2
VLDL
COMPOSITION:
Values
are expressed
Group
RATIO as average
OF LIPID/APO
B
(SD)
Triglyceride/ apo B
Cholesterol ester/ape B
Free cholesterol/apo
B
Phospholipid/ape
(1) NTG/NB
10.5
3.2
2.0
(2) NTG/HB
(5.2) 11.1
(1.2) 2.8
(0.8) 2.1
(0.7) NS 8.6
(1.1) NS 4.4
(3.6) 0.0005
(1.6) 0.0005 2.3
(7.1) 0.0025 9.7
(1:;) NS 0.0005 18.4 0.0001
(1.3) NS 0.0005 18.5 0.0001
(6.9) 0.01 0.0005 5.4 0.005
P (3) HTG/NB P (4) HTG/HB P P Group F value P
3
(5.2) NS 34.7 (12.2) 0.0005 19.4 (12.0) 0.0005 0.0005 11.4 0.0001
B
15.5 (11.7) 22.6 (14.0) 0.025 22.9
NTG = normotriglyceridemia (plasma triglycerides < 200 mg/dl); HTG = hypertriglyceridemia (plasma triglycerides > 200 mg/dl); NB = normal plasma apo B ( < 120 mg/dl); HB = high plasma apo B ( > 120 mg/dl); P values are calculated by t-test as compared to group 1: NTG/NB except where indicated. F value is calculated by one-way analysis of variance (ANOVA).
obtains with LDL apo B accounting for 91% (slope = 0.906) of the total apo B with an r2 of 0.983. These results indicate that.within the range of triglyceride values examined, the mass of apo B measured within VLDL accounts for little of the total apo B mass, and therefore, total apo B and LDL apo B retain a close relation even in moderately hypertriglyceridemic patients. Next, the composition of the d < 1.006 g/ml lipoproteins was examined. In all the hypertriglyceridemic patients, delipidated aliquots of this fraction were subjected to SDS 3-20% polyacrylamide gel electrophoresis; 100 pg of d < 1.006 protein was applied to the gel. In none of the samples was a band corresponding to apo B-48 identified; similarly in none of the samples tested was the presence of apo A-IV detected as compared to appropriate molecular weight standards. That is, in none was the presence of either chylomicrons or chylomicron remnants identified by this technique. Both apo B-48 and apo A-IV, however, were present in control chylomicron samples. Therefore, the d < 1.006 lipoproteins will be considered to be VLDL. Four subgroups were constructed: group 1 consisted of 18 individuals who were nor-
motriglyceridemic and had normal plasma apo B; group 2, 9 patients who were normotriglyceridemic and had elevated apo B; group 3, 15 patients who were hypertriglyceridemic with a normal apo B; and group 4, 15 patients who were hypertriglyceridemic with an elevated apo B. These results are given in Table 2 with all the lipid parameters expressed as a ratio to apo B. Since only one apo B molecule is present per VLDL particle, this allows the relative lipid load per particle to be compared. The average composition of the VLDL is the same in the two normotriglyceridemic patient groups while in the hypertriglyceridemic hyperapo B group (group 41, the only significant change is an increase in the triglyceride content per particle. This increase is relatively small, however, compared to the changes observed in the VLDL from the hypertriglyceridemic normal apo B subgroup (group 3). Here, the mass of all the lipid components is significantly increased per particle. That is, the average VLDL particle in this group contains more triglyceride, more cholesterol and more cholesterol ester than the VLDL in the other three groups. It is, therefore, larger and lipid loaded.
113 Discussion In most of the clinical studies which have compared the relative strengths of apo B and total and LDL cholesterol in separating those with from those without coronary disease, it is total, not LDL apo B which has been measured [5-111 and the relative importance of total versus LDL apo B in this regard has recently been questioned [22]. That is, in patients with elevated apo B, if a substantial portion of the apo B were in the d < 1.006 g/ml supernate, VLDL as well as LDL might be contributing importantly to the atherogenic process. However, the results of the present study establish that there is close correspondence between total and LDL apo B in subjects with plasma triglyceride levels up to 500 mg/dl. Indeed in all the groups examined, on average, LDL apo B accounted for more than 90% of the total apo B, and we conclude, therefore, that within the range of triglyceride values examined, total apo B is determined by LDL apo B with VLDL apo B making at most a relatively minor contribution. The atherogenic risk due to hypertriglyceridemia has been, and remains, controversial. Most epidemiologic analyses have considered it as a homogeneous disorder [23]. It is not, certainly with regard to apo B. This observation is not new; indeed it was one of the major findings of the first clinical study in which apo B was measured by immunoassay. In this seminal work, not only did Lees [24] establish that important variations in LDL composition as reflected by the LDL cholesterol/ape B ratio could occur, but in a subsequent study, he and his colleagues showed there was no predictable relation between plasma triglycerides and apo B in type IV hyperlipoproteinemia [25]. That is, in some hypertriglyceridemic patients it was elevated, while in others it was not. This finding was soon confirmed by others [15,16], and in the studies performed to date, hypertriglyceridemia with elevated apo B is much more strongly connected to atherosclerotic disease than hypertriglyceridemia with normal apo B [15-191. The evidence that the level of apo B correlates better with coronary risk than LDL cholesterol is now consistent and broad [4-111; there can,
therefore, be no doubt that the observations initially made in this regard by Avogaro and his colleagues [26] have been amply confirmed. The pathophysiologic basis for this rests in the now well-established differences in LDL composition that are so frequently found, particularly in patients with premature coronary artery disease. A variety of analytic techniques have established that patients with coronary disease frequently have increased numbers of smaller, denser, cholesterol-poor LDL particles [27-301. Since the content of apo B per LDL particle is fixed whereas that of cholesterol is not, LDL apo B is an accurate index of LDL particle number whereas LDL cholesterol frequently is not. Measurement of LDL apo B thus allows an elevated LDL particle number to be recognized in patients with a normal LDL cholesterol and either normal or elevated plasma triglycerides. Given the close agreement between total and LDL apo B demonstrated in this study, total apo B, a measure which is relatively easy to obtain clinically, would appear to be an acceptable clinical surrogate for LDL apo B, a measure which is not widely available. This study has limitations which must not be overlooked. The most important practical one is that severe hypertriglyceridemia was not examined. It should be noted, however, that severe hypertriglyceridemia is not common. and when it occurs, chylomicrons or chylomicron remnants are usually present in large amounts as well. The capture antibody used in this assay is directed at the carboxy terminal end of apo B, and therefore, would not recognize apo B-48 containing chylomicrons [2,3]. This antibody, however has been shown to recognize greater than 90% of the VLDL apo B even in patients with hypertriglyceridemia [32]. Moreover even had it cross-reacted completely, the mass of apo-B-48 in the d < 1.006 lipoproteins, under almost all circumstances constitutes only a minor proportion of the apo B in the d < 1.006 supernates present [33]. The other important issue is whether the apparent differences in VLDL composition in the two hypertriglyceridemic groups are valid. For example, chylomicron remnants, if present in sufficient quantities, could produce an elevated lipid to apo B ratio in the d < 1.006 g/ml supernate.
114 However, this seems unlikely for several reasons. First, gel electrophoresis failed to detect the presence of any such particles. Second, to explain the difference between groups, such remnant particles would have to be present in much greater quantities in the normal apo B hypertriglyceridemic group than in the hyperapo B hypertriglyceridemic group. Not only is there no reason to think this occurred but previous work suggests that the opposite would be more likely; that is, chylomicron clearance is related to the level of apo B as well as fasting plasma triglyceride level [34,35] and has been shown to be delayed even in normotriglyceridemic patients with Hyperapo B 1341. The data, therefore, suggest that hypertriglyceridemia might have resulted in these two patient groups by two pathogenetically distinguishable mechanisms. One possible explanation is that in the normal apo B group, normal numbers of lipid enriched hepatic apo B particles were secreted whereas in the hyperapo B group, increased numbers of VLDL particles of essentially normal composition were being secreted. Such differences have been seen in previous studies of different genetic forms of hypertriglyceridemia. For example, whereas there appears to be no important difference in the rate of triglyceride clearance in the two disorders [361, a normal secretion rate of triglyceride-enriched VLDL has been documented in familial hypertriglyceridemia while increased numbers of VLDL particles are secreted in familial combined hyperlipidemia [37]. Obviously no genetic diagnosis can be inferred in the present study; the point at hand, though, is that the measurements made in this study may allow recognition of the pathophysiologic basis of the hypertriglyceridemia in particular patients. More important still, they raise the issue as to what should be the objective, or objectives, of therapy in such hypertriglyceridemic patient groups. In the normal apo B hypertriglyceridemic group, lowering triglycerides and increasing HDL cholesterol are the obvious goals. But in the hyperapo B hypertriglyceridemic group, the issue is more complicated. This dilemma has recently been addressed by Vega and Grundy [381. In their study, hypertriglyceridemic patients with elevated apo B were treated with either gemfibrozil
or lovastatin: the former reduced triglycerides markedly but did not change the level of LDL apo B; by contrast, lovastatin sharply decreased LDL particle number. We would agree with the conclusion they drew, namely that given the evidence that the hypertriglyceridemic hyperapo B patients are at particular risk of coronary disease, the objectives of therapy should be examined in an appropriate clinical trial. In the interim, the best evidence at hand comes from the FATS trial [371. In that study, all patients had an elevated Apo B although their lipid phenotype varied. Clinical and angiographic improvement correlated best with decreases in Apo B and increases in HDL cholesterol, the same parameters which had previously been shown to be the best predictors of new disease progression in native coronary arteries or the appearance of lesions in saphenous vein bypass grafts [18]. The appropriate clinical conclusion for the moment appears to us to be that patients with elevated Apo B may benefit from pharmacologic therapy which lowers their plasma LDL particle number. The present study establishes that an elevated LDL particle number can be recognized in moderately hypertriglyceridemic as well as normotriglyceridemic individuals by measurement of total Apo B. Acknowledgements
This research was supported by grants from the Medical Research Council of Canada (M75480) and Merck Frosst Ltd. Canada. References Kannel, W.B., Castelli, W.P., Gordon, T. and McNamara, P.M., Serum cholesterol, lipoproteins and the risk of coronary heart disease. Ann. Intern. Med., 74 (1971) 1. Goldstein, J.L., Hazzard, W.R., Shrott, H.G., Bierman, E.L. and Motulsky, A.G., Hyperlipidemia in coronary heart disease, 1. Lipid levels in five survivors of myocardial infarction. J. Clin. Invest., 52 (1973) 1533. Gotto, A.M., Gorry, G.A., Thompson, J.R., Cole, J.S., Trost, R., Yeshurun, D. and DeBakry, M.R., Relationship between plasma lipid concentration and coronary artery disease in 496 patients. Circulation, 56 (1977) 875. Holmes, D.R. Jr., Elveback, S.R., Frye, R.S., Kottke, B.A. and Ellefson, R.D. Association of risk factor variables and coronary artery disease documented with angiography. Circulation, 63 (1981) 293.
115 H.D., Sniderman, A.D., Albers, J.J. and 5 Brunzell, Kwiterovich, P.O.. Jr.. Apoproteins B and A-l and coronary artery disease in humans. Arteriosclerosis, 4(2) (1984) 79. A.D., Apolipoprotein B and Apolipoprotein 6 Sniderman. AI as predictors of coronary artery disease. Can. J. Cardiol., 4 (SuppI. A) t 1988) 24A. N., Hayat, N. and Al-Khafaji, M., Lipopro7 Al-Muhtaseb. teins and apolipoproteins in young male survivors of myocardial infarction. Atherosclerosis, 77 (1989) 131. 8 Sullivan, D.R.. Marwick. T.H. and Freedman, S.B.. A new method of scoring coronary angiograms to reflect extent of coronary atherosclerosis and improve correlation with major risk factors. Am. Heart J., 119 (1990) 1262. Y Fujiwara, R.. Kutsumi. Y.. Hayashi, T., Kim. S.S., Misawa. T., Tada. H.. Nichio, H.. Toyota, K, Tamai, T.. Nakai, T and Miyabo. S.. Metabolic risk factors in the normolipidemic male patients with angiographically defined coronary artery disease. Jap. Circul. J., 54 (1990) 493. 10 Durrington. P.N., Ishola. M.. Hunt, L., Arrol, S. and Bhatnagar. D.. Apolipoproteins (a), AI and B and parental history in men with early onset ischaemic heart disease. Lancer 1 (19X8) 1070. 11 Reinhart, R.A.. Gani. K., Arndt. M.R. and Broste. SK., Apolipoproteins A-I and B as predictors of angiographitally defined coronary artery disease. Arch. Intern. Med., I50 (1990) 1629. 12 Blank. D.. Silberberg, J. and Sniderman, A.D., Trade-oTfs on cutpoints for the treatment of hyperlipidemia. Coron. Artery Dis. l(4) (1990) 455. 13 Albert, J.J.. Brunzell. J.D. and Knapp, Q.H.. Apoprotein measurements and their clinical application. Clin. Lab. Med.. 9 (1YXY) 137. 14 Teng. B.. Sniderman. A.D.. Soutar, A.K. and Thompson. G.R.. Metabolic basis of hyperapobetalipoproteinemia: turnover of apolipoprotein B in low-density lipoprotein and its precursors and subfractions compared with normal and familial hypercholesterolemia. J. Clin. Invest.. 77 ( 1986) h6.7. 15 Durrington. P.N., Bolton. C.H. and Hartog, M.. Serum and lipoprotein apolipoprotein B levels in normal subjects and patients with hyperlipoproteinaemia. Clin. Chim. Acta. X2 (1978) 151. 16 Sniderman. A.D.. Wolfson, C., Teng. B., Franklin, F.A., Bachorik. P.S. and Kwiterovich. P.O., Jr., Association of hyperapobetalipoproteinemia with endogenous hypertriglyceridemia and atherosclerosis. Ann. Intern. Med.. 7 (19X2) X33. 17 Durrington, P.N.. Hunt, L., Ishola, M., Kane, J. and Stepheno, W.P.. Serum Apohpoproteins Al and B and lipoproteins in middle-aged men with and without previous myocardial infarction. Br. Heart J., 56 (1986) 206. 1X Campcau. L.. Enjalbert. M.. Lesperance. J., Bourassa, M.G., Kwiterovich. P.O., Jr., Wacholder, S. and Sniderman. AD., The relation of risk factors to the development of atherosclerosis in saphenous-vein bypass grafts and the progression of disease in the native circulation: a study 10
19
20
21
22
23
24 25
26
27
2X
2Y
30 31
32
33
years after aortocoronary bypass surgery. New Engl. J. Med.. 311 (1984) 1329. Brunzell. J.D.. Schrott. H.G.. Motulsky, A.G. and Bierman, E.L., Myocardial infarction in the familial forms of hypertriglyceridemia. Metabolism, 25 (1976) 313. Havel, R.J., Eder, H. and Bragdon. J., The distribution and chemical composition of ultracentrifugally separated lipoproteins in human serum. J. Clin. Invest.. 34 (1953) 1345. Young. S.. Smith, R.S.. Hogle. D.M., Curtiss. L.K. and Witzum, J.L., Two new monoclonal antibody-based enzyme-linked assays of apolipoprotein B. Clin. Chem.. 32 (1986) 1484. Vega, G.L. and Grundy. SM., Does measurement of Apolipoprotein B have a place in cholesterol management? Arteriosclerosis. 10 (1990) 668. Halley. S.B., Rosenman. R.H.. Bawal. R.D. and Brand, R.J., Epidemiology as a guide to clinical decisions: the association between triglycerides and coronary heart diseasse. N. Engl. J. Med.. 302 (1980) 1383. Lees, R.S.. Immunoassay of plasma low densrty lipoproteins. Science. 169 (1970) 493. Schonfeld. G.. Lees. R.S.. George, P.K. and Pleger. B.. Assay of total plasma apolipoprotein B concentration in human subjects. J. Clin. Invest.. 53 (1974) 3358. Avogaro. P., Bittolo Bon. G.. Cazzolata. G. and Quinci, G.B., Are apolipoproteins better discriminators than lipids for atherosclerosis’? Lancet. 1 (1979) 90 I, Teng. B.. Thompson. G.R., Sniderman. A.D.. Forte. T.M., Kraus, R.M. and Kwiterovich, P.O., Jr.. Composition and distribution of low-density lipoprotein fractions in hyperapobetalipoproteinemia. normolipidemia and familial hypercholesterolemia. Proc. Natl. Acad. Sci. USA. X0 (lY83) 6662. Brunzell, J.D., Albers, J.J.. Chait. A.. Grundy. SM., Groszek. E. and McDonald. G.B., Plasma lipoproteins in familial combined hyperlipidemia and monogenic familial hypertriglyceridemia. J. Lipid Res.. 24 (lYX3) 147. Crouse. J.R.. Parks. J.S., Schey, H.M. and Kahl. F.R.. Studies of low density lipoprotein molecular weight in human beings with coronary artery disease. J. Lipid Res.. 36 (1985) 566. Austin, M.A. and Krauss, R.M.. Genetic control of lowdensity lipoprotein subclasses. Lancet. 2 (1986) 592. Knott. T.J., Pease, R.J.. Powell, L.M., Wallis. S.C.. Rail. SC.. Jr.. Innerarity, T.L. Blackhart. B.. Taylor, W.H.. Marcel, Y.. Milne. R.. Johnson, D.. Fuller. M.. Lusis, A.J.. McCarthy. B.J., Mahley, R.W., Levy-Wilson. B. and Scott, J.. Complete protein sequence and identification of structural domains of human apolipoprotein B. Nature. 323 (1986) 734. Albers. J.J., Lodge. M.S. and Curtiss, L.K., Evaluation of a monoclonal antibody-based enzyme-linked tmmunosorbent assay as a candidate reference method for the measurement of apolipoprotein B-100. J. Lipid Res.. 30 (lYX9) 1445. Cohn. J., McNamara. J.. Cohn. S., Ordovas. J. and Schae-
116
34
35
36
37
fer, F., Plasma apolipoprotein changes in the triglyceriderich lipoprotein fraction of human subjects fed a fat-rich meal. J. Lipid Res., 29 (1988) 925. Genest, J., Sniderman, A.D., Cianflone, K., Teng, B., Wacholder, S., Marcel, Y.L. and Kwiterovich, P.O., Jr., Hyperapobetalipoproteinemia: Plasma lipoprotein responses to oral fat load. Arteriosclerosis, 6 (1986) 297. Patsch, J.R., Hopferweiser, T.L., Muhlbergce, V., Knapp, E., Braunsteiner, H., Gotto, A.M., Jr., Dunn, K. and Patsch, W., Postprandial lipemia in patients with coronary artery disease. Circulation, 82 (1990) 111-284. Sane, T. and Nikkill, E.A., Very low density lipoprotein triglyceride metabolism in relatives of hypertriglyceridemic probands. Arteriosclerosis, 8 (1987) 217. Kissebah, A.H., Alfarsi, S. and Adams, P.W., Integrated
regulation of very low density lipoprotein triglyceride and apolipoprotein-B kinetics in man: normolipemic subjects, familial combined hyperlipidemia. Metabolism, 30 (1981) 856. 38 Vega, G.L. and Grundy, S.M., Primary hypertriglyceridemia with borderline high cholesterol and elevated apolipoprotein B concentrations: comparison of gemfibrozil vs lovastatin therapy. JAMA, 21 (1990) 2759. 39 Brown, G., Albers, J.J., Fisher, L.D., Schaefer, SM., Lin, J.T., Kaplan, C., Zhao, X.Q., Bisson, B.D., Fitzpatrick, V.F. and Dodge, H.T., Regression of coronary artery disease as a result of intensive lipid-lowering therapy in men with high levels of apolipoprotein B. New Engl. J. Med., 323 (1990) 1289.
109 Ireland,
Ltd. 0021.9150/91/$03.50
002191509100140E
ATHERQ
04660
Effect of moderate hypertriglyceridemia on the relation of plasma total and LDL apo B levels Allan Sniderman, Hai Vu and Katherine Cianflone .McGill Unit for the Prel,ention of Cardiocascular Disease, McGill Unilzrsity, Montreal, Quebec (Cunadal (Received 26 December, 1990) (Revised, received 27 February, 1991) (Accepted 25 March, 1991)
Summary The risk of premature coronary artery disease is related to an important degree to the number of particles of low density lipoproteins (LDL) in plasma, an estimate given by measurement of LDL apo B. In clinical practise, though, it is total, not LDL apo B, which is measured. The purpose of the present study therefore was to compare plasma total and LDL apo B in the presence and absence of moderate hypertriglyceridemia. The results demonstrate that within the range of plasma triglyceride levels examined, i.e., values of triglyceride up to 500 mg/dl, there is close correspondence between total and LDL apo B, with the latter more than 90% of the former. VLDL composition was also examined and two patterns found in hypertriglyceridemic patients: those with normal apo B had markedly lipid enriched VLDL while those with elevated apo B had VLDL which was normal in composition except for a moderate increase in triglyceride content. Thus total apo B within the circumstances studied reflects principally LDL apo B. Moreover measurement of apo B allows distinction between two different forms of hypertriglyceridemia, only one of which - that with an increased LDL particle number - has previous work shown to be associated with increased coronary risk. Total apo B, therefore, provides additional information not available from conventional plasma and lipoprotein lipids which allows more precise physiologic classification and may lead to more rational choice of pharmacologic therapy in normolipidemic and hypertriglyceridemic patients.
Key words:
Hypertriglyceridemia,
apo B; Hyperapobetalipoproteinemia
Correspondence to: Allan D. Sniderman, M.D., Cardiology Division. Royal Victoria Hospital, 687 Pine Avenue West, Montreal, Quebec, H3A 1Al Canada. Tel.: (514) 842-1231 ext. 4631; Fax: (514) 9X2-0686.
Introduction The present clinical practise to determine the atherogenic risk posed by lipoproteins is based on
110
measurement of serum and lipoprotein lipids. Thus if total, and more particularly, low density lipoprotein (LDL) cholesterol, is markedly elevated, risk is markedly elevated, and pharmacologic therapy is justified if dietary therapy fails. If plasma triglycerides are elevated, but LDL cholesterol is not, there is still no clear consensus as to how urgent or aggressive therapy should be. Marked elevation of LDL cholesterol to a level greater than the 90th percentile of the general population occurs in only a minority of patients with premature coronary artery disease [l-4]. Numerous prospective cross-sectional studies have shown apo B to be superior to total or LDL cholesterol in separating those with from those without premature coronary artery disease [4-l 11. That is, there is a substantially larger number of coronary patients with an apo B above the 90th percentile compared to those with an LDL cholesterol above this level [12,13]. However, apo B-100 is an integral constituent of 3 lipoprotein classes: very low density lipoproteins (VLDL), intermediate density lipoproteins (IDL), and low density lipoproteins (LDL). IDL and LDL are much smaller lipoprotein particles than are VLDL and this difference in size likely explains why IDL and LDL gain access to the arterial wall from the vessel lumen with greater ease than VLDL. Under most circumstances, by far the greatest proportion of the total apo B is found in LDL [14] and the same relations between apo B and risk of disease have been shown for LDL apo B as for total apo B [5,6]. While there is still little data on these relationships in hypertriglyceridemic patients, it is known that only a proportion of hypertriglyceridemic subjects have an elevated apo B, and that those who do appear to be at higher risk of coronary disease than those who do not [15-191. The primary objective of this study was to determine the relations between total and LDL apo B in a group of patients, some of whom were moderately hypertriglyceridemic. In addition, the composition of VLDL (that is, the composition of the d < 1.006 g/ml supernate lipoproteins) has been compared in those hypertriglyceridemic patients with, and those without, elevated apo B.
Methods
Fasting plasma samples were obtained from 56 patients. Their average age was 57.6 years; 32 were female, 24 male. Plasma was separated by centrifugation at 2500 x g at 4 ’ C. To obtain the d < 1.006 lipoproteins, 1 ml plasma was added to 4 ml of a salt solution of density 1.006 g/ml in an ultra clear centrifuge tube; VLDL was separated by ultracentrifugation at 40 000 rpm for 18 h in an SW50 rotor at 4” C [20] and the d < 1.006 g/ml supernate collected. The following parameters were measured: on plasma, total cholesterol, triglycerides, and apo B; on the d < 1.006 g/ml supernate, total and free cholesterol, triglycerides, phospholipids and apo B; on the d > 1.006 g/ml infranatant, apo B. Triglycerides, free and total cholesterol and phospholipids were measured by enzymatic kits (Boehringer, Mannheim) while apo B was measured by an ELISA immunoassay [21] as modified by Ortho Diagnostics, La Jolla, California. HDL cholesterol was measured by heparin manganese precipitation while LDL cholesterol was quantitated as the difference between total plasma cholesterol and the sum of VLDL and HDL cholesterol. VLDL apo B was defined as the apo B measured in the d < 1.006 g/ml supernate, while for the purposes of this study, LDL apo B was defined as the apo B measured in the d > 1.006 g/ml infranate. This would include intermediate density lipoproteins within the LDL fraction. LDL 3-20% gradient gel PAGE was performed by the method of Laemmli [22]. Statistical analysis was performed by r-test of 2 means or by ANOVA. Linear regression was performed by least-squares linear regression. Results
The average plasma lipid, lipoprotein lipid, and total apo B results for the total group are given in Table 1. The total group is also subdivided into those with a fasting plasma triglyceride < 200 mg/dl (the normotriglyceridemic patients) and those with a fasting plasma triglyceride > 200 mg/dl (the hypertriglyceridemic group). There
111 TABLE
I
Normo-triglyceridemia (n = 27)
Total (n = 57)
54.4 (18.8) 33F/24M 207.9 (101.9) 142.1 (96.3) 136.3 (66.8) 9.8
Age Sex Plasma TG VLDL TG Plasma
apo B
VLDL apo B
Chol
LDL Chol HDL Chol
53.3 (21.6) 16F/14M 289.6 (69.7) 214.1 (78.0) 155.1 (76.2) 12.1 (11.4) 143 (69.6) 271.3 (45.9) I65 (45.7) 35.6 (9.8)
55.5
(4.2) 108.1 (47.1) 264.7 (53.8) 185.9 (58.1) 45.1 (14.7)
0
r
a~08
lmg/d’l
100
50
150
PI-ASMA
200
250
300
, 353
apoB (mg/dl)
Fig. 2. Plasma apo B vs. LDL apo B in 27 normal triglyceridemic samples (plasma triglycerides < 200 mg/dl). Regression: LDL apo B = I.011 plasma apo B - 8.477; r’ = 0.992.
were 27 patients in the former group and 29 in the latter. The first issue examined was the relation between total apo B and LDL apo B in the entire group. These results are shown in Fig. 1. The
LDL
--
01
Normotriglyceridemia = plasma triglycerides < 200 mg/dl; Hypertriglyceridemia = plasma triglycerides > 200 mg/dl. TG = triglycerides (mg/dl); apo B = apoiipoprotein B (mg/dl); Chol = cholesterol (mg/dl).
35”
~DOB (mg/dli
Hyper-triglyceridemia (n = 30)
(15.6) 17F/lOM 117 (28.8) 62.1 (25.5) 115.3 (46.4) 7.2
(9.1) 126.5 (62.5) 268.2 (49.9) 174.9 (53.0) 40.1 (13.2)
LDL apo B Plasma
LDL ,-
350
i
linear regression obtained was y = 0.927x + 0.127 which indicates that, overall, LDL apo B accounted for just under 93% of the total apo B. The correlation coefficient was high with an r’ of 0.985 and inspection of the data indicates that this relationship held throughout the range of values. The results in the two subgroups are shown in Figs. 2 and 3. In the normotriglyceridemic group (Fig. 21, there is again a strong relation between plasma and LDL apo B with a slope of 1.011 and an r2 of 0.992. This, of course, is not unexpected. The results in Fig. 3 are more critical to the present study. Again however, a close relation
LDL
awB
(mg/dli
350,
300
i’>ll
I
250
:
zoo/ 150
100
, i_~L.._._ 0
50
/
I 100
150
PLASMA
200
250
300
350
apoB (mg/dl)
Fig. 1. Plasma apo B vs. LDL apo B in 57 normal and hypertriglyceridemic samples. Regression: LDL apo B = 0.927 plasma apo B + 0.128; r2 = 0.985.
_1
0
100
150
PLASMA
200
250
333
350
apoB (mg/dt)
Fig. 3. Plasma apo B vs. LDL apo B in 30 hypertriglyceridemic samples (plasma triglycerides > 200 mg/dl). Regression: LDL apo B = 0.9058 plasma apo B + 2.485; r’ = 0.983.
112 TABLE
2
VLDL
COMPOSITION:
Values
are expressed
Group
RATIO as average
OF LIPID/APO
B
(SD)
Triglyceride/ apo B
Cholesterol ester/ape B
Free cholesterol/apo
B
Phospholipid/ape
(1) NTG/NB
10.5
3.2
2.0
(2) NTG/HB
(5.2) 11.1
(1.2) 2.8
(0.8) 2.1
(0.7) NS 8.6
(1.1) NS 4.4
(3.6) 0.0005
(1.6) 0.0005 2.3
(7.1) 0.0025 9.7
(1:;) NS 0.0005 18.4 0.0001
(1.3) NS 0.0005 18.5 0.0001
(6.9) 0.01 0.0005 5.4 0.005
P (3) HTG/NB P (4) HTG/HB P P Group F value P
3
(5.2) NS 34.7 (12.2) 0.0005 19.4 (12.0) 0.0005 0.0005 11.4 0.0001
B
15.5 (11.7) 22.6 (14.0) 0.025 22.9
NTG = normotriglyceridemia (plasma triglycerides < 200 mg/dl); HTG = hypertriglyceridemia (plasma triglycerides > 200 mg/dl); NB = normal plasma apo B ( < 120 mg/dl); HB = high plasma apo B ( > 120 mg/dl); P values are calculated by t-test as compared to group 1: NTG/NB except where indicated. F value is calculated by one-way analysis of variance (ANOVA).
obtains with LDL apo B accounting for 91% (slope = 0.906) of the total apo B with an r2 of 0.983. These results indicate that.within the range of triglyceride values examined, the mass of apo B measured within VLDL accounts for little of the total apo B mass, and therefore, total apo B and LDL apo B retain a close relation even in moderately hypertriglyceridemic patients. Next, the composition of the d < 1.006 g/ml lipoproteins was examined. In all the hypertriglyceridemic patients, delipidated aliquots of this fraction were subjected to SDS 3-20% polyacrylamide gel electrophoresis; 100 pg of d < 1.006 protein was applied to the gel. In none of the samples was a band corresponding to apo B-48 identified; similarly in none of the samples tested was the presence of apo A-IV detected as compared to appropriate molecular weight standards. That is, in none was the presence of either chylomicrons or chylomicron remnants identified by this technique. Both apo B-48 and apo A-IV, however, were present in control chylomicron samples. Therefore, the d < 1.006 lipoproteins will be considered to be VLDL. Four subgroups were constructed: group 1 consisted of 18 individuals who were nor-
motriglyceridemic and had normal plasma apo B; group 2, 9 patients who were normotriglyceridemic and had elevated apo B; group 3, 15 patients who were hypertriglyceridemic with a normal apo B; and group 4, 15 patients who were hypertriglyceridemic with an elevated apo B. These results are given in Table 2 with all the lipid parameters expressed as a ratio to apo B. Since only one apo B molecule is present per VLDL particle, this allows the relative lipid load per particle to be compared. The average composition of the VLDL is the same in the two normotriglyceridemic patient groups while in the hypertriglyceridemic hyperapo B group (group 41, the only significant change is an increase in the triglyceride content per particle. This increase is relatively small, however, compared to the changes observed in the VLDL from the hypertriglyceridemic normal apo B subgroup (group 3). Here, the mass of all the lipid components is significantly increased per particle. That is, the average VLDL particle in this group contains more triglyceride, more cholesterol and more cholesterol ester than the VLDL in the other three groups. It is, therefore, larger and lipid loaded.
113 Discussion In most of the clinical studies which have compared the relative strengths of apo B and total and LDL cholesterol in separating those with from those without coronary disease, it is total, not LDL apo B which has been measured [5-111 and the relative importance of total versus LDL apo B in this regard has recently been questioned [22]. That is, in patients with elevated apo B, if a substantial portion of the apo B were in the d < 1.006 g/ml supernate, VLDL as well as LDL might be contributing importantly to the atherogenic process. However, the results of the present study establish that there is close correspondence between total and LDL apo B in subjects with plasma triglyceride levels up to 500 mg/dl. Indeed in all the groups examined, on average, LDL apo B accounted for more than 90% of the total apo B, and we conclude, therefore, that within the range of triglyceride values examined, total apo B is determined by LDL apo B with VLDL apo B making at most a relatively minor contribution. The atherogenic risk due to hypertriglyceridemia has been, and remains, controversial. Most epidemiologic analyses have considered it as a homogeneous disorder [23]. It is not, certainly with regard to apo B. This observation is not new; indeed it was one of the major findings of the first clinical study in which apo B was measured by immunoassay. In this seminal work, not only did Lees [24] establish that important variations in LDL composition as reflected by the LDL cholesterol/ape B ratio could occur, but in a subsequent study, he and his colleagues showed there was no predictable relation between plasma triglycerides and apo B in type IV hyperlipoproteinemia [25]. That is, in some hypertriglyceridemic patients it was elevated, while in others it was not. This finding was soon confirmed by others [15,16], and in the studies performed to date, hypertriglyceridemia with elevated apo B is much more strongly connected to atherosclerotic disease than hypertriglyceridemia with normal apo B [15-191. The evidence that the level of apo B correlates better with coronary risk than LDL cholesterol is now consistent and broad [4-111; there can,
therefore, be no doubt that the observations initially made in this regard by Avogaro and his colleagues [26] have been amply confirmed. The pathophysiologic basis for this rests in the now well-established differences in LDL composition that are so frequently found, particularly in patients with premature coronary artery disease. A variety of analytic techniques have established that patients with coronary disease frequently have increased numbers of smaller, denser, cholesterol-poor LDL particles [27-301. Since the content of apo B per LDL particle is fixed whereas that of cholesterol is not, LDL apo B is an accurate index of LDL particle number whereas LDL cholesterol frequently is not. Measurement of LDL apo B thus allows an elevated LDL particle number to be recognized in patients with a normal LDL cholesterol and either normal or elevated plasma triglycerides. Given the close agreement between total and LDL apo B demonstrated in this study, total apo B, a measure which is relatively easy to obtain clinically, would appear to be an acceptable clinical surrogate for LDL apo B, a measure which is not widely available. This study has limitations which must not be overlooked. The most important practical one is that severe hypertriglyceridemia was not examined. It should be noted, however, that severe hypertriglyceridemia is not common. and when it occurs, chylomicrons or chylomicron remnants are usually present in large amounts as well. The capture antibody used in this assay is directed at the carboxy terminal end of apo B, and therefore, would not recognize apo B-48 containing chylomicrons [2,3]. This antibody, however has been shown to recognize greater than 90% of the VLDL apo B even in patients with hypertriglyceridemia [32]. Moreover even had it cross-reacted completely, the mass of apo-B-48 in the d < 1.006 lipoproteins, under almost all circumstances constitutes only a minor proportion of the apo B in the d < 1.006 supernates present [33]. The other important issue is whether the apparent differences in VLDL composition in the two hypertriglyceridemic groups are valid. For example, chylomicron remnants, if present in sufficient quantities, could produce an elevated lipid to apo B ratio in the d < 1.006 g/ml supernate.
114 However, this seems unlikely for several reasons. First, gel electrophoresis failed to detect the presence of any such particles. Second, to explain the difference between groups, such remnant particles would have to be present in much greater quantities in the normal apo B hypertriglyceridemic group than in the hyperapo B hypertriglyceridemic group. Not only is there no reason to think this occurred but previous work suggests that the opposite would be more likely; that is, chylomicron clearance is related to the level of apo B as well as fasting plasma triglyceride level [34,35] and has been shown to be delayed even in normotriglyceridemic patients with Hyperapo B 1341. The data, therefore, suggest that hypertriglyceridemia might have resulted in these two patient groups by two pathogenetically distinguishable mechanisms. One possible explanation is that in the normal apo B group, normal numbers of lipid enriched hepatic apo B particles were secreted whereas in the hyperapo B group, increased numbers of VLDL particles of essentially normal composition were being secreted. Such differences have been seen in previous studies of different genetic forms of hypertriglyceridemia. For example, whereas there appears to be no important difference in the rate of triglyceride clearance in the two disorders [361, a normal secretion rate of triglyceride-enriched VLDL has been documented in familial hypertriglyceridemia while increased numbers of VLDL particles are secreted in familial combined hyperlipidemia [37]. Obviously no genetic diagnosis can be inferred in the present study; the point at hand, though, is that the measurements made in this study may allow recognition of the pathophysiologic basis of the hypertriglyceridemia in particular patients. More important still, they raise the issue as to what should be the objective, or objectives, of therapy in such hypertriglyceridemic patient groups. In the normal apo B hypertriglyceridemic group, lowering triglycerides and increasing HDL cholesterol are the obvious goals. But in the hyperapo B hypertriglyceridemic group, the issue is more complicated. This dilemma has recently been addressed by Vega and Grundy [381. In their study, hypertriglyceridemic patients with elevated apo B were treated with either gemfibrozil
or lovastatin: the former reduced triglycerides markedly but did not change the level of LDL apo B; by contrast, lovastatin sharply decreased LDL particle number. We would agree with the conclusion they drew, namely that given the evidence that the hypertriglyceridemic hyperapo B patients are at particular risk of coronary disease, the objectives of therapy should be examined in an appropriate clinical trial. In the interim, the best evidence at hand comes from the FATS trial [371. In that study, all patients had an elevated Apo B although their lipid phenotype varied. Clinical and angiographic improvement correlated best with decreases in Apo B and increases in HDL cholesterol, the same parameters which had previously been shown to be the best predictors of new disease progression in native coronary arteries or the appearance of lesions in saphenous vein bypass grafts [18]. The appropriate clinical conclusion for the moment appears to us to be that patients with elevated Apo B may benefit from pharmacologic therapy which lowers their plasma LDL particle number. The present study establishes that an elevated LDL particle number can be recognized in moderately hypertriglyceridemic as well as normotriglyceridemic individuals by measurement of total Apo B. Acknowledgements
This research was supported by grants from the Medical Research Council of Canada (M75480) and Merck Frosst Ltd. Canada. References Kannel, W.B., Castelli, W.P., Gordon, T. and McNamara, P.M., Serum cholesterol, lipoproteins and the risk of coronary heart disease. Ann. Intern. Med., 74 (1971) 1. Goldstein, J.L., Hazzard, W.R., Shrott, H.G., Bierman, E.L. and Motulsky, A.G., Hyperlipidemia in coronary heart disease, 1. Lipid levels in five survivors of myocardial infarction. J. Clin. Invest., 52 (1973) 1533. Gotto, A.M., Gorry, G.A., Thompson, J.R., Cole, J.S., Trost, R., Yeshurun, D. and DeBakry, M.R., Relationship between plasma lipid concentration and coronary artery disease in 496 patients. Circulation, 56 (1977) 875. Holmes, D.R. Jr., Elveback, S.R., Frye, R.S., Kottke, B.A. and Ellefson, R.D. Association of risk factor variables and coronary artery disease documented with angiography. Circulation, 63 (1981) 293.
115 H.D., Sniderman, A.D., Albers, J.J. and 5 Brunzell, Kwiterovich, P.O.. Jr.. Apoproteins B and A-l and coronary artery disease in humans. Arteriosclerosis, 4(2) (1984) 79. A.D., Apolipoprotein B and Apolipoprotein 6 Sniderman. AI as predictors of coronary artery disease. Can. J. Cardiol., 4 (SuppI. A) t 1988) 24A. N., Hayat, N. and Al-Khafaji, M., Lipopro7 Al-Muhtaseb. teins and apolipoproteins in young male survivors of myocardial infarction. Atherosclerosis, 77 (1989) 131. 8 Sullivan, D.R.. Marwick. T.H. and Freedman, S.B.. A new method of scoring coronary angiograms to reflect extent of coronary atherosclerosis and improve correlation with major risk factors. Am. Heart J., 119 (1990) 1262. Y Fujiwara, R.. Kutsumi. Y.. Hayashi, T., Kim. S.S., Misawa. T., Tada. H.. Nichio, H.. Toyota, K, Tamai, T.. Nakai, T and Miyabo. S.. Metabolic risk factors in the normolipidemic male patients with angiographically defined coronary artery disease. Jap. Circul. J., 54 (1990) 493. 10 Durrington. P.N., Ishola. M.. Hunt, L., Arrol, S. and Bhatnagar. D.. Apolipoproteins (a), AI and B and parental history in men with early onset ischaemic heart disease. Lancer 1 (19X8) 1070. 11 Reinhart, R.A.. Gani. K., Arndt. M.R. and Broste. SK., Apolipoproteins A-I and B as predictors of angiographitally defined coronary artery disease. Arch. Intern. Med., I50 (1990) 1629. 12 Blank. D.. Silberberg, J. and Sniderman, A.D., Trade-oTfs on cutpoints for the treatment of hyperlipidemia. Coron. Artery Dis. l(4) (1990) 455. 13 Albert, J.J.. Brunzell. J.D. and Knapp, Q.H.. Apoprotein measurements and their clinical application. Clin. Lab. Med.. 9 (1YXY) 137. 14 Teng. B.. Sniderman. A.D.. Soutar, A.K. and Thompson. G.R.. Metabolic basis of hyperapobetalipoproteinemia: turnover of apolipoprotein B in low-density lipoprotein and its precursors and subfractions compared with normal and familial hypercholesterolemia. J. Clin. Invest.. 77 ( 1986) h6.7. 15 Durrington. P.N., Bolton. C.H. and Hartog, M.. Serum and lipoprotein apolipoprotein B levels in normal subjects and patients with hyperlipoproteinaemia. Clin. Chim. Acta. X2 (1978) 151. 16 Sniderman. A.D.. Wolfson, C., Teng. B., Franklin, F.A., Bachorik. P.S. and Kwiterovich. P.O., Jr., Association of hyperapobetalipoproteinemia with endogenous hypertriglyceridemia and atherosclerosis. Ann. Intern. Med.. 7 (19X2) X33. 17 Durrington, P.N.. Hunt, L., Ishola, M., Kane, J. and Stepheno, W.P.. Serum Apohpoproteins Al and B and lipoproteins in middle-aged men with and without previous myocardial infarction. Br. Heart J., 56 (1986) 206. 1X Campcau. L.. Enjalbert. M.. Lesperance. J., Bourassa, M.G., Kwiterovich. P.O., Jr., Wacholder, S. and Sniderman. AD., The relation of risk factors to the development of atherosclerosis in saphenous-vein bypass grafts and the progression of disease in the native circulation: a study 10
19
20
21
22
23
24 25
26
27
2X
2Y
30 31
32
33
years after aortocoronary bypass surgery. New Engl. J. Med.. 311 (1984) 1329. Brunzell. J.D.. Schrott. H.G.. Motulsky, A.G. and Bierman, E.L., Myocardial infarction in the familial forms of hypertriglyceridemia. Metabolism, 25 (1976) 313. Havel, R.J., Eder, H. and Bragdon. J., The distribution and chemical composition of ultracentrifugally separated lipoproteins in human serum. J. Clin. Invest.. 34 (1953) 1345. Young. S.. Smith, R.S.. Hogle. D.M., Curtiss. L.K. and Witzum, J.L., Two new monoclonal antibody-based enzyme-linked assays of apolipoprotein B. Clin. Chem.. 32 (1986) 1484. Vega, G.L. and Grundy. SM., Does measurement of Apolipoprotein B have a place in cholesterol management? Arteriosclerosis. 10 (1990) 668. Halley. S.B., Rosenman. R.H.. Bawal. R.D. and Brand, R.J., Epidemiology as a guide to clinical decisions: the association between triglycerides and coronary heart diseasse. N. Engl. J. Med.. 302 (1980) 1383. Lees, R.S.. Immunoassay of plasma low densrty lipoproteins. Science. 169 (1970) 493. Schonfeld. G.. Lees. R.S.. George, P.K. and Pleger. B.. Assay of total plasma apolipoprotein B concentration in human subjects. J. Clin. Invest.. 53 (1974) 3358. Avogaro. P., Bittolo Bon. G.. Cazzolata. G. and Quinci, G.B., Are apolipoproteins better discriminators than lipids for atherosclerosis’? Lancet. 1 (1979) 90 I, Teng. B.. Thompson. G.R., Sniderman. A.D.. Forte. T.M., Kraus, R.M. and Kwiterovich, P.O., Jr.. Composition and distribution of low-density lipoprotein fractions in hyperapobetalipoproteinemia. normolipidemia and familial hypercholesterolemia. Proc. Natl. Acad. Sci. USA. X0 (lY83) 6662. Brunzell, J.D., Albers, J.J.. Chait. A.. Grundy. SM., Groszek. E. and McDonald. G.B., Plasma lipoproteins in familial combined hyperlipidemia and monogenic familial hypertriglyceridemia. J. Lipid Res.. 24 (lYX3) 147. Crouse. J.R.. Parks. J.S., Schey, H.M. and Kahl. F.R.. Studies of low density lipoprotein molecular weight in human beings with coronary artery disease. J. Lipid Res.. 36 (1985) 566. Austin, M.A. and Krauss, R.M.. Genetic control of lowdensity lipoprotein subclasses. Lancet. 2 (1986) 592. Knott. T.J., Pease, R.J.. Powell, L.M., Wallis. S.C.. Rail. SC.. Jr.. Innerarity, T.L. Blackhart. B.. Taylor, W.H.. Marcel, Y.. Milne. R.. Johnson, D.. Fuller. M.. Lusis, A.J.. McCarthy. B.J., Mahley, R.W., Levy-Wilson. B. and Scott, J.. Complete protein sequence and identification of structural domains of human apolipoprotein B. Nature. 323 (1986) 734. Albers. J.J., Lodge. M.S. and Curtiss, L.K., Evaluation of a monoclonal antibody-based enzyme-linked tmmunosorbent assay as a candidate reference method for the measurement of apolipoprotein B-100. J. Lipid Res.. 30 (lYX9) 1445. Cohn. J., McNamara. J.. Cohn. S., Ordovas. J. and Schae-
116
34
35
36
37
fer, F., Plasma apolipoprotein changes in the triglyceriderich lipoprotein fraction of human subjects fed a fat-rich meal. J. Lipid Res., 29 (1988) 925. Genest, J., Sniderman, A.D., Cianflone, K., Teng, B., Wacholder, S., Marcel, Y.L. and Kwiterovich, P.O., Jr., Hyperapobetalipoproteinemia: Plasma lipoprotein responses to oral fat load. Arteriosclerosis, 6 (1986) 297. Patsch, J.R., Hopferweiser, T.L., Muhlbergce, V., Knapp, E., Braunsteiner, H., Gotto, A.M., Jr., Dunn, K. and Patsch, W., Postprandial lipemia in patients with coronary artery disease. Circulation, 82 (1990) 111-284. Sane, T. and Nikkill, E.A., Very low density lipoprotein triglyceride metabolism in relatives of hypertriglyceridemic probands. Arteriosclerosis, 8 (1987) 217. Kissebah, A.H., Alfarsi, S. and Adams, P.W., Integrated
regulation of very low density lipoprotein triglyceride and apolipoprotein-B kinetics in man: normolipemic subjects, familial combined hyperlipidemia. Metabolism, 30 (1981) 856. 38 Vega, G.L. and Grundy, S.M., Primary hypertriglyceridemia with borderline high cholesterol and elevated apolipoprotein B concentrations: comparison of gemfibrozil vs lovastatin therapy. JAMA, 21 (1990) 2759. 39 Brown, G., Albers, J.J., Fisher, L.D., Schaefer, SM., Lin, J.T., Kaplan, C., Zhao, X.Q., Bisson, B.D., Fitzpatrick, V.F. and Dodge, H.T., Regression of coronary artery disease as a result of intensive lipid-lowering therapy in men with high levels of apolipoprotein B. New Engl. J. Med., 323 (1990) 1289.
